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Sample records for engineered chaperonin protein

  1. Ordered nanoparticle arrays formed on engineered chaperonin protein templates

    Science.gov (United States)

    McMillan, R. Andrew; Paavola, Chad D.; Howard, Jeanie; Chan, Suzanne L.; Zaluzec, Nestor J.; Trent, Jonathan D.

    2002-01-01

    Traditional methods for fabricating nanoscale arrays are usually based on lithographic techniques. Alternative new approaches rely on the use of nanoscale templates made of synthetic or biological materials. Some proteins, for example, have been used to form ordered two-dimensional arrays. Here, we fabricated nanoscale ordered arrays of metal and semiconductor quantum dots by binding preformed nanoparticles onto crystalline protein templates made from genetically engineered hollow double-ring structures called chaperonins. Using structural information as a guide, a thermostable recombinant chaperonin subunit was modified to assemble into chaperonins with either 3 nm or 9 nm apical pores surrounded by chemically reactive thiols. These engineered chaperonins were crystallized into two-dimensional templates up to 20 microm in diameter. The periodic solvent-exposed thiols within these crystalline templates were used to size-selectively bind and organize either gold (1.4, 5 or 10nm) or CdSe-ZnS semiconductor (4.5 nm) quantum dots into arrays. The order within the arrays was defined by the lattice of the underlying protein crystal. By combining the self-assembling properties of chaperonins with mutations guided by structural modelling, we demonstrate that quantum dots can be manipulated using modified chaperonins and organized into arrays for use in next-generation electronic and photonic devices.

  2. In silico engineering of aggregation-prone recombinant proteins for substrate recognition by the chaperonin GroEL

    National Research Council Canada - National Science Library

    Kumar, Vipul; Punetha, Ankita; Sundar, Durai; Chaudhuri, Tapan K

    2012-01-01

    Molecular chaperones appear to have been evolved to facilitate protein folding in the cell through entrapment of folding intermediates on the interior of a large cavity formed between GroEL and its co-chaperonin GroES...

  3. Chloroplast Chaperonin: An Intricate Protein Folding Machine for Photosynthesis

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    Qian Zhao

    2018-01-01

    Full Text Available Group I chaperonins are large cylindrical-shaped nano-machines that function as a central hub in the protein quality control system in the bacterial cytosol, mitochondria and chloroplasts. In chloroplasts, proteins newly synthesized by chloroplast ribosomes, unfolded by diverse stresses, or translocated from the cytosol run the risk of aberrant folding and aggregation. The chloroplast chaperonin system assists these proteins in folding into their native states. A widely known protein folded by chloroplast chaperonin is the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco, an enzyme responsible for the fixation of inorganic CO2 into organic carbohydrates during photosynthesis. Chloroplast chaperonin was initially identified as a Rubisco-binding protein. All photosynthetic eucaryotes genomes encode multiple chaperonin genes which can be divided into α and β subtypes. Unlike the homo-oligomeric chaperonins from bacteria and mitochondria, chloroplast chaperonins are more complex and exists as intricate hetero-oligomers containing both subtypes. The Group I chaperonin requires proper interaction with a detachable lid-like co-chaperonin in the presence of ATP and Mg2+ for substrate encapsulation and conformational transition. Besides the typical Cpn10-like co-chaperonin, a unique co-chaperonin consisting of two tandem Cpn10-like domains joined head-to-tail exists in chloroplasts. Since chloroplasts were proposed as sensors to various environmental stresses, this diversified chloroplast chaperonin system has the potential to adapt to complex conditions by accommodating specific substrates or through regulation at both the transcriptional and post-translational levels. In this review, we discuss recent progress on the unique structure and function of the chloroplast chaperonin system based on model organisms Chlamydomonas reinhardtii and Arabidopsis thaliana. Knowledge of the chloroplast chaperonin system may ultimately lead

  4. Substrate protein recognition mechanism of archaeal and eukaryotic chaperonins.

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    Shrestha, Pooja; Jayasinghe, Manori; Stan, George

    2009-03-01

    Chaperonins are double ring-shaped biological nanomachines that assist protein folding. Spectacular conformational changes take place within each chaperonin ring using energy derived from ATP hydrolysis. These changes result in transitions from the open to the closed ring. Substrate proteins bind to the open ring and are encapsulated within the closed ring cavity. We focus on the substrate protein recognition mechanism of archaeal and eukaryotic chaperonins. We predict substrate protein binding sites using structural and bioinformatic analyses of functional states during the chaperonin cycle. Based on large changes in solvent accessible surface area and contact maps we glean the functional role of chaperonin amino acids. During the transition between open to closed chaperonin ring, the largest change in accessible surface area of amino acids is found in helical protrusion and two helices located at the cavity opening. Our calculations suggest that the helical protrusion and two helices constitute the substrate protein binding site.

  5. Chaperonin Structure - The Large Multi-Subunit Protein Complex

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    Irena Roterman

    2009-03-01

    Full Text Available The multi sub-unit protein structure representing the chaperonins group is analyzed with respect to its hydrophobicity distribution. The proteins of this group assist protein folding supported by ATP. The specific axial symmetry GroEL structure (two rings of seven units stacked back to back - 524 aa each and the GroES (single ring of seven units - 97 aa each polypeptide chains are analyzed using the hydrophobicity distribution expressed as excess/deficiency all over the molecule to search for structure-to-function relationships. The empirically observed distribution of hydrophobic residues is confronted with the theoretical one representing the idealized hydrophobic core with hydrophilic residues exposure on the surface. The observed discrepancy between these two distributions seems to be aim-oriented, determining the structure-to-function relation. The hydrophobic force field structure generated by the chaperonin capsule is presented. Its possible influence on substrate folding is suggested.

  6. Dataset concerning GroEL chaperonin interaction with proteins

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    V.V. Marchenkov

    2016-03-01

    Full Text Available GroEL chaperonin is well-known to interact with a wide variety of polypeptide chains. Here we show the data related to our previous work (http://dx.doi.org/10.1016/j.pep.2015.11.020 [1], and concerning the interaction of GroEL with native (lysozyme, α-lactalbumin and denatured (lysozyme, α-lactalbumin and pepsin proteins in solution. The use of affinity chromatography on the base of denatured pepsin for GroEL purification from fluorescent impurities is represented as well.

  7. Chaperonins fight aminoglycoside-induced protein misfolding and promote short-term tolerance in Escherichia coli

    DEFF Research Database (Denmark)

    Goltermann, Lise; Good, Liam; Bentin, Thomas

    2013-01-01

    For almost half of a century, we have known that aminoglycoside antibiotics corrupt ribosomes, causing translational misreading, yet it remains unclear whether or not misreading triggers protein misfolding, and possible effects of chaperone action on drug susceptibilities are poorly understood....... Here, we show that aminoglycosides cause cytosolic protein misfolding and that chaperonin GroEL/GroES overexpression counters this defect. During aminoglycoside exposure to exponential cultures, chaperonin overexpression protected the bacterial membrane potential, rescued cell growth, and facilitated...

  8. Circular Permutation of a Chaperonin Protein: Biophysics and Application to Nanotechnology

    Science.gov (United States)

    Paavola, Chad; Chan, Suzanne; Li, Yi-Fen; McMillan, R. Andrew; Trent, Jonathan

    2004-01-01

    We have designed five circular permutants of a chaperonin protein derived from the hyperthermophilic organism Sulfolobus shibatae. These permuted proteins were expressed in E. coli and are well-folded. Furthermore, all the permutants assemble into 18-mer double rings of the same form as the wild-type protein. We characterized the thermodynamics of folding for each permutant by both guanidine denaturation and differential scanning calorimetry. We also examined the assembly of chaperonin rings into higher order structures that may be used as nanoscale templates. The results show that circular permutation can be used to tune the thermodynamic properties of a protein template as well as facilitating the fusion of peptides, binding proteins or enzymes onto nanostructured templates.

  9. Knotting and unknotting proteins in the chaperonin cage: Effects of the excluded volume.

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    Szymon Niewieczerzal

    Full Text Available Molecular dynamics simulations are used to explore the effects of chaperonin-like cages on knotted proteins with very low sequence similarity, different depths of a knot but with a similar fold, and the same type of topology. The investigated proteins are VirC2, DndE and MJ0366 with two depths of a knot. A comprehensive picture how encapsulation influences folding rates is provided based on the analysis of different cage sizes and temperature conditions. Neither of these two effects with regard to knotted proteins has been studied by means of molecular dynamics simulations with coarse-grained structure-based models before. We show that encapsulation in a chaperonin is sufficient to self-tie and untie small knotted proteins (VirC2, DndE, for which the equilibrium process is not accessible in the bulk solvent. Furthermore, we find that encapsulation reduces backtracking that arises from the destabilisation of nucleation sites, smoothing the free energy landscape. However, this effect can also be coupled with temperature rise. Encapsulation facilitates knotting at the early stage of folding and can enhance an alternative folding route. Comparison to unknotted proteins with the same fold shows directly how encapsulation influences the free energy landscape. In addition, we find that as the size of the cage decreases, folding times increase almost exponentially in a certain range of cage sizes, in accordance with confinement theory and experimental data for unknotted proteins.

  10. Multivalent binding of nonnative substrate proteins by the chaperonin GroEL.

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    Farr, G W; Furtak, K; Rowland, M B; Ranson, N A; Saibil, H R; Kirchhausen, T; Horwich, A L

    2000-03-03

    The chaperonin GroEL binds nonnative substrate protein in the central cavity of an open ring through exposed hydrophobic residues at the inside aspect of the apical domains and then mediates productive folding upon binding ATP and the cochaperonin GroES. Whether nonnative proteins bind to more than one of the seven apical domains of a GroEL ring is unknown. We have addressed this using rings with various combinations of wild-type and binding-defective mutant apical domains, enabled by their production as single polypeptides. A wild-type extent of binary complex formation with two stringent substrate proteins, malate dehydrogenase or Rubisco, required a minimum of three consecutive binding-proficient apical domains. Rhodanese, a less-stringent substrate, required only two wild-type domains and was insensitive to their arrangement. As a physical correlate, multivalent binding of Rubisco was directly observed in an oxidative cross-linking experiment.

  11. Versatile platform for nanotechnology based on circular permutations of chaperonin protein

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    Paavola, Chad D. (Inventor); Trent, Jonathan D. (Inventor); Chan, Suzanne L. (Inventor); Li, Yi-Fen (Inventor); McMillan, R. Andrew (Inventor); Kagawa, Hiromi (Inventor)

    2010-01-01

    The present invention provides chaperonin polypeptides which are modified to include N-terminal and C-terminal ends that are relocated from the central pore region to various different positions in the polypeptide which are located on the exterior of the folded modified chaperonin polypeptide. In the modified chaperonin polypeptide, the naturally-occurring N-terminal and C-terminal ends are joined together directly or with an intervening linker peptide sequence. The relocated N-terminal or C-terminal ends can be covalently joined to, or bound with another molecule such as a nucleic acid molecule, a lipid, a carbohydrate, a second polypeptide, or a nanoparticle. The modified chaperonin polypeptides can assemble into double-ringed chaperonin structures. Further, the chaperonin structures can organize into higher order structures such as nanofilaments or nanoarrays which can be used to produce nanodevices and nanocoatings.

  12. Chaperonin filaments: The archael cytoskeleton

    Energy Technology Data Exchange (ETDEWEB)

    Trent, J.D.; Kagawa, H.K.; Yaoi, Takuro; Olle, E.; Zaluzec, N.J.

    1997-08-01

    Chaperonins are multi-subunit double-ring complexed composed of 60-kDa proteins that are believed to mediate protein folding in vivo. The chaperonins in the hyperthermophilic archaeon Sulfolobus shibatae are composed of the organism`s two most abundant proteins, which represent 4% of its total protein and have an intracellular concentration of {ge} 3.0 mg/ml. At concentrations of 1.0 mg/ml, purified chaperonin proteins aggregate to form ordered filaments. Filament formation, which requires Mg{sup ++} and nucleotide binding (not hydrolysis), occurs at physiological temperatures under conditions suggesting filaments may exist in vivo. If the estimated 4,600 chaperonins per cell, formed filaments in vivo, they could create a matrix of filaments that would span the diameter of an average S. shibatae cell 100 times. Direct observations of unfixed, minimally treated cells by intermediate voltage electron microscopy (300 kV) revealed an intracellular network of filaments that resembles chaperonin filaments produced in vitro. The hypothesis that the intracellular network contains chaperonins is supported by immunogold analyses. The authors propose that chaperonin activity may be regulated in vivo by filament formation and that chaperonin filaments may serve a cytoskeleton-like function in archaea and perhaps in other prokaryotes.

  13. Chaperonin-based biolayer interferometry to assess the kinetic stability of metastable, aggregation-prone proteins

    Science.gov (United States)

    Lea, Wendy A.; Naik, Subhashchandra; Chaudhri, Tapan; Machen, Alexandra J.; O’Neil, Pierce T.; McGinn-Straub, Wesley; Tischer, Alexander; Auton, Matthew T.; Burns, Joshua R.; Baldwin, Michael R.; Khar, Karen R.; Karanicolas, John; Fisher, Mark T.

    2017-01-01

    Stabilizing the folded state of metastable and/or aggregation-prone proteins through exogenous ligand binding is an appealing strategy to decrease disease pathologies brought on by protein folding defects or deleterious kinetic transitions. Current methods of examining ligand binding to these marginally stable native states are limited, because protein aggregation typically interferes with analysis. Here, we describe a rapid method for assessing the kinetic stability of folded proteins and monitoring the effects of ligand stabilization for both intrinsically stable proteins (monomers, oligomers, multi-domain) and metastable proteins (e.g. low Tm) that uses a new GroEL chaperonin-based biolayer interferometry (BLI) denaturant-pulse platform. A kinetically controlled denaturation isotherm is generated by exposing a target protein immobilized on a BLI biosensor to increasing denaturant concentrations (urea or GnHCl) in a pulsatile manner to induce partial or complete unfolding of the attached protein population. Following the rapid removal of the denaturant, the extent of hydrophobic unfolded/partially folded species that remain is detected by increased GroEL binding. Since this kinetic denaturant pulse is brief, the amplitude of the GroEL binding to the immobilized protein depends on the duration of exposure to denaturant, the concentration of denaturant, wash times, and the underlying protein unfolding/refolding kinetics; fixing all other parameters and plotting GroEL binding amplitude versus denaturant pulse concentration results in a kinetically controlled denaturation isotherm. When folding osmolytes or stabilizing ligands are added to the immobilized target proteins before and during the denaturant pulse, the diminished population of unfolded/partially folded protein is manifested by a decreased GroEL binding and/or a marked shift in these kinetically controlled denaturation profiles to higher denaturant concentrations. This particular platform approach can be

  14. Potential of mean force between a large solute and a biomolecular complex: A model analysis on protein flux through chaperonin system

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    Amano, Ken-ich; Oshima, Hiraku; Kinoshita, Masahiro

    2011-11-01

    Insertion of a large solute into an even larger vessel comprising biopolymers followed by release of the same solute from it is one of the important functions sustaining life. As a typical example, an unfolded protein is inserted into a chaperonin from bulk aqueous solution, a cochaperonin acting as a lid is attached to the chaperonin rim and the protein folds into its native structure within the closed cavity, the cochaperonin is detached after the folding is finished, and the folded protein is released back to the bulk solution. On the basis of the experimental observations manifesting that the basic aspects of the protein flux through the chaperonin system is independent of the chaperonin, cochaperonin, and protein species, we adopt a simple model system with which we can cover the whole cycle of the protein flux. We calculate the spatial distribution of the solvent-mediated potential of mean force (PMF) between a spherical solute and a cylindrical vessel or vessel/lid complex. The calculation is performed using the three-dimensional integral equation theory, and the PMF is decomposed into energetic and entropic components. We argue that an unfolded protein with a larger excluded volume (EV) and weak hydrophobicity is entropically inserted into the chaperonin cavity and constrained within a small space almost in its center. The switch from insertion to release is achieved by decreasing the EV and turning the protein surface hydrophilic in the folding process. For this release, in which the energetic component is a requisite, the feature that the chaperonin inner surface in the absence of the cochaperonin is not hydrophilic plays essential roles. On the other hand, the inner surface of the chaperonin/cochaperonin complex is hydrophilic, and the protein is energetically repelled from it: The protein remains constrained within the small space mentioned above without contacting the inner surface for correct folding. The structural and inner-surface properties of the

  15. Chaperonin filaments: The archaeal cytoskeleton?

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    Trent, Jonathan D.; Kagawa, Hiromi K.; Yaoi, Takuro; Olle, Eric; Zaluzec, Nestor J.

    1997-01-01

    Chaperonins are high molecular mass double-ring structures composed of 60-kDa protein subunits. In the hyperthermophilic archaeon Sulfolobus shibatae the two chaperonin proteins represent ≈4% of its total protein and have a combined intracellular concentration of >30 mg/ml. At concentrations ≥ 0.5 mg/ml purified chaperonins form filaments in the presence of Mg2+ and nucleotides. Filament formation requires nucleotide binding (not hydrolysis), and occurs at physiological temperatures in biologically relevant buffers, including a buffer made from cell extracts. These observations suggest that chaperonin filaments may exist in vivo and the estimated 4600 chaperonins per cell suggest that such filaments could form an extensive cytostructure. We observed filamentous structures in unfixed, uranyl-acetate-stained S. shibatae cells, which resemble the chaperonin filaments in size and appearance. ImmunoGold (Janssen) labeling using chaperonin antibodies indicated that many chaperonins are associated with insoluble cellular structures and these structures appear to be filamentous in some areas, although they could not be uranyl-acetate-stained. The existence of chaperonin filaments in vivo suggests a mechanism whereby their protein-folding activities can be regulated. More generally, the filaments themselves may play a cytoskeletal role in Archaea. PMID:9144246

  16. Folding of newly translated membrane protein CCR5 is assisted by the chaperonin GroEL-GroES

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    Chi, Haixia; Wang, Xiaoqiang; Li, Jiqiang; Ren, Hao; Huang, Fang

    2015-11-01

    The in vitro folding of newly translated human CC chemokine receptor type 5 (CCR5), which belongs to the physiologically important family of G protein-coupled receptors (GPCRs), has been studied in a cell-free system supplemented with the surfactant Brij-35. The freshly synthesized CCR5 can spontaneously fold into its biologically active state but only slowly and inefficiently. However, on addition of the GroEL-GroES molecular chaperone system, the folding of the nascent CCR5 was significantly enhanced, as was the structural stability and functional expression of the soluble form of CCR5. The chaperonin GroEL was partially effective on its own, but for maximum efficiency both the GroEL and its GroES lid were necessary. These results are direct evidence for chaperone-assisted membrane protein folding and therefore demonstrate that GroEL-GroES may be implicated in the folding of membrane proteins.

  17. Purification and characterization of chaperonin 60 and heat-shock protein 70 from chromoplasts of Narcissus pseudonarcissus.

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    Bonk, M; Tadros, M; Vandekerckhove, J; Al-Babili, S; Beyer, P

    1996-01-01

    In chromoplast differentiation during flower formation in Narcissus pseudonarcissus, the molecular chaperones chaperonin 60 (Cpn60; alpha and beta) and heat-shock protein 70 (Hsp70) greatly increase in abundance. Both were purified and shown to be present in a functional form in chromoplasts, indicating their requirement in the extensive structural rearrangements during the chloroplast-to-chromoplast transition. The purified proteins, sequenced N terminally and from internal peptides, showed strong homology to plastid Cpn60 and Hsp 70 representatives from other plant species. During chromoplast differentiation, the carotenoid biosynthetic pathway is strongly induced. The corresponding enzymes are all nuclear encoded and form a large, soluble, hetero-oligomeric protein complex after import but prior to their membrane attachment. By immunoprecipitations we have shown that the plastid Hsp70 is a structural constituent of a soluble entity also containing phytoene desaturase. PMID:8754688

  18. Bardet-Biedl Syndrome as a Chaperonopathy: Dissecting the Major Role of Chaperonin-Like BBS Proteins (BBS6-BBS10-BBS12

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    María Álvarez-Satta

    2017-07-01

    Full Text Available Bardet-Biedl syndrome (BBS is a rare genetic disorder that belongs to the group of ciliopathies, defined as diseases caused by defects in cilia structure and/or function. The six diagnostic features considered for this syndrome include retinal dystrophy, obesity, polydactyly, cognitive impairment and renal and urogenital anomalies. Furthermore, three of the 21 genes currently known to be involved in BBS encode chaperonin-like proteins (MKKS/BBS6, BBS10, and BBS12, so BBS can be also considered a member of the growing group of chaperonopathies. Remarkably, up to 50% of clinically-diagnosed BBS families can harbor disease-causing variants in these three genes, which highlights the importance of chaperone defects as pathogenic factors even for genetically heterogeneous syndromes such as BBS. In addition, it is interesting to note that BBS families with deleterious variants in MKKS/BBS6, BBS10 or BBS12 genes generally display more severe phenotypes than families with changes in other BBS genes. The chaperonin-like BBS proteins have structural homology to the CCT family of group II chaperonins, although they are believed to conserve neither the ATP-dependent folding activity of canonical CCT chaperonins nor the ability to form CCT-like oligomeric complexes. Thus, they play an important role in the initial steps of assembly of the BBSome, which is a multiprotein complex essential for mediating the ciliary trafficking activity. In this review, we present a comprehensive review of those genetic, functional and evolutionary aspects concerning chaperonin-like BBS proteins, trying to provide a new perspective that expands the classical conception of BBS only from a ciliary point of view.

  19. Assisted protein folding at low temperature: evolutionary adaptation of the Antarctic fish chaperonin CCT and its client proteins

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    Cuellar, Jorge; Yébenes, Hugo; Parker, Sandra K.; Carranza, Gerardo; Serna, Marina; Valpuesta, José María; Zabala, Juan Carlos; Detrich, H. William

    2014-01-01

    ABSTRACT Eukaryotic ectotherms of the Southern Ocean face energetic challenges to protein folding assisted by the cytosolic chaperonin CCT. We hypothesize that CCT and its client proteins (CPs) have co-evolved molecular adaptations that facilitate CCT–CP interaction and the ATP-driven folding cycle at low temperature. To test this hypothesis, we compared the functional and structural properties of CCT–CP systems from testis tissues of an Antarctic fish, Gobionotothen gibberifrons (Lönnberg) (habitat/body T = −1.9 to +2°C), and of the cow (body T = 37°C). We examined the temperature dependence of the binding of denatured CPs (β-actin, β-tubulin) by fish and bovine CCTs, both in homologous and heterologous combinations and at temperatures between −4°C and 20°C, in a buffer conducive to binding of the denatured CP to the open conformation of CCT. In homologous combination, the percentage of G. gibberifrons CCT bound to CP declined linearly with increasing temperature, whereas the converse was true for bovine CCT. Binding of CCT to heterologous CPs was low, irrespective of temperature. When reactions were supplemented with ATP, G. gibberifrons CCT catalyzed the folding and release of actin at 2°C. The ATPase activity of apo-CCT from G. gibberifrons at 4°C was ∼2.5-fold greater than that of apo-bovine CCT, whereas equivalent activities were observed at 20°C. Based on these results, we conclude that the catalytic folding cycle of CCT from Antarctic fishes is partially compensated at their habitat temperature, probably by means of enhanced CP-binding affinity and increased flexibility of the CCT subunits. PMID:24659247

  20. Cloning, characterization and sub-cellular localization of gamma subunit of T-complex protein-1 (chaperonin) from Leishmania donovani

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    Bhaskar,; Kumari, Neeti [Division of Biochemistry, CSIR-Central Drug Research Institute, Chattar Manzil Palace, PO Box 173, Lucknow (India); Goyal, Neena, E-mail: neenacdri@yahoo.com [Division of Biochemistry, CSIR-Central Drug Research Institute, Chattar Manzil Palace, PO Box 173, Lucknow (India)

    2012-12-07

    Highlights: Black-Right-Pointing-Pointer The study presents cloning and characterization of TCP1{gamma} gene from L. donovani. Black-Right-Pointing-Pointer TCP1{gamma} is a subunit of T-complex protein-1 (TCP1), a chaperonin class of protein. Black-Right-Pointing-Pointer LdTCP{gamma} exhibited differential expression in different stages of promastigotes. Black-Right-Pointing-Pointer LdTCP{gamma} co-localized with actin, a cytoskeleton protein. Black-Right-Pointing-Pointer The data suggests that this gene may have a role in differentiation/biogenesis. Black-Right-Pointing-Pointer First report on this chapronin in Leishmania. -- Abstract: T-complex protein-1 (TCP1) complex, a chaperonin class of protein, ubiquitous in all genera of life, is involved in intracellular assembly and folding of various proteins. The gamma subunit of TCP1 complex (TCP1{gamma}), plays a pivotal role in the folding and assembly of cytoskeleton protein(s) as an individual or complexed with other subunits. Here, we report for the first time cloning, characterization and expression of the TCP1{gamma} of Leishmania donovani (LdTCP1{gamma}), the causative agent of Indian Kala-azar. Primary sequence analysis of LdTCP1{gamma} revealed the presence of all the characteristic features of TCP1{gamma}. However, leishmanial TCP1{gamma} represents a distinct kinetoplastid group, clustered in a separate branch of the phylogenic tree. LdTCP1{gamma} exhibited differential expression in different stages of promastigotes. The non-dividing stationary phase promastigotes exhibited 2.5-fold less expression of LdTCP1{gamma} as compared to rapidly dividing log phase parasites. The sub-cellular distribution of LdTCP1{gamma} was studied in log phase promastigotes by employing indirect immunofluorescence microscopy. The protein was present not only in cytoplasm but it was also localized in nucleus, peri-nuclear region, flagella, flagellar pocket and apical region. Co-localization of LdTCP1{gamma} with actin suggests

  1. Difference in the distribution pattern of substrate enzymes in the metabolic network of Escherichia coli, according to chaperonin requirement.

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    Takemoto, Kazuhiro; Niwa, Tatsuya; Taguchi, Hideki

    2011-06-24

    Chaperonins are important in living systems because they play a role in the folding of proteins. Earlier comprehensive analyses identified substrate proteins for which folding requires the chaperonin GroEL/GroES (GroE) in Escherichia coli, and they revealed that many chaperonin substrates are metabolic enzymes. This result implies the importance of chaperonins in metabolism. However, the relationship between chaperonins and metabolism is still unclear. We investigated the distribution of chaperonin substrate enzymes in the metabolic network using network analysis techniques as a first step towards revealing this relationship, and found that as chaperonin requirement increases, substrate enzymes are more laterally distributed in the metabolic. In addition, comparative genome analysis showed that the chaperonin-dependent substrates were less conserved, suggesting that these substrates were acquired later on in evolutionary history. This result implies the expansion of metabolic networks due to this chaperonin, and it supports the existing hypothesis of acceleration of evolution by chaperonins. The distribution of chaperonin substrate enzymes in the metabolic network is inexplicable because it does not seem to be associated with individual protein features such as protein abundance, which has been observed characteristically in chaperonin substrates in previous works. However, it becomes clear by considering this expansion process due to chaperonin. This finding provides new insights into metabolic evolution and the roles of chaperonins in living systems.

  2. Difference in the distribution pattern of substrate enzymes in the metabolic network of Escherichia coli, according to chaperonin requirement

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    Niwa Tatsuya

    2011-06-01

    Full Text Available Abstract Background Chaperonins are important in living systems because they play a role in the folding of proteins. Earlier comprehensive analyses identified substrate proteins for which folding requires the chaperonin GroEL/GroES (GroE in Escherichia coli, and they revealed that many chaperonin substrates are metabolic enzymes. This result implies the importance of chaperonins in metabolism. However, the relationship between chaperonins and metabolism is still unclear. Results We investigated the distribution of chaperonin substrate enzymes in the metabolic network using network analysis techniques as a first step towards revealing this relationship, and found that as chaperonin requirement increases, substrate enzymes are more laterally distributed in the metabolic. In addition, comparative genome analysis showed that the chaperonin-dependent substrates were less conserved, suggesting that these substrates were acquired later on in evolutionary history. Conclusions This result implies the expansion of metabolic networks due to this chaperonin, and it supports the existing hypothesis of acceleration of evolution by chaperonins. The distribution of chaperonin substrate enzymes in the metabolic network is inexplicable because it does not seem to be associated with individual protein features such as protein abundance, which has been observed characteristically in chaperonin substrates in previous works. However, it becomes clear by considering this expansion process due to chaperonin. This finding provides new insights into metabolic evolution and the roles of chaperonins in living systems.

  3. Affinity chromatography of GroEL chaperonin based on denatured proteins: role of electrostatic interactions in regulation of GroEL affinity for protein substrates.

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    Marchenko, N Iu; Marchenkov, V V; Kaĭsheva, A L; Kashparov, I A; Kotova, N V; Kaliman, P A; Semisotnov, G V

    2006-12-01

    The chaperonin GroEL of the heat shock protein family from Escherichia coli cells can bind various polypeptides lacking rigid tertiary structure and thus prevent their nonspecific association and provide for acquisition of native conformation. In the present work we studied the interaction of GroEL with six denatured proteins (alpha-lactalbumin, ribonuclease A, egg lysozyme in the presence of dithiothreitol, pepsin, beta-casein, and apocytochrome c) possessing negative or positive total charge at neutral pH values and different in hydrophobicity (affinity for a hydrophobic probe ANS). To prevent the influence of nonspecific association of non-native proteins on their interaction with GroEL and make easier the recording of the complexing, the proteins were covalently attached to BrCN-activated Sepharose. At low ionic strength (lower than 60 mM), tight binding of the negatively charged denatured proteins with GroEL (which is also negatively charged) needed relatively low concentrations (approximately 10 mM) of bivalent cations Mg2+ or Ca2+. At the high ionic strength (approximately 600 mM), a tight complex was produced also in the absence of bivalent cations. In contrast, positively charged denatured proteins tightly interacted with GroEL irrespectively of the presence of bivalent cations and ionic strength of the solution (from 20 to 600 mM). These features of GroEL interaction with positively and negatively charged denatured proteins were confirmed by polarized fluorescence (fluorescence anisotropy). The findings suggest that the affinity of GroEL for denatured proteins can be determined by the balance of hydrophobic and electrostatic interactions.

  4. Chaperonin GroEL uses asymmetric and symmetric reaction cycles in response to the concentration of non-native substrate proteins.

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    Iizuka, Ryo; Funatsu, Takashi

    2016-01-01

    The Escherichia coli chaperonin GroEL is an essential molecular chaperone that mediates protein folding in association with its cofactor, GroES. It is widely accepted that GroEL alternates the GroES-sealed folding-active rings during the reaction cycle. In other words, an asymmetric GroEL-GroES complex is formed during the cycle, whereas a symmetric GroEL-(GroES)2 complex is not formed. However, this conventional view has been challenged by the recent reports indicating that such symmetric complexes can be formed in the GroEL-GroES reaction cycle. In this review, we discuss the studies of the symmetric GroEL-(GroES)2 complex, focusing on the molecular mechanism underlying its formation. We also suggest that GroEL can be involved in two types of reaction cycles (asymmetric or symmetric) and the type of cycle used depends on the concentration of non-native substrate proteins.

  5. Identification of a Novel BBS Gene (BBS12) Highlights the Major Role of a Vertebrate-Specific Branch of Chaperonin-Related Proteins in Bardet-Biedl Syndrome

    Science.gov (United States)

    Stoetzel, Corinne; Muller, Jean; Laurier, Virginie; Davis, Erica E.; Zaghloul, Norann A.; Vicaire, Serge; Jacquelin, Cécile; Plewniak, Frédéric; Leitch, Carmen C.; Sarda, Pierre; Hamel, Christian; de Ravel, Thomy J. L.; Lewis, Richard Alan; Friederich, Evelyne; Thibault, Christelle; Danse, Jean-Marc; Verloes, Alain; Bonneau, Dominique; Katsanis, Nicholas; Poch, Olivier; Mandel, Jean-Louis; Dollfus, Hélène

    2007-01-01

    Bardet-Biedl syndrome (BBS) is primarily an autosomal recessive ciliopathy characterized by progressive retinal degeneration, obesity, cognitive impairment, polydactyly, and kidney anomalies. The disorder is genetically heterogeneous, with 11 BBS genes identified to date, which account for ∼70% of affected families. We have combined single-nucleotide–polymorphism array homozygosity mapping with in silico analysis to identify a new BBS gene, BBS12. Patients from two Gypsy families were homozygous and haploidentical in a 6-Mb region of chromosome 4q27. FLJ35630 was selected as a candidate gene, because it was predicted to encode a protein with similarity to members of the type II chaperonin superfamily, which includes BBS6 and BBS10. We found pathogenic mutations in both Gypsy families, as well as in 14 other families of various ethnic backgrounds, indicating that BBS12 accounts for ∼5% of all BBS cases. BBS12 is vertebrate specific and, together with BBS6 and BBS10, defines a novel branch of the type II chaperonin superfamily. These three genes are characterized by unusually rapid evolution and are likely to perform ciliary functions specific to vertebrates that are important in the pathophysiology of the syndrome, and together they account for about one-third of the total BBS mutational load. Consistent with this notion, suppression of each family member in zebrafish yielded gastrulation-movement defects characteristic of other BBS morphants, whereas simultaneous suppression of all three members resulted in severely affected embryos, possibly hinting at partial functional redundancy within this protein family. PMID:17160889

  6. Chaperonin filaments : their formation and an evaluation of methods for studying them.

    Energy Technology Data Exchange (ETDEWEB)

    Yaoi, T.; Kagawa, K. H.; Trent, J. D.; Center for Mechanistic Biology and Biotechnology

    1998-08-01

    Chaperonins are multisubunit protein complexes that can be isolated from cells as high-molecular-weight structures that appear as double rings in the electron microscope. We recently discovered that chaperonin double rings isolated from the hyperthermophilic archaeon Sulfolobus shibatae, when incubated at physiological temperatures in the presence of ATP and Mg{sup 2+}, stacked into filaments; we hypothesized that these filaments are related to filaments seen inside S. shibatae cells and that chaperonins exist as filaments in vivo. This paper elucidates the conditions under which we have observed S. shibatae chaperonins to form filaments and evaluates native polyacrylamide gel electrophoresis (PAGE), TEM, spectrophotometry, and centrifugation as methods for studying these filaments. We observed that in the presence of Mg{sup 2+} combined with ATP, ADP, ATP{gamma}S, or GTP, native PAGE indicated that chaperonin subunits assembled into double rings and that the conformation of these double rings was effected by nucleotide binding, but we saw no indication of chaperonin filament formation. Under these same conditions, however, TEM, spectroscopy, and centrifugation methods indicated that chaperonin subunits and double rings had assembled into filaments. We determined that this discrepancy in the representation of the chaperonin structure was due to the native PAGE method itself. When we exposed chaperonin filaments to the electrophoretic field used in native PAGE, the filaments dissociated into double rings. This suggests that TEM, spectrophotometry, and centrifugation are the preferred methods for studying the higher-order structures of chaperonins, which are likely to be of biological significance.

  7. A Model for the Molecular Mechanism of an Engineered Light-Driven Protein Machine.

    Science.gov (United States)

    Hoersch, Daniel; Kortemme, Tanja

    2016-04-05

    Controllable protein-based machines and materials are of considerable interest for diverse biotechnological applications. We previously re-engineered an ATP-driven protein machine, a group II chaperonin, to function as a light-gated nanocage. Here we develop and test a model for the molecular mechanism of the re-engineered chaperonin, which undergoes a large-scale closed to open conformational change triggered by reversible photo-isomerization of a site-specifically attached azobenzene crosslinker. In silico experiments using all-atom simulations suggest that rigid body motions of protein subdomains couple the length changes of the crosslinker to rearrangements of the nucleotide-binding pocket, leading to cage opening. We tested this model by designing a mutant for which the orientation of the two protein subdomains forming the nucleotide-binding pocket is directly controlled by the crosslinker, and confirmed successful reversible photoswitching in vitro. The model probes the conformational cycle of group II chaperonins and offers a design principle for engineering other light-driven protein-based molecular machines. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Identification of elements that dictate the specificity of mitochondrial Hsp60 for its co-chaperonin.

    Directory of Open Access Journals (Sweden)

    Avital Parnas

    Full Text Available Type I chaperonins (cpn60/Hsp60 are essential proteins that mediate the folding of proteins in bacteria, chloroplast and mitochondria. Despite the high sequence homology among chaperonins, the mitochondrial chaperonin system has developed unique properties that distinguish it from the widely-studied bacterial system (GroEL and GroES. The most relevant difference to this study is that mitochondrial chaperonins are able to refold denatured proteins only with the assistance of the mitochondrial co-chaperonin. This is in contrast to the bacterial chaperonin, which is able to function with the help of co-chaperonin from any source. The goal of our work was to determine structural elements that govern the specificity between chaperonin and co-chaperonin pairs using mitochondrial Hsp60 as model system. We used a mutagenesis approach to obtain human mitochondrial Hsp60 mutants that are able to function with the bacterial co-chaperonin, GroES. We isolated two mutants, a single mutant (E321K and a double mutant (R264K/E358K that, together with GroES, were able to rescue an E. coli strain, in which the endogenous chaperonin system was silenced. Although the mutations are located in the apical domain of the chaperonin, where the interaction with co-chaperonin takes place, none of the residues are located in positions that are directly responsible for co-chaperonin binding. Moreover, while both mutants were able to function with GroES, they showed distinct functional and structural properties. Our results indicate that the phenotype of the E321K mutant is caused mainly by a profound increase in the binding affinity to all co-chaperonins, while the phenotype of R264K/E358K is caused by a slight increase in affinity toward co-chaperonins that is accompanied by an alteration in the allosteric signal transmitted upon nucleotide binding. The latter changes lead to a great increase in affinity for GroES, with only a minor increase in affinity toward the mammalian

  9. Dynamics, flexibility, and allostery in molecular chaperonins.

    Science.gov (United States)

    Skjærven, Lars; Cuellar, Jorge; Martinez, Aurora; Valpuesta, José María

    2015-09-14

    The chaperonins are a family of molecular chaperones present in all three kingdoms of life. They are classified into Group I and Group II. Group I consists of the bacterial variants (GroEL) and the eukaryotic ones from mitochondria and chloroplasts (Hsp60), while Group II consists of the archaeal (thermosomes) and eukaryotic cytosolic variants (CCT or TRiC). Both groups assemble into a dual ring structure, with each ring providing a protective folding chamber for nascent and denatured proteins. Their functional cycle is powered by ATP binding and hydrolysis, which drives a series of structural rearrangements that enable encapsulation and subsequent release of the substrate protein. Chaperonins have elaborate allosteric mechanisms to regulate their functional cycle. Long-range negative cooperativity between the two rings ensures alternation of the folding chambers. Positive intra-ring cooperativity, which facilitates concerted conformational transitions within the protein subunits of one ring, has only been demonstrated for Group I chaperonins. In this review, we describe our present understanding of the underlying mechanisms and the structure-function relationships in these complex protein systems with a particular focus on the structural dynamics, allostery, and associated conformational rearrangements. Copyright © 2015. Published by Elsevier B.V.

  10. Novel chaperonins are prevalent in the virioplankton and demonstrate links to viral biology and ecology

    Science.gov (United States)

    Marine, Rachel L; Nasko, Daniel J; Wray, Jeffrey; Polson, Shawn W; Wommack, K Eric

    2017-01-01

    Chaperonins are protein-folding machinery found in all cellular life. Chaperonin genes have been documented within a few viruses, yet, surprisingly, analysis of metagenome sequence data indicated that chaperonin-carrying viruses are common and geographically widespread in marine ecosystems. Also unexpected was the discovery of viral chaperonin sequences related to thermosome proteins of archaea, indicating the presence of virioplankton populations infecting marine archaeal hosts. Virioplankton large subunit chaperonin sequences (GroELs) were divergent from bacterial sequences, indicating that viruses have carried this gene over long evolutionary time. Analysis of viral metagenome contigs indicated that: the order of large and small subunit genes was linked to the phylogeny of GroEL; both lytic and temperate phages may carry group I chaperonin genes; and viruses carrying a GroEL gene likely have large double-stranded DNA (dsDNA) genomes (>70 kb). Given these connections, it is likely that chaperonins are critical to the biology and ecology of virioplankton populations that carry these genes. Moreover, these discoveries raise the intriguing possibility that viral chaperonins may more broadly alter the structure and function of viral and cellular proteins in infected host cells. PMID:28731469

  11. Novel chaperonins are prevalent in the virioplankton and demonstrate links to viral biology and ecology.

    Science.gov (United States)

    Marine, Rachel L; Nasko, Daniel J; Wray, Jeffrey; Polson, Shawn W; Wommack, K Eric

    2017-11-01

    Chaperonins are protein-folding machinery found in all cellular life. Chaperonin genes have been documented within a few viruses, yet, surprisingly, analysis of metagenome sequence data indicated that chaperonin-carrying viruses are common and geographically widespread in marine ecosystems. Also unexpected was the discovery of viral chaperonin sequences related to thermosome proteins of archaea, indicating the presence of virioplankton populations infecting marine archaeal hosts. Virioplankton large subunit chaperonin sequences (GroELs) were divergent from bacterial sequences, indicating that viruses have carried this gene over long evolutionary time. Analysis of viral metagenome contigs indicated that: the order of large and small subunit genes was linked to the phylogeny of GroEL; both lytic and temperate phages may carry group I chaperonin genes; and viruses carrying a GroEL gene likely have large double-stranded DNA (dsDNA) genomes (>70 kb). Given these connections, it is likely that chaperonins are critical to the biology and ecology of virioplankton populations that carry these genes. Moreover, these discoveries raise the intriguing possibility that viral chaperonins may more broadly alter the structure and function of viral and cellular proteins in infected host cells.

  12. CHAPERONIN 20 mediates iron superoxide dismutase (FeSOD) activity independent of its co-chaperonin role in Arabidopsis chloroplasts.

    Science.gov (United States)

    Kuo, W Y; Huang, C H; Liu, A C; Cheng, C P; Li, S H; Chang, W C; Weiss, C; Azem, A; Jinn, T L

    2013-01-01

    Iron superoxide dismutases (FeSODs; FSDs) are primary antioxidant enzymes in Arabidopsis thaliana chloroplasts. The stromal FSD1 conferred the only detectable FeSOD activity, whereas the thylakoid membrane- and nucleoid-co-localized FSD2 and FSD3 double mutant showed arrested chloroplast development. FeSOD requires cofactor Fe for its activity, but its mechanism of activation is unclear. We used reversed-phase high-performance liquid chromatography (HPLC), gel filtration chromatography, LC-MS/MS, protoplast transient expression and virus-induced gene silencing (VIGS) analyses to identify and characterize a factor involved in FeSOD activation. We identified the chloroplast-localized co-chaperonin CHAPERONIN 20 (CPN20) as a mediator of FeSOD activation by direct interaction. The relationship between CPN20 and FeSOD was confirmed by in vitro experiments showing that CPN20 alone could enhance FSD1, FSD2 and FSD3 activity. The in vivo results showed that CPN20-overexpressing mutants and mutants with defective co-chaperonin activity increased FSD1 activity, without changing the chaperonin CPN60 protein level, and VIGS-induced downregulation of CPN20 also led to decreased FeSOD activity. Our findings reveal that CPN20 can mediate FeSOD activation in chloroplasts, a role independent of its known function in the chaperonin system. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

  13. Chaperonin filaments: their formation and an evaluation of methods for studying them.

    Science.gov (United States)

    Yaoi, T; Kagawa, H K; Trent, J D

    1998-08-01

    Chaperonins are multisubunit protein complexes that can be isolated from cells as high-molecular-weight structures that appear as double rings in the electron microscope. We recently discovered that chaperonin double rings isolated from the hyperthermophilic archaeon Sulfolobus shibatae, when incubated at physiological temperatures in the presence of ATP and Mg2+, stacked into filaments; we hypothesized that these filaments are related to filaments seen inside S. shibatae cells and that chaperonins exist as filaments in vivo (J. D. Trent et al., 1997, Proc. Natl. Acad. Sci. USA 94, 5383-5388). This paper elucidates the conditions under which we have observed S. shibatae chaperonins to form filaments and evaluates native polyacrylamide gel electrophoresis (PAGE), TEM, spectrophotometry, and centrifugation as methods for studying these filaments. We observed that in the presence of Mg2+ combined with ATP, ADP, ATPgammaS, or GTP, native PAGE indicated that chaperonin subunits assembled into double rings and that the conformation of these double rings was effected by nucleotide binding, but we saw no indication of chaperonin filament formation. Under these same conditions, however, TEM, spectroscopy, and centrifugation methods indicated that chaperonin subunits and double rings had assembled into filaments. We determined that this discrepancy in the representation of the chaperonin structure was due to the native PAGE method itself. When we exposed chaperonin filaments to the electrophoretic field used in native PAGE, the filaments dissociated into double rings. This suggests that TEM, spectrophotometry, and centrifugation are the preferred methods for studying the higher-order structures of chaperonins, which are likely to be of biological significance. Copyright 1998 Academic Press.

  14. Protein disulfide engineering.

    Science.gov (United States)

    Dombkowski, Alan A; Sultana, Kazi Zakia; Craig, Douglas B

    2014-01-21

    Improving the stability of proteins is an important goal in many biomedical and industrial applications. A logical approach is to emulate stabilizing molecular interactions found in nature. Disulfide bonds are covalent interactions that provide substantial stability to many proteins and conform to well-defined geometric conformations, thus making them appealing candidates in protein engineering efforts. Disulfide engineering is the directed design of novel disulfide bonds into target proteins. This important biotechnological tool has achieved considerable success in a wide range of applications, yet the rules that govern the stabilizing effects of disulfide bonds are not fully characterized. Contrary to expectations, many designed disulfide bonds have resulted in decreased stability of the modified protein. We review progress in disulfide engineering, with an emphasis on the issue of stability and computational methods that facilitate engineering efforts. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  15. Symmetric GroEL:GroES2 complexes are the protein-folding functional form of the chaperonin nanomachine.

    Science.gov (United States)

    Yang, Dong; Ye, Xiang; Lorimer, George H

    2013-11-12

    Using calibrated FRET, we show that the simultaneous occupancy of both rings of GroEL by ATP and GroES occurs, leading to the rapid formation of symmetric GroEL:GroES2 "football" particles regardless of the presence or absence of substrate protein (SP). In the absence of SP, these symmetric particles revert to asymmetric GroEL:GroES1 "bullet" particles. The breakage of GroES symmetry requires the stochastic hydrolysis of ATP and the breakage of nucleotide symmetry. These asymmetric particles are both persistent and dynamic; they turnover via the asymmetric cycle. When challenged with SP, however, they revert to symmetric particles within a second. In the presence of SP, the symmetric particles are also persistent and dynamic. They turn over via the symmetric cycle. Under these conditions, the stochastic hydrolysis of ATP and the breakage of nucleotide symmetry also occur within the ensemble of particles. However, on account of SP-catalyzed ADP/ATP exchange, GroES symmetry is rapidly restored. The residence time of both GroES and SP on functional GroEL is reduced to ∼1 s, enabling many more iterations than was previously believed possible, consistent with the iterative annealing mechanism. This result is inconsistent with currently accepted models. Using a foldable SP, we show that as the SP folds to the native state and the population of unfolded SP declines, the population of symmetric particles reverts to asymmetric particles in parallel, a result that is consistent with the former being the folding functional form.

  16. Artificially Engineered Protein Polymers.

    Science.gov (United States)

    Yang, Yun Jung; Holmberg, Angela L; Olsen, Bradley D

    2017-06-07

    Modern polymer science increasingly requires precise control over macromolecular structure and properties for engineering advanced materials and biomedical systems. The application of biological processes to design and synthesize artificial protein polymers offers a means for furthering macromolecular tunability, enabling polymers with dispersities of ∼1.0 and monomer-level sequence control. Taking inspiration from materials evolved in nature, scientists have created modular building blocks with simplified monomer sequences that replicate the function of natural systems. The corresponding protein engineering toolbox has enabled the systematic development of complex functional polymeric materials across areas as diverse as adhesives, responsive polymers, and medical materials. This review discusses the natural proteins that have inspired the development of key building blocks for protein polymer engineering and the function of these elements in material design. The prospects and progress for scalable commercialization of protein polymers are reviewed, discussing both technology needs and opportunities.

  17. Engineering therapeutic protein disaggregases.

    Science.gov (United States)

    Shorter, James

    2016-05-15

    Therapeutic agents are urgently required to cure several common and fatal neurodegenerative disorders caused by protein misfolding and aggregation, including amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), and Alzheimer's disease (AD). Protein disaggregases that reverse protein misfolding and restore proteins to native structure, function, and localization could mitigate neurodegeneration by simultaneously reversing 1) any toxic gain of function of the misfolded form and 2) any loss of function due to misfolding. Potentiated variants of Hsp104, a hexameric AAA+ ATPase and protein disaggregase from yeast, have been engineered to robustly disaggregate misfolded proteins connected with ALS (e.g., TDP-43 and FUS) and PD (e.g., α-synuclein). However, Hsp104 has no metazoan homologue. Metazoa possess protein disaggregase systems distinct from Hsp104, including Hsp110, Hsp70, and Hsp40, as well as HtrA1, which might be harnessed to reverse deleterious protein misfolding. Nevertheless, vicissitudes of aging, environment, or genetics conspire to negate these disaggregase systems in neurodegenerative disease. Thus, engineering potentiated human protein disaggregases or isolating small-molecule enhancers of their activity could yield transformative therapeutics for ALS, PD, and AD. © 2016 Shorter. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  18. Ordered biological nanostructures formed from chaperonin polypeptides

    Science.gov (United States)

    Trent, Jonathan D. (Inventor); McMillan, R. Andrew (Inventor); Kagawa, Hiromi (Inventor); Paavola, Chad D. (Inventor)

    2010-01-01

    The following application relates to nanotemplates, nanostructures, nanoarrays and nanodevices formed from wild-type and mutated chaperonin polypeptides, methods of producing such compositions, methods of using such compositions and particular chaperonin polypeptides that can be utilized in producing such compositions.

  19. Isolation and characterization of a Paracentrotus lividus cDNA encoding a stress-inducible chaperonin

    Science.gov (United States)

    Gianguzza, Fabrizio; Antonietta Ragusa, Maria; Roccheri, Maria Carmela; Liegro, Italia Di; Rinaldi, Anna Maria

    2000-01-01

    Chaperonins are ubiquitous proteins that facilitate protein folding in an adenosine triphosphate–dependent manner. Here we report the isolation of a sea urchin cDNA (Plhsp60) coding for mitochondrial chaperonin (Cpn60), whose basal expression is further enhanced by heat shock. The described cDNA corresponds to a full-length mRNA encoding a protein of 582 amino acids, the first 32 of which constitute a putative mitochondrial targeting leader sequence. Comparative analysis has demonstrated that this protein is highly conserved in evolution. PMID:11147969

  20. Chaperonin-Containing t-Complex Protein-1 Subunit β as a Possible Biomarker for the Phase of Glomerular Hyperfiltration of Diabetic Nephropathy

    Directory of Open Access Journals (Sweden)

    Chung-Ze Wu

    2015-01-01

    Full Text Available In cell model, we discovered the association between chaperonin-containing t-complex polypeptide 1 subunit β (TCP-1β and early diabetic nephropathy (DN. In this study, we further explored the relationships between TCP-1β and type 2 diabetic mellitus (DM. To mimic the clinical hyperfiltration state, a type 2 DM mice model was established by feeding a high-fat diet in combination with treatment of streptozotocin and nicotinamide. Blood and urine were collected to determine creatinine clearance (Ccr, and kidney tissues were harvested for evaluation of TCP-1β expression by immunohistochemistry and Western blot. Meanwhile, clinical subjects of healthy controls and type 2 DM were recruited to strengthen the evidence with urine TCP-1β. Results showed that Ccr and the expression of TCP-1β in kidney were significantly higher one week after hyperglycemia development, suggesting that the hyperfiltration state was successfully established in the mice model. TCP-1β was expressed predominantly on renal tubules. By using the estimated glomerular filtration rate to index progression in clinical investigation, urine TCP-1β level was associated with the hyperfiltration phase in type 2 DM patients. Conclusively, we confirmed that TCP-1β is a possible biomarker for early nephropathy of type 2 DM, but further mechanistic study to elucidate its cause and pathway is needed.

  1. Engineering ancestral protein hyperstability.

    Science.gov (United States)

    Romero-Romero, M Luisa; Risso, Valeria A; Martinez-Rodriguez, Sergio; Ibarra-Molero, Beatriz; Sanchez-Ruiz, Jose M

    2016-10-15

    Many experimental analyses and proposed scenarios support that ancient life was thermophilic. In congruence with this hypothesis, proteins encoded by reconstructed sequences corresponding to ancient phylogenetic nodes often display very high stability. Here, we show that such 'reconstructed ancestral hyperstability' can be further engineered on the basis of a straightforward approach that uses exclusively information afforded by the ancestral reconstruction process itself. Since evolution does not imply continuous progression, screening of the mutations between two evolutionarily related resurrected ancestral proteins may identify mutations that further stabilize the most stable one. To explore this approach, we have used a resurrected thioredoxin corresponding to the last common ancestor of the cyanobacterial, Deinococcus and Thermus groups (LPBCA thioredoxin), which has a denaturation temperature of ∼123°C. This high value is within the top 0.1% of the denaturation temperatures in the ProTherm database and, therefore, achieving further stabilization appears a priori as a challenging task. Nevertheless, experimental comparison with a resurrected thioredoxin corresponding to the last common ancestor of bacteria (denaturation temperature of ∼115°C) immediately identifies three mutations that increase the denaturation temperature of LPBCA thioredoxin to ∼128°C. Comparison between evolutionarily related resurrected ancestral proteins thus emerges as a simple approach to expand the capability of ancestral reconstruction to search sequence space for extreme protein properties of biotechnological interest. The fact that ancestral sequences for many phylogenetic nodes can be derived from a single alignment of modern sequences should contribute to the general applicability of this approach. © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  2. Hydrogels Constructed from Engineered Proteins.

    Science.gov (United States)

    Li, Hongbin; Kong, Na; Laver, Bryce; Liu, Junqiu

    2016-02-24

    Due to their various potential biomedical applications, hydrogels based on engineered proteins have attracted considerable interest. Benefitting from significant progress in recombinant DNA technology and protein engineering/design techniques, the field of protein hydrogels has made amazing progress. The latest progress of hydrogels constructed from engineered recombinant proteins are presented, mainly focused on biorecognition-driven physical hydrogels as well as chemically crosslinked hydrogels. The various bio-recognition based physical crosslinking strategies are discussed, as well as chemical crosslinking chemistries used to engineer protein hydrogels, and protein hydrogels' various biomedical applications. The future perspectives of this fast evolving field of biomaterials are also discussed. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Chaperonin cofactors, Cpn10 and Cpn20, of green algae and plants function as hetero-oligomeric ring complexes.

    Science.gov (United States)

    Tsai, Yi-Chin C; Mueller-Cajar, Oliver; Saschenbrecker, Sandra; Hartl, F Ulrich; Hayer-Hartl, Manajit

    2012-06-08

    The chloroplast chaperonin system of plants and green algae is a curiosity as both the chaperonin cage and its lid are encoded by multiple genes, in contrast to the single genes encoding the two components of the bacterial and mitochondrial systems. In the green alga Chlamydomonas reinhardtii (Cr), three genes encode chaperonin cofactors, with cpn10 encoding a single ∼10-kDa domain and cpn20 and cpn23 encoding tandem cpn10 domains. Here, we characterized the functional interaction of these proteins with the Escherichia coli chaperonin, GroEL, which normally cooperates with GroES, a heptamer of ∼10-kDa subunits. The C. reinhardtii cofactor proteins alone were all unable to assist GroEL-mediated refolding of bacterial ribulose-bisphosphate carboxylase/oxygenase but gained this ability when CrCpn20 and/or CrCpn23 was combined with CrCpn10. Native mass spectrometry indicated the formation of hetero-oligomeric species, consisting of seven ∼10-kDa domains. The cofactor "heptamers" interacted with GroEL and encapsulated substrate protein in a nucleotide-dependent manner. Different hetero-oligomer arrangements, generated by constructing cofactor concatamers, indicated a preferential heptamer configuration for the functional CrCpn10-CrCpn23 complex. Formation of heptamer Cpn10/Cpn20 hetero-oligomers was also observed with the Arabidopsis thaliana (At) cofactors, which functioned with the chloroplast chaperonin, AtCpn60α(7)β(7). It appears that hetero-oligomer formation occurs more generally for chloroplast chaperonin cofactors, perhaps adapting the chaperonin system for the folding of specific client proteins.

  4. Chaperonin GroEL/GroES over-expression promotes multi-drug resistance in E. coli following exposure to aminoglycoside antibiotics

    Directory of Open Access Journals (Sweden)

    Lise eGoltermann

    2016-01-01

    Full Text Available Antibiotic resistance is an increasing challenge to modern healthcare. Aminoglycoside antiobiotics cause translation corruption and protein misfolding and aggregation in Escherichia coli. We previously showed that chaperonin GroEL/GroES depletion and overexpression sensitize and promote short-term tolerance, respectively, to this drug class. Here we show that chaperonin GroEL/GroES over-expression accelerates acquisition of aminoglycoside resistance and multi-drug resistance following sub-lethal aminoglycoside antibiotic exposure. Chaperonin buffering could provide a novel mechanism for antibiotic resistance and multi-drug resistance development.

  5. Protein design for pathway engineering

    Energy Technology Data Exchange (ETDEWEB)

    Eriksen, DT; Lian, JZ; Zhao, HM

    2014-02-01

    Design and construction of biochemical pathways has increased the complexity of biosynthetically-produced compounds when compared to single enzyme biocatalysis. However, the coordination of multiple enzymes can introduce a complicated set of obstacles to overcome in order to achieve a high titer and yield of the desired compound. Metabolic engineering has made great strides in developing tools to optimize the flux through a target pathway, but the inherent characteristics of a particular enzyme within the pathway can still limit the productivity. Thus, judicious protein design is critical for metabolic and pathway engineering. This review will describe various strategies and examples of applying protein design to pathway engineering to optimize the flux through the pathway. The proteins can be engineered for altered substrate specificity/selectivity, increased catalytic activity, reduced mass transfer limitations through specific protein localization, and reduced substrate/product inhibition. Protein engineering can also be expanded to design biosensors to enable high through-put screening and to customize cell signaling networks. These strategies have successfully engineered pathways for significantly increased productivity of the desired product or in the production of novel compounds. (C) 2013 Elsevier Inc. All rights reserved.

  6. Protein Design for Pathway Engineering

    Science.gov (United States)

    Eriksen, Dawn T.; Lian, Jiazhang; Zhao, Huimin

    2013-01-01

    Design and construction of biochemical pathways has increased the complexity of biosynthetically-produced compounds when compared to single enzyme biocatalysis. However, the coordination of multiple enzymes can introduce a complicated set of obstacles to overcome in order to achieve a high titer and yield of the desired compound. Metabolic engineering has made great strides in developing tools to optimize the flux through a target pathway, but the inherent characteristics of a particular enzyme within the pathway can still limit the productivity. Thus, judicious protein design is critical for metabolic and pathway engineering. This review will describe various strategies and examples of applying protein design to pathway engineering to optimize the flux through the pathway. The proteins can be engineered for altered substrate specificity/selectivity, increased catalytic activity, reduced mass transfer limitations through specific protein localization, and reduced substrate/product inhibition. Protein engineering can also be expanded to design biosensors to enable high through-put screening and to customize cell signaling networks. These strategies have successfully engineered pathways for significantly increased productivity of the desired product or in the production of novel compounds. PMID:23558037

  7. Cross Kingdom Glyco Protein Engineering

    DEFF Research Database (Denmark)

    Möller, Svenning Rune

    -glycosylation structures and enzyme machineries primarily in mammalian cells and plants. The second part of the review, Chapter 2, ‘Trans kingdom glyco engineering - the biotechnological and pharmacological potential’, focuses on past and current efforts of controlling the two main types of protein glycosylation including...

  8. Single-molecule fluorescence polarization study of conformational change in archaeal group II chaperonin.

    Directory of Open Access Journals (Sweden)

    Ryo Iizuka

    Full Text Available Group II chaperonins found in archaea and in eukaryotic cytosol mediate protein folding without a GroES-like cofactor. The function of the cofactor is substituted by the helical protrusion at the tip of the apical domain, which forms a built-in lid on the central cavity. Although many studies on the change in lid conformation coupled to the binding and hydrolysis of nucleotides have been conducted, the molecular mechanism of lid closure remains poorly understood. Here, we performed a single-molecule polarization modulation to probe the rotation of the helical protrusion of a chaperonin from a hyperthermophilic archaeum, Thermococcus sp. strain KS-1. We detected approximately 35° rotation of the helical protrusion immediately after photorelease of ATP. The result suggests that the conformational change from the open lid to the closed lid state is responsible for the approximately 35° rotation of the helical protrusion.

  9. Cross Kingdom Glyco Protein Engineering

    DEFF Research Database (Denmark)

    Möller, Svenning Rune

    -glycosylation structures and enzyme machineries primarily in mammalian cells and plants. The second part of the review, Chapter 2, ‘Trans kingdom glyco engineering - the biotechnological and pharmacological potential’, focuses on past and current efforts of controlling the two main types of protein glycosylation including...... glycoengineering is detailed knowledge of the glycogenes and resulting structures involved which is provided in the two introductory chapters, where Chapter 1 ‘Plant and Mammalian Cell Protein N- and O-glycosylation – Recent Developments and Future Prospects’ provides a descriptive overview of N- and O...... is a prerequisite for the subsequent hydroxy proline arabinosylations. In Chapter 5, ‘Glycoproteomic analysis of seven major allergenic proteins reveals novel post-translational modifications ‘, we have, as part of a cross-disciplinary effort, participated in an in depth glycoprofiling analysis of seven major plant...

  10. NewProt - a protein engineering portal

    NARCIS (Netherlands)

    Schwarte, A.; Genz, M.; Skalden, L.; Nobili, A.; Vickers, C.; Melse, O.; Kuipers, R.; Joosten, H.J.; Stourac, J.; Bendl, J.; Black, J.R.; Haase, P.; Baakman, C.A.; Damborsky, J.; Bornscheuer, U.; Vriend, G.; Venselaar, H.

    2017-01-01

    The NewProt protein engineering portal is a one-stop-shop for in silico protein engineering. It gives access to a large number of servers that compute a wide variety of protein structure characteristics supporting work on the modification of proteins through the introduction of (multiple) point

  11. The structural basis of substrate recognition by the eukaryotic chaperonin TRiC/CCT.

    Science.gov (United States)

    Joachimiak, Lukasz A; Walzthoeni, Thomas; Liu, Corey W; Aebersold, Ruedi; Frydman, Judith

    2014-11-20

    The eukaryotic chaperonin TRiC (also called CCT) is the obligate chaperone for many essential proteins. TRiC is hetero-oligomeric, comprising two stacked rings of eight different subunits each. Subunit diversification from simpler archaeal chaperonins appears linked to proteome expansion. Here, we integrate structural, biophysical, and modeling approaches to identify the hitherto unknown substrate-binding site in TRiC and uncover the basis of substrate recognition. NMR and modeling provided a structural model of a chaperonin-substrate complex. Mutagenesis and crosslinking-mass spectrometry validated the identified substrate-binding interface and demonstrate that TRiC contacts full-length substrates combinatorially in a subunit-specific manner. The binding site of each subunit has a distinct, evolutionarily conserved pattern of polar and hydrophobic residues specifying recognition of discrete substrate motifs. The combinatorial recognition of polypeptides broadens the specificity of TRiC and may direct the topology of bound polypeptides along a productive folding trajectory, contributing to TRiC's unique ability to fold obligate substrates.

  12. Disease-Associated Mutations in the HSPD1 Gene Encoding the Large Subunit of the Mitochondrial HSP60/HSP10 Chaperonin Complex

    DEFF Research Database (Denmark)

    Bross, Peter; Fernandez-Guerra, Paula

    2016-01-01

    Heat shock protein 60 (HSP60) forms together with heat shock protein 10 (HSP10) double-barrel chaperonin complexes that are essential for folding to the native state of proteins in the mitochondrial matrix space. Two extremely rare monogenic disorders have been described that are caused by missen...

  13. Protein Engineering: Case Studies of Commercialized Engineered Products

    Science.gov (United States)

    Walsh, Gary

    2007-01-01

    Programs in biochemistry invariably encompass the principles of protein engineering. Students often display increased understanding and enthusiasm when theoretical concepts are underpinned by practical example. Herein are presented five case studies, each focusing upon a commercial protein product engineered to enhance its application-relevant…

  14. Protein engineering techniques gateways to synthetic protein universe

    CERN Document Server

    Poluri, Krishna Mohan

    2017-01-01

    This brief provides a broad overview of protein-engineering research, offering a glimpse of the most common experimental methods. It also presents various computational programs with applications that are widely used in directed evolution, computational and de novo protein design. Further, it sheds light on the advantages and pitfalls of existing methodologies and future perspectives of protein engineering techniques.

  15. Conversion of an inactive xylose isomerase into a functional enzyme by co-expression of GroEL-GroES chaperonins in Saccharomyces cerevisiae.

    Science.gov (United States)

    Temer, Beatriz; Dos Santos, Leandro Vieira; Negri, Victor Augusti; Galhardo, Juliana Pimentel; Magalhães, Pedro Henrique Mello; José, Juliana; Marschalk, Cidnei; Corrêa, Thamy Lívia Ribeiro; Carazzolle, Marcelo Falsarella; Pereira, Gonçalo Amarante Guimarães

    2017-09-09

    Second-generation ethanol production is a clean bioenergy source with potential to mitigate fossil fuel emissions. The engineering of Saccharomyces cerevisiae for xylose utilization is an essential step towards the production of this biofuel. Though xylose isomerase (XI) is the key enzyme for xylose conversion, almost half of the XI genes are not functional when expressed in S. cerevisiae. To date, protein misfolding is the most plausible hypothesis to explain this phenomenon. This study demonstrated that XI from the bacterium Propionibacterium acidipropionici becomes functional in S. cerevisiae when co-expressed with GroEL-GroES chaperonin complex from Escherichia coli. The developed strain BTY34, harboring the chaperonin complex, is able to efficiently convert xylose to ethanol with a yield of 0.44 g ethanol/g xylose. Furthermore, the BTY34 strain presents a xylose consumption rate similar to those observed for strains carrying the widely used XI from the fungus Orpinomyces sp. In addition, the tetrameric XI structure from P. acidipropionici showed an elevated number of hydrophobic amino acid residues on the surface of protein when compared to XI commonly expressed in S. cerevisiae. Based on our results, we elaborate an extensive discussion concerning the uncertainties that surround heterologous expression of xylose isomerases in S. cerevisiae. Probably, a correct folding promoted by GroEL-GroES could solve some issues regarding a limited or absent XI activity in S. cerevisiae. The strains developed in this work have promising industrial characteristics, and the designed strategy could be an interesting approach to overcome the non-functionality of bacterial protein expression in yeasts.

  16. Conformational states of ribulosebisphosphate carboxylase and their interaction with chaperonin 60.

    Science.gov (United States)

    van der Vies, S M; Viitanen, P V; Gatenby, A A; Lorimer, G H; Jaenicke, R

    1992-04-14

    Conformational states of ribulosebisphosphate carboxylase (Rubisco) from Rhodospirillum rubrum were examined by far-UV circular dichroism (CD), tryptophan fluorescence, and 1-anilino-naphthalenesulfonate (ANS) binding. At pH 2 and low ionic strength (I = 0.01), Rubisco adopts an unfolded, monomeric conformation (UA1 state) as judged by far-UV CD and tryptophan fluorescence. As with other acid-unfolded proteins [Goto, Y., Calciano, L. J., & Fink, A. L. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 573-577], an intermediate conformation (A1 state) is observed at pH 2 and high ionic strength. The A1 state has an alpha-helical content equivalent to 64% of that present in the native dimer (N2 state). However, fluorescence measurements indicate that the tertiary structure of the A1 state is largely disordered. A site-directed mutant, K168E, which exists as a stable monomer [Mural, R. J., Soper, T. S., Larimer, F. W., & Hartman, F. C. (1990) J. Biol. Chem. 265, 6501-6505] was used to characterize the "native" monomer (N1 state). The far-UV CD spectra of the N1 and N2 states are almost identical, indicating a similar secondary structure content. However, the tertiary structure of the N1 state is less ordered than that of the N2 state. Nevertheless, when appropriately complemented in vitro, K168E forms an active heterodimer. Upon neutralization of acid-denatured Rubisco or dilution of guanidine hydrochloride-denatured Rubisco, unstable folding intermediates (I1 state) are rapidly formed. At concentrations at or below the "critical aggregation concentration" (CAC), the I1 state reverts spontaneously but slowly to the native states with high yield (greater than 65%). The CAC is temperature-dependent. At concentrations above the CAC, the I1 and the A1 states undergo irreversible aggregation. The commitment to aggregation is rapid [ef. Goldberg, M. E., Rudolph, R., & Jaenicke, R. (1991) Biochemistry 30, 2790-2797] and proceeds until the concentration of folding intermediate(s) has

  17. Engineering cells to improve protein expression.

    Science.gov (United States)

    Xiao, Su; Shiloach, Joseph; Betenbaugh, Michael J

    2014-06-01

    Cellular engineering of bacteria, fungi, insect cells and mammalian cells is a promising methodology to improve recombinant protein production for structural, biochemical, and commercial applications. Increased understanding of the host organism biology has suggested engineering strategies targeting bottlenecks in transcription, translation, protein processing and secretory pathways, as well as cell growth and survival. A combination of metabolic engineering and synthetic biology has been used to improve the properties of cells for protein production, which has resulted in enhanced yields of multiple protein classes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Protein engineering approaches to chemical biotechnology.

    Science.gov (United States)

    Chen, Zhen; Zeng, An-Ping

    2016-12-01

    Protein engineering for the improvement of properties of biocatalysts and for the generation of novel metabolic pathways plays more and more important roles in chemical biotechnology aiming at the production of chemicals from biomass. Although widely used in single-enzyme catalysis process, protein engineering is only being increasingly explored in recent years to achieve more complex in vitro and in vivo biocatalytic processes. This review focuses on major contributions of protein engineering to chemical biotechnology in the field of multi-enzymatic cascade catalysis and metabolic engineering. Especially, we discuss and highlight recent strategies for combining pathway design and protein engineering for the production of novel products. Copyright © 2016. Published by Elsevier Ltd.

  19. Structural and mechanistic characterization of an archaeal-like chaperonin from a thermophilic bacterium

    OpenAIRE

    An, Young Jun; Rowland, Sara E.; Na, Jung-Hyun; Spigolon, Dario; Hong, Seung Kon; Yoon, Yeo Joon; Lee, Jung-Hyun; Robb, Frank T.; Cha, Sun-Shin

    2017-01-01

    The chaperonins (CPNs) are megadalton sized hollow complexes with two cavities that open and close to encapsulate non-native proteins. CPNs are assigned to two sequence-related groups that have distinct allosteric mechanisms. In Group I CPNs a detachable co-chaperone, GroES, closes the chambers whereas in Group II a built-in lid closes the chambers. Group I CPNs have a bacterial ancestry, whereas Group II CPNs are archaeal in origin. Here we describe open and closed crystal structures represe...

  20. Heterologous production of active ribonuclease inhibitor in Escherichia coli by redox state control and chaperonin coexpression

    Directory of Open Access Journals (Sweden)

    Neubauer Peter

    2011-08-01

    Full Text Available Abstract Background Eukaryotic Ribonuclease inhibitor (RI, belonging to the RNH1 family, is distinguished by unique features - a high sensitivity to oxidation due to the large number of reduced cysteins and a high hydrophobicity, which made most production approaches so far unsuccessful or resulted in very low yields. In this work efficient in vivo folding of native RI in the Escherichia coli cytoplasm was obtained by external addition of a reducing agent in tandem with oxygen limitation and overproduction of a molecular chaperonin. After optimisation of the production conditions in the shake flask scale the process was scaled up to high cell densities by applying a glucose limited fed-batch procedure. Results RI production in a T7 RNA polymerase based system results in accumulation of aggregated inactive product in inclusion bodies. Combination of addition of the reductant DTT, low production temperature and coexpression of the chaperonin GroELS resulted in high level production of approximately 25 mg g-1 CDW active RI in E. coli ER2566 pET21b, corresponding to approximately 800 kU g-1 cell wet weight. Further conditional screening under fed-batch-like conditions with the EnBase® technology and scale up into the bioreactor scale resulted in an efficient high cell density glucose and oxygen limited fed-batch process with a final cell dry weight of 25 g L-1 and a total RI yield of app. 625 mg L-1 (volumetric activity of 80,000 kU L-1. The E. coli based production constructs showed a very high robustness. The recombinant culture maintained its productivity despite the combination of the toxic growth conditions, the substrate limited production mode in tandem with a high level expression of several recombinant proteins, the set of molecular chaperonins and the target protein (RI. Conclusions High level production of active RI in E. coli in a T7 RNA polymerase expression system depends on the following factors: (i addition of a reducing agent, (ii

  1. Engineering RNA-binding proteins for biology.

    Science.gov (United States)

    Chen, Yu; Varani, Gabriele

    2013-08-01

    RNA-binding proteins play essential roles in the regulation of gene expression. Many have modular structures and combine relatively few common domains in various arrangements to recognize RNA sequences and/or structures. Recent progress in engineering the specificity of the PUF class RNA-binding proteins has shown that RNA-binding domains may be combined with various effector or functional domains to regulate the metabolism of targeted RNAs. Designer RNA-binding proteins with tailored sequence specificity will provide valuable tools for biochemical research as well as potential therapeutic applications. In this review, we discuss the suitability of various RNA-binding domains for engineering RNA-binding specificity, based on the structural basis for their recognition. We also compare various protein engineering and design methods applied to RNA-binding proteins, and discuss future applications of these proteins. © 2013 FEBS.

  2. Effects of a Mutation in the HSPE1 Gene Encoding the Mitochondrial Co-chaperonin HSP10 and Its Potential Association with a Neurological and Developmental Disorder

    DEFF Research Database (Denmark)

    Bie, Anne S; Fernandez-Guerra, Paula; Birkler, Rune I D

    2016-01-01

    We here report molecular investigations of a missense mutation in the HSPE1 gene encoding the HSP10 subunit of the HSP60/ HSP10 chaperonin complex that assists protein folding in the mitochondrial matrix. The mutation was identified in an infant who came to clinical attention due to infantile spa...

  3. Chaperonin GroEL/GroES Over-Expression Promotes Aminoglycoside Resistance and Reduces Drug Susceptibilities in Escherichia coli Following Exposure to Sublethal Aminoglycoside Doses

    DEFF Research Database (Denmark)

    Goltermann, Lise; Sarusie, Menachem V; Bentin, Thomas

    2016-01-01

    Antibiotic resistance is an increasing challenge to modern healthcare. Aminoglycoside antibiotics cause translation corruption and protein misfolding and aggregation in Escherichia coli. We previously showed that chaperonin GroEL/GroES depletion and over-expression sensitize and promote short...

  4. Protein-Based Hydrogels for Tissue Engineering.

    Science.gov (United States)

    Schloss, Ashley C; Williams, Danielle M; Regan, Lynne J

    2016-01-01

    The tunable mechanical and structural properties of protein-based hydrogels make them excellent scaffolds for tissue engineering and repair. Moreover, using protein-based components provides the option to insert sequences associated with promoting both cellular adhesion to the substrate and overall cell growth. Protein-based hydrogel components are appealing for their structural designability, specific biological functionality, and stimuli-responsiveness. Here we present highlights in the field of protein-based hydrogels for tissue engineering applications including design requirements, components, and gel types.

  5. Generic comparison of protein inference engines.

    Science.gov (United States)

    Claassen, Manfred; Reiter, Lukas; Hengartner, Michael O; Buhmann, Joachim M; Aebersold, Ruedi

    2012-04-01

    Protein identifications, instead of peptide-spectrum matches, constitute the biologically relevant result of shotgun proteomics studies. How to appropriately infer and report protein identifications has triggered a still ongoing debate. This debate has so far suffered from the lack of appropriate performance measures that allow us to objectively assess protein inference approaches. This study describes an intuitive, generic and yet formal performance measure and demonstrates how it enables experimentalists to select an optimal protein inference strategy for a given collection of fragment ion spectra. We applied the performance measure to systematically explore the benefit of excluding possibly unreliable protein identifications, such as single-hit wonders. Therefore, we defined a family of protein inference engines by extending a simple inference engine by thousands of pruning variants, each excluding a different specified set of possibly unreliable identifications. We benchmarked these protein inference engines on several data sets representing different proteomes and mass spectrometry platforms. Optimally performing inference engines retained all high confidence spectral evidence, without posterior exclusion of any type of protein identifications. Despite the diversity of studied data sets consistently supporting this rule, other data sets might behave differently. In order to ensure maximal reliable proteome coverage for data sets arising in other studies we advocate abstaining from rigid protein inference rules, such as exclusion of single-hit wonders, and instead consider several protein inference approaches and assess these with respect to the presented performance measure in the specific application context.

  6. Generic Comparison of Protein Inference Engines*

    Science.gov (United States)

    Claassen, Manfred; Reiter, Lukas; Hengartner, Michael O.; Buhmann, Joachim M.; Aebersold, Ruedi

    2012-01-01

    Protein identifications, instead of peptide-spectrum matches, constitute the biologically relevant result of shotgun proteomics studies. How to appropriately infer and report protein identifications has triggered a still ongoing debate. This debate has so far suffered from the lack of appropriate performance measures that allow us to objectively assess protein inference approaches. This study describes an intuitive, generic and yet formal performance measure and demonstrates how it enables experimentalists to select an optimal protein inference strategy for a given collection of fragment ion spectra. We applied the performance measure to systematically explore the benefit of excluding possibly unreliable protein identifications, such as single-hit wonders. Therefore, we defined a family of protein inference engines by extending a simple inference engine by thousands of pruning variants, each excluding a different specified set of possibly unreliable identifications. We benchmarked these protein inference engines on several data sets representing different proteomes and mass spectrometry platforms. Optimally performing inference engines retained all high confidence spectral evidence, without posterior exclusion of any type of protein identifications. Despite the diversity of studied data sets consistently supporting this rule, other data sets might behave differently. In order to ensure maximal reliable proteome coverage for data sets arising in other studies we advocate abstaining from rigid protein inference rules, such as exclusion of single-hit wonders, and instead consider several protein inference approaches and assess these with respect to the presented performance measure in the specific application context. PMID:22057310

  7. Protein engineering with unnatural amino acids.

    Science.gov (United States)

    Zhang, William H; Otting, Gottfried; Jackson, Colin J

    2013-08-01

    Protein engineering has become an extensively used tool in many fields, allowing us to probe protein function, characterize proteins using a range of biophysical techniques, chemically modify proteins and improve protein function for medical and industrial applications. It is now possible to site-specifically incorporate unnatural, or non-canonical, amino acids (uAAs) into proteins, which has had a major impact on protein engineering. In this review, we discuss the recent technical developments in the field and how uAA-protein engineering is becoming an increasingly valuable molecular tool, with the unique chemical functionalities of some uAAs allowing a range of otherwise impossible experiments to be performed. Finally, the impediments that have resulted in a relatively small number of recent studies in which uAA-protein engineering has been used to improve protein function are discussed, alongside some of the recent technical developments that may serve to overcome these obstacles. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

  8. Properties of the alpha subunit of a Chaperonin from the hyperthermophilic Crenarchaeon Aeropyrum pernix K1.

    Science.gov (United States)

    Son, Hae-Jin; Shin, Eun-Jung; Nam, Soo-Wan; Kim, Dong-Eun; Jeon, Sung-Jong

    2007-01-01

    The gene encoding for a putative thermosome from the hyperthermophilic crenarchaeon Aeropyrum pernix K1 (ApcpnA) was cloned and the biochemical characteristics of the resulting recombinant protein were examined. The gene (accession no. APE0907) from A. pernix K1 showed some homology with other group II chaperonins from archaea. The recombinant ApcpnA protein has a molecular mass of 60 kDa, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and exhibited ATPase activity with an optimum temperature and pH of 90 degrees C and 5.0, respectively. The ATPase activity was found to be dependent on manganese and potassium ions, but not magnesium ion. The K(m) for ATP at pH 5.0 and 90 degrees C was 10.04 (+/- 1.31) microM, and k(cat) was determined to be 2.21 (+/- 0.11) min(-1) for the ApcpnA monomer. The recombinant ApcpnA prevents thermal aggregation of bovine rhodanese and enhances the thermal stability of alcohol dehydrogenase in vitro, indicating that the protein is suitable as a molecular chaperonin in the high-temperature environment.

  9. Cooperativity of alpha- and beta-subunits of group II chaperonin from the hyperthermophilic archaeum Aeropyrum pernix K1.

    Science.gov (United States)

    Kim, Jeong-Hwan; Lee, Jin-Woo; Shin, Eun-Jung; Nam, Soo-Wan

    2011-02-01

    alpha- and beta-subunits (ApCpnA and ApCpnB) are group II chaperonins from the hyperthermophilic archaeum Aeropyrum pernix K1, specialized in preventing the aggregation and inactivation of substrate proteins under conditions of transient heat stress. In the present study, the cooperativity of alpha- and beta-subunits from the A. pernix K1 was investigated. The ApCpnA and ApCpnB chaperonin genes were overexpressed in E. coli Rosetta and Codonplus (DE3), respectively. Each of the recombinant alpha- and beta- subunits was purified to 92% and 94% by using anionexchange chromatography. The cooperative activity between purified alpha- and beta-subunits was examined using citrate synthase (CS), alcohol dehydrogenase (ADH), and malate dehydrogenase (MDH) as substrate proteins. The addition of both alpha- and beta-subunits could effectively protect CS and ADH from thermal aggregation and inactivation at 43 degreesC and 50 degreesC, respectively, and MDH from thermal inactivation at 80 degreesC and 85 degreesC. Moreover, in the presence of ATP, the protective effects of alpha- and beta-subunits on CS from thermal aggregation and inactivation, and ADH from thermal aggregation, were more enhanced, whereas cooperation between chaperonins and ATP in protection activity on ADH and MDH (at 85 degreesC) from thermal inactivation was not observed. Specifically, the presence of both alpha- and beta- subunits could effectively protect MDH from thermal inactivation at 80 degreesC in an ATP-dependent manner.

  10. Engineered Proteins: Redox Properties and Their Applications

    Science.gov (United States)

    Prabhulkar, Shradha; Tian, Hui; Wang, Xiaotang; Zhu, Jun-Jie

    2012-01-01

    Abstract Oxidoreductases and metalloproteins, representing more than one third of all known proteins, serve as significant catalysts for numerous biological processes that involve electron transfers such as photosynthesis, respiration, metabolism, and molecular signaling. The functional properties of the oxidoreductases/metalloproteins are determined by the nature of their redox centers. Protein engineering is a powerful approach that is used to incorporate biological and abiological redox cofactors as well as novel enzymes and redox proteins with predictable structures and desirable functions for important biological and chemical applications. The methods of protein engineering, mainly rational design, directed evolution, protein surface modifications, and domain shuffling, have allowed the creation and study of a number of redox proteins. This review presents a selection of engineered redox proteins achieved through these methods, resulting in a manipulation in redox potentials, an increase in electron-transfer efficiency, and an expansion of native proteins by de novo design. Such engineered/modified redox proteins with desired properties have led to a broad spectrum of practical applications, ranging from biosensors, biofuel cells, to pharmaceuticals and hybrid catalysis. Glucose biosensors are one of the most successful products in enzyme electrochemistry, with reconstituted glucose oxidase achieving effective electrical communication with the sensor electrode; direct electron-transfer-type biofuel cells are developed to avoid thermodynamic loss and mediator leakage; and fusion proteins of P450s and redox partners make the biocatalytic generation of drug metabolites possible. In summary, this review includes the properties and applications of the engineered redox proteins as well as their significance and great potential in the exploration of bioelectrochemical sensing devices. Antioxid. Redox Signal. 17, 1796–1822. PMID:22435347

  11. Replacing antibodies: engineering new binding proteins.

    Science.gov (United States)

    Banta, Scott; Dooley, Kevin; Shur, Oren

    2013-01-01

    Nature's reliance on proteins to carry out nearly all biological processes has led to the evolution of biomolecules that exhibit a seemingly endless range of functions. Much research has been devoted toward advancing this process in the laboratory in order to create new proteins with improved or unique capabilities. The protein-engineering field has rapidly evolved from pioneering studies in engineering protein stability and activity to an application-driven powerhouse on the forefront of emerging technologies in biomedical engineering and biotechnology. A classic protein-engineering technique in the medical field has focused on manipulating antibodies and antibody fragments for various applications. New classes of alternative scaffolds have recently challenged this paradigm, and these structures have been successfully engineered for applications including targeted cancer therapy, regulated drug delivery, in vivo imaging, and a host of others. This review aims to capture recent advances in the engineering of nonimmunoglobulin scaffolds as well as some of the applications for these molecular recognition elements in the biomedical field.

  12. Natural and Genetically Engineered Proteins for Tissue Engineering

    Science.gov (United States)

    Gomes, Sílvia; Leonor, Isabel B.; Mano, João F.; Reis, Rui L.

    2011-01-01

    To overcome the limitations of traditionally used autografts, allografts and, to a lesser extent, synthetic materials, there is the need to develop a new generation of scaffolds with adequate mechanical and structural support, control of cell attachment, migration, proliferation and differentiation and with bio-resorbable features. This suite of properties would allow the body to heal itself at the same rate as implant degradation. Genetic engineering offers a route to this level of control of biomaterial systems. The possibility of expressing biological components in nature and to modify or bioengineer them further, offers a path towards multifunctional biomaterial systems. This includes opportunities to generate new protein sequences, new self-assembling peptides or fusions of different bioactive domains or protein motifs. New protein sequences with tunable properties can be generated that can be used as new biomaterials. In this review we address some of the most frequently used proteins for tissue engineering and biomedical applications and describe the techniques most commonly used to functionalize protein-based biomaterials by combining them with bioactive molecules to enhance biological performance. We also highlight the use of genetic engineering, for protein heterologous expression and the synthesis of new protein-based biopolymers, focusing the advantages of these functionalized biopolymers when compared with their counterparts extracted directly from nature and modified by techniques such as physical adsorption or chemical modification. PMID:22058578

  13. Probing water density and dynamics in the chaperonin GroEL cavity.

    Science.gov (United States)

    Franck, John M; Sokolovski, Miri; Kessler, Naama; Matalon, Erez; Gordon-Grossman, Michal; Han, Song-I; Goldfarb, Daniella; Horovitz, Amnon

    2014-07-02

    ATP-dependent binding of the chaperonin GroEL to its cofactor GroES forms a cavity in which encapsulated substrate proteins can fold in isolation from bulk solution. It has been suggested that folding in the cavity may differ from that in bulk solution owing to steric confinement, interactions with the cavity walls, and differences between the properties of cavity-confined and bulk water. However, experimental data regarding the cavity-confined water are lacking. Here, we report measurements of water density and diffusion dynamics in the vicinity of a spin label attached to a cysteine in the Tyr71 → Cys GroES mutant obtained using two magnetic resonance techniques: electron-spin echo envelope modulation and Overhauser dynamic nuclear polarization. Residue 71 in GroES is fully exposed to bulk water in free GroES and to confined water within the cavity of the GroEL-GroES complex. Our data show that water density and translational dynamics in the vicinity of the label do not change upon complex formation, thus indicating that bulk water-exposed and cavity-confined GroES surface water share similar properties. Interestingly, the diffusion dynamics of water near the GroES surface are found to be unusually fast relative to other protein surfaces studied. The implications of these findings for chaperonin-assisted folding mechanisms are discussed.

  14. Structural and mechanistic characterization of an archaeal-like chaperonin from a thermophilic bacterium.

    Science.gov (United States)

    An, Young Jun; Rowland, Sara E; Na, Jung-Hyun; Spigolon, Dario; Hong, Seung Kon; Yoon, Yeo Joon; Lee, Jung-Hyun; Robb, Frank T; Cha, Sun-Shin

    2017-10-10

    The chaperonins (CPNs) are megadalton sized hollow complexes with two cavities that open and close to encapsulate non-native proteins. CPNs are assigned to two sequence-related groups that have distinct allosteric mechanisms. In Group I CPNs a detachable co-chaperone, GroES, closes the chambers whereas in Group II a built-in lid closes the chambers. Group I CPNs have a bacterial ancestry, whereas Group II CPNs are archaeal in origin. Here we describe open and closed crystal structures representing a new phylogenetic branch of CPNs. These Group III CPNs are divergent in sequence and structure from extant CPNs, but are closed by a built-in lid like Group II CPNs. A nucleotide-sensing loop, present in both Group I and Group II CPNs, is notably absent. We identified inter-ring pivot joints that articulate during ring closure. These Group III CPNs likely represent a relic from the ancestral CPN that formed distinct bacterial and archaeal branches.Chaperonins (CPNs) are ATP-dependent protein-folding machines. Here the authors present the open and closed crystal structures of a Group III CPN from the thermophilic bacterium Carboxydothermus hydrogenoformans, discuss its mechanism and structurally compare it with Group I and II CPNs.

  15. Engineering of protein assemblies within cells.

    Science.gov (United States)

    Nguyen, Tien K; Ueno, Takafumi

    2018-01-06

    Recent achievements in development of protein assembles within cells have extended biosupramolecular composites into a new era with versatile applications in the fields of biomaterial and biotechnology. Using methods with biological and physicochemical routes has made this era of research more interesting and challenging. Further advances in protein engineering have facilitated efficient fabrication of supramolecular complexes within living cells. Here, we provide a review of recent efforts to engineer protein assemblies within cells and describe the promising properties of these assemblies. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Recombinant protein scaffolds for tissue engineering.

    Science.gov (United States)

    Werkmeister, Jerome A; Ramshaw, John A M

    2012-02-01

    New biological materials for tissue engineering are now being developed using common genetic engineering capabilities to clone and express a variety of genetic elements that allow cost-effective purification and scaffold fabrication from these recombinant proteins, peptides or from chimeric combinations of these. The field is limitless as long as the gene sequences are known. The utility is dependent on the ease, product yield and adaptability of these protein products to the biomedical field. The development of recombinant proteins as scaffolds, while still an emerging technology with respect to commercial products, is scientifically superior to current use of natural materials or synthetic polymer scaffolds, in terms of designing specific structures with desired degrees of biological complexities and motifs. In the field of tissue engineering, next generation scaffolds will be the key to directing appropriate tissue regeneration. The initial period of biodegradable synthetic scaffolds that provided shape and mechanical integrity, but no biological information, is phasing out. The era of protein scaffolds offers distinct advantages, particularly with the combination of powerful tools of molecular biology. These include, for example, the production of human proteins of uniform quality that are free of infectious agents and the ability to make suitable quantities of proteins that are found in low quantity or are hard to isolate from tissue. For the particular needs of tissue engineering scaffolds, fibrous proteins like collagens, elastin, silks and combinations of these offer further advantages of natural well-defined structural scaffolds as well as endless possibilities of controlling functionality by genetic manipulation.

  17. NewProt - a protein engineering portal.

    Science.gov (United States)

    Schwarte, Andreas; Genz, Maika; Skalden, Lilly; Nobili, Alberto; Vickers, Clare; Melse, Okke; Kuipers, Remko; Joosten, Henk-Jan; Stourac, Jan; Bendl, Jaroslav; Black, Jon; Haase, Peter; Baakman, Coos; Damborsky, Jiri; Bornscheuer, Uwe; Vriend, Gert; Venselaar, Hanka

    2017-06-01

    The NewProt protein engineering portal is a one-stop-shop for in silico protein engineering. It gives access to a large number of servers that compute a wide variety of protein structure characteristics supporting work on the modification of proteins through the introduction of (multiple) point mutations. The results can be inspected through multiple visualizers. The HOPE software is included to indicate mutations with possible undesired side effects. The Hotspot Wizard software is embedded for the design of mutations that modify a proteins' activity, specificity, or stability. The NewProt portal is freely accessible at http://newprot.cmbi.umcn.nl/ and http://newprot.fluidops.net/. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  18. Modulating Mobility: a Paradigm for Protein Engineering?

    Science.gov (United States)

    McAuley, Margaret; Timson, David J

    2017-01-01

    Proteins are highly mobile structures. In addition to gross conformational changes occurring on, for example, ligand binding, they are also subject to constant thermal motion. The mobility of a protein varies through its structure and can be modulated by ligand binding and other events. It is becoming increasingly clear that this mobility plays an important role in key functions of proteins including catalysis, allostery, cooperativity, and regulation. Thus, in addition to an optimum structure, proteins most likely also require an optimal dynamic state. Alteration of this dynamic state through protein engineering will affect protein function. A dramatic example of this is seen in some inherited metabolic diseases where alternation of residues distant from the active site affects the mobility of the protein and impairs function. We postulate that using molecular dynamics simulations, experimental data or a combination of the two, it should be possible to engineer the mobility of active sites. This may be useful in, for example, increasing the promiscuity of enzymes. Thus, a paradigm for protein engineering is suggested in which the mobility of the active site is rationally modified. This might be combined with more "traditional" approaches such as altering functional groups in the active site.

  19. ENGINEERED FLUORESCENT PROTEINS ILLUMINATE THE BACTERIAL PERIPLASM

    Directory of Open Access Journals (Sweden)

    Thorben Dammeyer

    2012-10-01

    Full Text Available The bacterial periplasm is of special interest whenever cell factories are designed and engineered. Recombinantely produced proteins are targeted to the periplasmic space of Gram negative bacteria to take advantage of the authentic N-termini, disulfide bridge formation and easy accessibility for purification with less contaminating cellular proteins. The oxidizing environment of the periplasm promotes disulfide bridge formation – a prerequisite for proper folding of many proteins into their active conformation. In contrast, the most popular reporter protein in all of cell biology, Green Fluorescent Protein (GFP, remains inactive if translocated to the periplasmic space prior to folding. Here, the self-catalyzed chromophore maturation is blocked by formation of covalent oligomers via interchain disulfide bonds in the oxidizing environment. However, different protein engineering approaches addressing folding and stability of GFP resulted in improved proteins with enhanced folding properties. Recent studies describe GFP variants that are not only active if translocated in their folded form via the twin-arginine translocation (Tat pathway, but actively fold in the periplasm following general secretory pathway (Sec and signal recognition particle (SRP mediated secretion. This mini-review highlights the progress that enables new insights into bacterial export and periplasmic protein organization, as well as new biotechnological applications combining the advantages of the periplasmic production and the Aequorea-based fluorescent reporter proteins.

  20. Engineered fluorescent proteins illuminate the bacterial periplasm

    Directory of Open Access Journals (Sweden)

    Thorben Dammeyer

    2012-10-01

    Full Text Available The bacterial periplasm is of special interest whenever cell factories are designed and engineered. Recombinantely produced proteins are targeted to the periplasmic space of Gram negative bacteria to take advantage of the authentic N-termini, disulfide bridge formation and easy accessibility for purification with less contaminating cellular proteins. The oxidizing environment of the periplasm promotes disulfide bridge formation - a prerequisite for proper folding of many proteins into their active conformation. In contrast, the most popular reporter protein in all of cell biology, Green Fluorescent Protein (GFP, remains inactive if translocated to the periplasmic space prior to folding. Here, the self-catalyzed chromophore maturation is blocked by formation of covalent oligomers via interchain disulfide bonds in the oxidizing environment. However, different protein engineering approaches addressing folding and stability of GFP resulted in improved proteins with enhanced folding properties. Recent studies describe GFP variants that are not only active if translocated in their folded form via the twin-arginine translocation (Tat pathway, but actively fold in the periplasm following general secretory pathway (Sec and signal recognition particle (SRP mediated secretion. This mini-review highlights the progress that enables new insights into bacterial export and periplasmic protein organization, as well as new biotechnological applications combining the advantages of the periplasmic production and the Aequorea-based fluorescent reporter proteins.

  1. Engineering of filamentous bacteriophage for protein sensing

    Science.gov (United States)

    Brasino, Michael

    Methods of high throughput, sensitive and cost effective quantification of proteins enables personalized medicine by allowing healthcare professionals to better monitor patient condition and response to treatment. My doctoral research has attempted to advance these methods through the use of filamentous bacteriophage (phage). These bacterial viruses are particularly amenable to both genetic and chemical engineering and can be produced efficiently in large amounts. Here, I discuss several strategies for modifying phage for use in protein sensing assays. These include the expression of bio-orthogonal conjugation handles on the phage coat, the incorporation of specific recognition sequences within the phage genome, and the creation of antibody-phage conjugates via a photo-crosslinking non-canonical amino acid. The physical and chemical characterization of these engineered phage and the results of their use in modified protein sensing assays will be presented.

  2. Dentin Matrix Proteins in Bone Tissue Engineering.

    Science.gov (United States)

    Ravindran, Sriram; George, Anne

    2015-01-01

    Dentin and bone are mineralized tissue matrices comprised of collagen fibrils and reinforced with oriented crystalline hydroxyapatite. Although both tissues perform different functionalities, they are assembled and orchestrated by mesenchymal cells that synthesize both collagenous and noncollagenous proteins albeit in different proportions. The dentin matrix proteins (DMPs) have been studied in great detail in recent years due to its inherent calcium binding properties in the extracellular matrix resulting in tissue calcification. Recent studies have shown that these proteins can serve both as intracellular signaling proteins leading to induction of stem cell differentiation and also function as nucleating proteins in the extracellular matrix. These properties make the DMPs attractive candidates for bone and dentin tissue regeneration. This chapter will provide an overview of the DMPs, their functionality and their proven and possible applications with respect to bone tissue engineering.

  3. Protein engineering and its applications in food industry.

    Science.gov (United States)

    Kapoor, Swati; Rafiq, Aasima; Sharma, Savita

    2017-07-24

    Protein engineering is a young discipline that has been branched out from the field of genetic engineering. Protein engineering is based on the available knowledge about the proteins structure/function(s), tools/instruments, software, bioinformatics database, available cloned gene, knowledge about available protein, vectors, recombinant strains and other materials that could lead to change in the protein backbone. Protein produced properly from genetic engineering process means a protein that is able to fold correctly and to do particular function(s) efficiently even after being subjected to engineering practices. Protein is modified through its gene or chemically. However, modification of protein through gene is easier. There is no specific limitation of Protein Engineering tools; any technique that can lead to change the protein constituent of amino acid and result in the modification of protein structure/function is in the frame of Protein Engineering. Meanwhile, there are some common tools used to reach a specific target. More active industrial and pharmaceutical based proteins have been invented by the field of Protein Engineering to introduce new function as well as to change its interaction with surrounding environment. A variety of protein engineering applications have been reported in the literature. These applications range from biocatalysis for food and industry to environmental, medical and nanobiotechnology applications. Successful combinations of various protein engineering methods had led to successful results in food industries and have created a scope to maintain the quality of finished product after processing.

  4. Correlated positions in protein evolution and engineering.

    Science.gov (United States)

    Franceus, Jorick; Verhaeghe, Tom; Desmet, Tom

    2017-05-01

    Statistical analysis of a protein multiple sequence alignment can reveal groups of positions that undergo interdependent mutations throughout evolution. At these so-called correlated positions, only certain combinations of amino acids appear to be viable for maintaining proper folding, stability, catalytic activity or specificity. Therefore, it is often speculated that they could be interesting guides for semi-rational protein engineering purposes. Because they are a fingerprint from protein evolution, their analysis may provide valuable insight into a protein's structure or function and furthermore, they may also be suitable target positions for mutagenesis. Unfortunately, little is currently known about the properties of these correlation networks and how they should be used in practice. This review summarises the recent findings, opportunities and pitfalls of the concept.

  5. Engineering enhanced protein disaggregases for neurodegenerative disease.

    Science.gov (United States)

    Jackrel, Meredith E; Shorter, James

    2015-01-01

    Protein misfolding and aggregation underpin several fatal neurodegenerative diseases, including Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD). There are no treatments that directly antagonize the protein-misfolding events that cause these disorders. Agents that reverse protein misfolding and restore proteins to native form and function could simultaneously eliminate any deleterious loss-of-function or toxic gain-of-function caused by misfolded conformers. Moreover, a disruptive technology of this nature would eliminate self-templating conformers that spread pathology and catalyze formation of toxic, soluble oligomers. Here, we highlight our efforts to engineer Hsp104, a protein disaggregase from yeast, to more effectively disaggregate misfolded proteins connected with PD, ALS, and FTD. Remarkably subtle modifications of Hsp104 primary sequence yielded large gains in protective activity against deleterious α-synuclein, TDP-43, FUS, and TAF15 misfolding. Unusually, in many cases loss of amino acid identity at select positions in Hsp104 rather than specific mutation conferred a robust therapeutic gain-of-function. Nevertheless, the misfolding and toxicity of EWSR1, an RNA-binding protein with a prion-like domain linked to ALS and FTD, could not be buffered by potentiated Hsp104 variants, indicating that further amelioration of disaggregase activity or sharpening of substrate specificity is warranted. We suggest that neuroprotection is achievable for diverse neurodegenerative conditions via surprisingly subtle structural modifications of existing chaperones.

  6. Applications of yeast surface display for protein engineering

    Science.gov (United States)

    Cherf, Gerald M.; Cochran, Jennifer R.

    2015-01-01

    The method of displaying recombinant proteins on the surface of Saccharomyces cerevisiae via genetic fusion to an abundant cell wall protein, a technology known as yeast surface display, or simply, yeast display, has become a valuable protein engineering tool for a broad spectrum of biotechnology and biomedical applications. This review focuses on the use of yeast display for engineering protein affinity, stability, and enzymatic activity. Strategies and examples for each protein engineering goal are discussed. Additional applications of yeast display are also briefly presented, including protein epitope mapping, identification of protein-protein interactions, and uses of displayed proteins in industry and medicine. PMID:26060074

  7. Lentiviral Delivery of Proteins for Genome Engineering.

    Science.gov (United States)

    Cai, Yujia; Mikkelsen, Jacob Giehm

    2016-01-01

    Viruses have evolved to traverse cellular barriers and travel to the nucleus by mechanisms that involve active transport through the cytoplasm and viral quirks to resist cellular restriction factors and innate immune responses. Virus-derived vector systems exploit the capacity of viruses to ferry genetic information into cells, and now - more than three decades after the discovery of HIV - lentiviral vectors based on HIV-1 have become instrumental in biomedical research and gene therapies that require genomic insertion of transgenes. By now, the efficacy of lentiviral gene delivery to stem cells, cells of the immune system including T cells, hepatic cells, and many other therapeutically relevant cell types is well established. Along with nucleic acids, HIV-1 virions carry the enzymatic tools that are essential for early steps of infection. Such capacity to package enzymes, even proteins of nonviral origin, has unveiled new ways of exploiting cellular intrusion of HIV-1. Based on early findings demonstrating the packaging of heterologous proteins into virus particles as part of the Gag and GagPol polypeptides, we have established lentiviral protein transduction for delivery of DNA transposases and designer nucleases. This strategy for delivering genome-engineering proteins facilitates high enzymatic activity within a short time frame and may potentially improve the safety of genome editing. Exploiting the full potential of lentiviral vectors, incorporation of foreign protein can be combined with the delivery of DNA transposons or a donor sequence for homology-directed repair in so-called 'all-in-one' lentiviral vectors. Here, we briefly describe intracellular restrictions that may affect lentiviral gene and protein delivery and review the current status of lentiviral particles as carriers of tool kits for genome engineering.

  8. Protein Engineering Approaches in the Post-Genomic Era.

    Science.gov (United States)

    Singh, Raushan K; Lee, Jung-Kul; Selvaraj, Chandrabose; Singh, Ranjitha; Li, Jinglin; Kim, Sang-Yong; Kalia, Vipin C

    2018-01-01

    Proteins are one of the most multifaceted macromolecules in living systems. Proteins have evolved to function under physiological conditions and, therefore, are not usually tolerant of harsh experimental and environmental conditions. The growing use of proteins in industrial processes as a greener alternative to chemical catalysts often demands constant innovation to improve their performance. Protein engineering aims to design new proteins or modify the sequence of a protein to create proteins with new or desirable functions. With the emergence of structural and functional genomics, protein engineering has been invigorated in the post-genomic era. The three-dimensional structures of proteins with known functions facilitate protein engineering approaches to design variants with desired properties. There are three major approaches of protein engineering research, namely, directed evolution, rational design, and de novo design. Rational design is an effective method of protein engineering when the threedimensional structure and mechanism of the protein is well known. In contrast, directed evolution does not require extensive information and a three-dimensional structure of the protein of interest. Instead, it involves random mutagenesis and selection to screen enzymes with desired properties. De novo design uses computational protein design algorithms to tailor synthetic proteins by using the three-dimensional structures of natural proteins and their folding rules. The present review highlights and summarizes recent protein engineering approaches, and their challenges and limitations in the post-genomic era. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  9. Pharmaceutical and industrial protein engineering: where we are?

    Science.gov (United States)

    Amara, Amro Abd-Al-Fattah

    2013-01-01

    The huge amount of information, the big number of scientists and their efforts, labs, man/hrs, fund, companies all and others factors build the success of the amazing new branch of genetic engineering the 'protein engineering' (PE). It concerns with the modification of protein structure/function(s) or building protein from scratch. The engineered proteins usually have new criteria(s). Engineering proteins can be mediated on the level of genes or proteins. PE fined its way in different important sectors including industrial, pharmaceutical and medicinal ones. Aspects about PE and its applications will be discussed with this review. The concept, tools, and the industrial applications of the protein, engineered proteins and PE will be under focus. In order to get up to date knowledge about the applications of PE in basic protein and molecular biology, several examples are discussed. PE can play a significant role in different industrial and pharmaceutical sectors if used wisely and selectively.

  10. Protein Engineering: A New Frontier for Biological Therapeutics

    Science.gov (United States)

    Tobin, Peter H.; Richards, David H.; Callender, Randolph A.

    2016-01-01

    Protein engineering holds the potential to transform the metabolic drug landscape through the development of smart, stimulus-responsive drug systems. Protein therapeutics are a rapidly expanding segment of Food and Drug Administration approved drugs that will improve clinical outcomes over the long run. Engineering of protein therapeutics is still in its infancy, but recent general advances in protein engineering capabilities are being leveraged to yield improved control over both pharmacokinetics and pharmacodynamics. Stimulus-responsive protein therapeutics are drugs which have been designed to be metabolized under targeted conditions. Protein engineering is being utilized to develop tailored smart therapeutics with biochemical logic. This review focuses on applications of targeted drug neutralization, stimulus-responsive engineered protein prodrugs, and emerging multicomponent smart drug systems (e.g., antibody-drug conjugates, responsive engineered zymogens, prospective biochemical logic smart drug systems, drug buffers, and network medicine applications). PMID:25495737

  11. Toward protein engineering for phytoremediation: possibilities and challenges.

    Science.gov (United States)

    Jez, Joseph M

    2011-01-01

    The combination of rational protein engineering and directed evolution techniques allow for the redesign of enzymes with tailored properties for use in environmental remediation. This review summarizes current molecular methods for either altering or improving protein function and highlights examples of how these methods can address bioremediation problems. Although much of the protein engineering applied to environmental clean-up employs microbial systems, there is great potential for and significant challenges to translating these approaches to plant systems for phytoremediation purposes. Protein engineering technologies combined with genomic information and metabolic engineering strategies hold promise for the design of plants and microbes to remediate organic and inorganic pollutants.

  12. Engineering protein scaffolds for protein separation, biocatalysis and nanotechnology applications

    Science.gov (United States)

    Liu, Fang

    Globally, there is growing appreciation for developing a sustainable economy that uses eco-efficient bio-processes. Biotechnology provides an increasing range of tools for industry to help reduce cost and improve environmental performance. Inspired by the naturally evolved machineries of protein scaffolds and their binding ligands, synthetic protein scaffolds were engineered based on cohesin-dockerin interactions and metal chelating peptides to tackle the challenges and make improvements in three specific areas: (1) protein purification, (2) biofuel cells, and (3) nanomaterial synthesis. The first objective was to develop efficient and cost-effective non-chromatographic purification processes to purify recombinant proteins in an effort to meet the dramatically growing market of protein drugs. In our design, the target protein was genetically fused with a dockerin domain from Clostridium thermocellum and direct purification and recovery was achieved using thermo-responsive elastin-like polypeptide (ELP) scaffold containing the cohesin domain from the same species. By exploiting the highly specific interaction between the dockerin and cohesin domain and the reversible aggregation property of ELP, highly purified and active dockerin-tagged proteins, such as endoglucanase CelA, chloramphenicol acetyl transferase (CAT) and enhanced green fluorescence protein (EGFP), were recovered directly from crude cell extracts in a single purification step with yields achieving over 90%. Incorporation of a self-cleaving intein domain enabled rapid removal of the affinity tag from the target proteins by another cycle of thermal precipitation. The purification cost can be further reduced by regenerating and recycling the ELP-cohesin capturing scaffolds. However, due to the high binding affinity between cohesin and dockerin domains, the bound dockerin-intein tag cannot be completely disassociated from ELP-cohesin scaffold after binding. Therefore, a truncated dockerin with the calcium

  13. Chaperonin 20 might be an iron chaperone for superoxide dismutase in activating iron superoxide dismutase (FeSOD).

    Science.gov (United States)

    Kuo, Wen-Yu; Huang, Chien-Hsun; Jinn, Tsung-Luo

    2013-02-01

    Activation of Cu/Zn superoxide dismutases (CuZnSODs) is aided by Cu incorporation and disulfide isomerization by Cu chaperone of SOD (CCS). As well, an Fe-S cluster scaffold protein, ISU, might alter the incorporation of Fe or Mn into yeast MnSOD (ySOD2), thus leading to active or inactive ySOD2. However, metallochaperones involved in the activation of FeSODs are unknown. Recently, we found that a chloroplastic chaperonin cofactor, CPN20, could mediate FeSOD activity. To investigate whether Fe incorporation in FeSOD is affected by CPN20, we used inductively coupled plasma mass spectrometry to analyze the ability of CPN20 to bind Fe. CPN20 could bind Fe, and the Fe binding to FeSOD was increased with CPN20 incubation. Thus, CPN20 might be an Fe chaperone for FeSOD activation, a role independent of its well-known co-chaperonin activity.

  14. Engineering Proteins for Thermostability with iRDP Web Server

    National Research Council Canada - National Science Library

    Panigrahi, Priyabrata; Sule, Manas; Ghanate, Avinash; Ramasamy, Sureshkumar; Suresh, C G

    2015-01-01

    ... been targeted through rational protein engineering. Although a range of factors contributing to thermal stability have been identified and widely researched, the in silico implementation of these as strategies directed towards enhancement of protein...

  15. Mimicking the action of GroEL in molecular dynamics simulations : Application to the refinement of protein structures

    NARCIS (Netherlands)

    Fan, H; Mark, AE

    Bacterial chaperonin, GroEL, together with its co-chaperonin, GroES, facilitates the folding of a variety of polypeptides. Experiments suggest that GroEL stimulates protein folding by multiple cycles of binding and release. Misfolded proteins first bind to an exposed hydrophobic surface on GroEL.

  16. Protein engineering for metabolic engineering: current and next-generation tools

    Science.gov (United States)

    Marcheschi, Ryan J.; Gronenberg, Luisa S.; Liao, James C.

    2014-01-01

    Protein engineering in the context of metabolic engineering is increasingly important to the field of industrial biotechnology. As the demand for biologically-produced food, fuels, chemicals, food additives, and pharmaceuticals continues to grow, the ability to design and modify proteins to accomplish new functions will be required to meet the high productivity demands for the metabolism of engineered organisms. This article reviews advances of selecting, modeling, and engineering proteins to improve or alter their activity. Some of the methods have only recently been developed for general use and are just beginning to find greater application in the metabolic engineering community. We also discuss methods of generating random and targeted diversity in proteins to generate mutant libraries for analysis. Recent uses of these techniques to alter cofactor use, produce non-natural amino acids, alcohols, and carboxylic acids, and alter organism phenotypes are presented and discussed as examples of the successful engineering of proteins for metabolic engineering purposes. PMID:23589443

  17. Enhanced crystallizability by protein engineering approaches: a general overview.

    Science.gov (United States)

    Ruggiero, Alessia; Smaldone, Giovanni; Squeglia, Flavia; Berisio, Rita

    2012-07-01

    The limiting step in macromolecular crystallography is the preparation protein crystals suitable for X-ray diffraction studies. A strong prerequisite for the success of crystallization experiments is the ability to produce monodisperse and properly folded protein samples. Since the production of most protein is usually achieved using recombinant methods, it has become possible to engineer target proteins with increased propensities to form well diffracting crystals. Recent advances in bioinformatics, which takes advantage from an enhanced information in the protein databases, are of enormous help for the design of modified proteins. Based on bioinformatics analyses, the reduction of the structural complexity of proteins or their site-specific mutagenesis has proven to have a dramatic impact on both the yield of heterologous protein expression and its crystallizability. Therefore, protein engineering represents a valid tool which supports the classical crystallization screenings with a more rational approach. This review describes key methods of protein-engineering and provides a number of examples of their successful use in crystallization. Scope of proposed topic: This Topic is focused on state-of-art protein engineering techniques to increase the propensity of proteins to form crystals with suitable X-ray diffraction properties. Protein engineering methods have proven to be of great help for the crystallization of difficult targets. We herein review molecular biology and chemical methods to help protein crystallization.

  18. Characterization of a novel stable biocatalyst obtained by protein engineering

    NARCIS (Netherlands)

    Van den Burg, B; de Kreij, A; Van der Veek, P; Mansfeld, J; Venema, G

    Protein engineering is a powerful tool for the improvement of the properties of biocatalysts, Previously we have applied protein engineering technologies to obtain an extremely stable variant of the thermolysin-like protease from Bacillus stearothermophilus [Van den Burg, Vriend, Veltman, Venema and

  19. A reliability measure of protein-protein interactions and a reliability measure-based search engine.

    Science.gov (United States)

    Park, Byungkyu; Han, Kyungsook

    2010-02-01

    Many methods developed for estimating the reliability of protein-protein interactions are based on the topology of protein-protein interaction networks. This paper describes a new reliability measure for protein-protein interactions, which does not rely on the topology of protein interaction networks, but expresses biological information on functional roles, sub-cellular localisations and protein classes as a scoring schema. The new measure is useful for filtering many spurious interactions, as well as for estimating the reliability of protein interaction data. In particular, the reliability measure can be used to search protein-protein interactions with the desired reliability in databases. The reliability-based search engine is available at http://yeast.hpid.org. We believe this is the first search engine for interacting proteins, which is made available to public. The search engine and the reliability measure of protein interactions should provide useful information for determining proteins to focus on.

  20. A discussion of molecular biology methods for protein engineering.

    Science.gov (United States)

    Zawaira, Alexander; Pooran, Anil; Barichievy, Samantha; Chopera, Denis

    2012-05-01

    A number of molecular biology techniques are available to generate variants from a particular start gene for eventual protein expression. We discuss the basic principles of these methods in a repertoire that may be used to achieve the elemental steps in protein engineering. These include site-directed, deletion and insertion mutagenesis. We provide detailed case studies, drawn from our own experiences, packaged together with conceptual discussions and include an analysis of the techniques presented with regards to their uses in protein engineering.

  1. Engineering membrane protein overproduction in Escherichia coli

    Science.gov (United States)

    Molina, Daniel Martinez; Cornvik, Tobias; Eshaghi, Said; Haeggström, Jesper Z.; Nordlund, Pär; Sabet, Marina Ignatushchenko

    2008-01-01

    Membrane proteins play a fundamental role in human disease and therapy, but suffer from a lack of structural and functional information compared to their soluble counterparts. The paucity of membrane protein structures is primarily due to the unparalleled difficulties in obtaining detergent-solubilized membrane proteins at sufficient levels and quality. We have developed an in vitro evolution strategy for optimizing the levels of detergent-solubilized membrane protein that can be overexpressed and purified from recombinant Escherichia coli. Libraries of random mutants for nine membrane proteins were screened for expression using a novel implementation of the colony filtration blot. In only one cycle of directed evolution were significant improvements of membrane protein yield obtained for five out of nine proteins. In one case, the yield of detergent-solubilized membrane protein was increased 40-fold. PMID:18305199

  2. Engineering Protein Hydrogels Using SpyCatcher-SpyTag Chemistry.

    Science.gov (United States)

    Gao, Xiaoye; Fang, Jie; Xue, Bin; Fu, Linglan; Li, Hongbin

    2016-09-12

    Constructing hydrogels from engineered proteins has attracted significant attention within the material sciences, owing to their myriad potential applications in biomedical engineering. Developing efficient methods to cross-link tailored protein building blocks into hydrogels with desirable mechanical, physical, and functional properties is of paramount importance. By making use of the recently developed SpyCatcher-SpyTag chemistry, we successfully engineered protein hydrogels on the basis of engineered tandem modular elastomeric proteins. Our resultant protein hydrogels are soft but stable, and show excellent biocompatibility. As the first step, we tested the use of these hydrogels as a drug carrier, as well as in encapsulating human lung fibroblast cells. Our results demonstrate the robustness of the SpyCatcher-SpyTag chemistry, even when the SpyTag (or SpyCatcher) is flanked by folded globular domains. These results demonstrate that SpyCatcher-SpyTag chemistry can be used to engineer protein hydrogels from tandem modular elastomeric proteins that can find applications in tissue engineering, in fundamental mechano-biological studies, and as a controlled drug release vehicle.

  3. Engineering enhanced protein disaggregases for neurodegenerative disease

    OpenAIRE

    Jackrel, Meredith E.; Shorter, James

    2015-01-01

    Abstract Protein misfolding and aggregation underpin several fatal neurodegenerative diseases, including Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD). There are no treatments that directly antagonize the protein-misfolding events that cause these disorders. Agents that reverse protein misfolding and restore proteins to native form and function could simultaneously eliminate any deleterious loss-of-function or toxic gain-of-function caused by...

  4. Engineering membrane protein overproduction in Escherichia coli

    OpenAIRE

    Molina, Daniel Martinez; Cornvik, Tobias; Eshaghi, Said; Haeggström, Jesper Z.; Nordlund, Pär; Sabet, Marina Ignatushchenko

    2008-01-01

    Membrane proteins play a fundamental role in human disease and therapy, but suffer from a lack of structural and functional information compared to their soluble counterparts. The paucity of membrane protein structures is primarily due to the unparalleled difficulties in obtaining detergent-solubilized membrane proteins at sufficient levels and quality. We have developed an in vitro evolution strategy for optimizing the levels of detergent-solubilized membrane protein that can be overexpresse...

  5. OsCpn60α1, encoding the plastid chaperonin 60α subunit, is essential for folding of rbcL.

    Science.gov (United States)

    Kim, Sung-Ryul; Yang, Jung-Il; An, Gynheung

    2013-05-01

    Chaperonins are involved in protein-folding. The rice genome encodes six plastid chaperonin subunits (Cpn60) - three α and three β. Our study showed that they were differentially expressed during normal plant development. Moreover, five were induced by heat stress (42°C) but not by cold (10°C). The oscpn60α1 mutant had a pale-green phenotype at the seedling stage and development ceased after the fourth leaf appeared. Transiently expressed OsCpn60α1:GFP fusion protein was localized to the chloroplast stroma. Immuno-blot analysis indicated that the level of Rubisco large subunit (rbcL) was severely reduced in the mutant while levels were unchanged for some imported proteins, e.g., stromal heat shock protein 70 (Hsp70) and chlorophyll a/b binding protein 1 (Lhcb1). This demonstrated that OsCpn60α1 is required for the folding of rbcL and that failure of that process is seedling-lethal.

  6. Protein Production in the Milk of Genetically Engineered Animals

    OpenAIRE

    Bates, Katherine M.

    1998-01-01

    There are numerous proteins that have potential uses in commercial and scientific applications that are not utilized to their full potential. this is partly because it is not economically feasible to isolate some of these proteins from their natural sources or to produce them using bacterial fermentation methods. The purpose of this research was to target recombinant protein expression to the mammary glands of genetically engineered or transgenic animals. Foreign protein expression has been a...

  7. Engineering Proteins for Thermostability with iRDP Web Server

    Science.gov (United States)

    Ghanate, Avinash; Ramasamy, Sureshkumar; Suresh, C. G.

    2015-01-01

    Engineering protein molecules with desired structure and biological functions has been an elusive goal. Development of industrially viable proteins with improved properties such as stability, catalytic activity and altered specificity by modifying the structure of an existing protein has widely been targeted through rational protein engineering. Although a range of factors contributing to thermal stability have been identified and widely researched, the in silico implementation of these as strategies directed towards enhancement of protein stability has not yet been explored extensively. A wide range of structural analysis tools is currently available for in silico protein engineering. However these tools concentrate on only a limited number of factors or individual protein structures, resulting in cumbersome and time-consuming analysis. The iRDP web server presented here provides a unified platform comprising of iCAPS, iStability and iMutants modules. Each module addresses different facets of effective rational engineering of proteins aiming towards enhanced stability. While iCAPS aids in selection of target protein based on factors contributing to structural stability, iStability uniquely offers in silico implementation of known thermostabilization strategies in proteins for identification and stability prediction of potential stabilizing mutation sites. iMutants aims to assess mutants based on changes in local interaction network and degree of residue conservation at the mutation sites. Each module was validated using an extensively diverse dataset. The server is freely accessible at http://irdp.ncl.res.in and has no login requirements. PMID:26436543

  8. Engineering Proteins for Thermostability with iRDP Web Server.

    Directory of Open Access Journals (Sweden)

    Priyabrata Panigrahi

    Full Text Available Engineering protein molecules with desired structure and biological functions has been an elusive goal. Development of industrially viable proteins with improved properties such as stability, catalytic activity and altered specificity by modifying the structure of an existing protein has widely been targeted through rational protein engineering. Although a range of factors contributing to thermal stability have been identified and widely researched, the in silico implementation of these as strategies directed towards enhancement of protein stability has not yet been explored extensively. A wide range of structural analysis tools is currently available for in silico protein engineering. However these tools concentrate on only a limited number of factors or individual protein structures, resulting in cumbersome and time-consuming analysis. The iRDP web server presented here provides a unified platform comprising of iCAPS, iStability and iMutants modules. Each module addresses different facets of effective rational engineering of proteins aiming towards enhanced stability. While iCAPS aids in selection of target protein based on factors contributing to structural stability, iStability uniquely offers in silico implementation of known thermostabilization strategies in proteins for identification and stability prediction of potential stabilizing mutation sites. iMutants aims to assess mutants based on changes in local interaction network and degree of residue conservation at the mutation sites. Each module was validated using an extensively diverse dataset. The server is freely accessible at http://irdp.ncl.res.in and has no login requirements.

  9. Overexpression, purification, and characterization of beta-subunit group II chaperonin from hyperthermophilic Aeropyrum pernix K1.

    Science.gov (United States)

    Shin, Eun-Jung; Lee, Jin-Woo; Kim, Jeong-Hwan; Jeon, Sung-Jong; Kim, Yeon-Hee; Nam, Soo-Wan

    2010-03-01

    In the present study, overexpression, purification, and characterization of Aeropyrum pernix K1 chaperonin B in E. coli were investigated. The chaperonin beta-subunit gene (ApCpnB, 1,665 bp ORF) from the hyperthermophilic archaeon A. pernix K1 was amplified by PCR and subcloned into vector pET21a. The constructed pET21a-ApCpnB (6.9 kb) was transformed into E. coli BL21 Codonplus (DE3). The transformant cell successfully expressed ApCpnB, and the expression of ApCpnB (61.2 kDa) was identified through analysis of the fractions by SDS-PAGE (14% gel). The recombinant ApCpnB was purified to higher than 94% by using heat-shock treatment at 90 degrees C for 20 min and fast protein liquid chromatography on a HiTrap Q column step. The purified ApCpnB showed ATPase activity and its activity was dependent on temperature. In the presence of ATP, ApCpnB effectively protected citrate synthase (CS) and alcohol dehydrogenase (ADH) from thermal aggregation and inactivation at 43 degrees C and 50 degrees C, respectively. Specifically, the activity of malate dehydrogenase (MDH) at 85 degrees C was greatly stabilized by the addition of ApCpnB and ATP. Coexpression of procarboxypeptidase B (pro-CPB) and ApCpnB in E. coli BL21 Codonplus (DE3) had a marked effect on the yield of pro-CPB as a soluble and active form, speculating that ApCpnB facilitates the correct folding of pro-CPB. These results suggest that ApCpnB has both foldase and holdase activities and can be used as a powerful molecular machinery for the production of recombinant proteins as soluble and active forms in E. coli.

  10. Modular protein domains: an engineering approach toward functional biomaterials.

    Science.gov (United States)

    Lin, Charng-Yu; Liu, Julie C

    2016-08-01

    Protein domains and peptide sequences are a powerful tool for conferring specific functions to engineered biomaterials. Protein sequences with a wide variety of functionalities, including structure, bioactivity, protein-protein interactions, and stimuli responsiveness, have been identified, and advances in molecular biology continue to pinpoint new sequences. Protein domains can be combined to make recombinant proteins with multiple functionalities. The high fidelity of the protein translation machinery results in exquisite control over the sequence of recombinant proteins and the resulting properties of protein-based materials. In this review, we discuss protein domains and peptide sequences in the context of functional protein-based materials, composite materials, and their biological applications. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. COMPUTATIONAL TOOLS FOR RATIONAL PROTEIN ENGINEERING OF ALDOLASES

    OpenAIRE

    Widmann, Michael; Pleiss, Jürgen; Samland, Anne K.

    2012-01-01

    In this mini-review we describe the different strategies for rational protein engineering and summarize the computational tools available. Computational tools can either be used to design focused libraries, to predict sequence-function relationships or for structure-based molecular modelling. This also includes de novo design of enzymes. Examples for protein engineering of aldolases and transaldolases are given in the second part of the mini-review.

  12. Computational tools for rational protein engineering of aldolases

    Directory of Open Access Journals (Sweden)

    Michael Widmann

    2012-09-01

    Full Text Available In this mini-review we describe the different strategies for rational protein engineering and summarize the computational tools available. Computational tools can either be used to design focused libraries, to predict sequence-function relationships or for structure-based molecular modelling. This also includes de novo design of enzymes. Examples for protein engineering of aldolases and transaldolases are given in the second part of the mini-review.

  13. Strain engineering for improved expression of recombinant proteins in bacteria

    Science.gov (United States)

    2011-01-01

    Protein expression in Escherichia coli represents the most facile approach for the preparation of non-glycosylated proteins for analytical and preparative purposes. So far, the optimization of recombinant expression has largely remained a matter of trial and error and has relied upon varying parameters, such as expression vector, media composition, growth temperature and chaperone co-expression. Recently several new approaches for the genome-scale engineering of E. coli to enhance recombinant protein expression have been developed. These methodologies now enable the generation of optimized E. coli expression strains in a manner analogous to metabolic engineering for the synthesis of low-molecular-weight compounds. In this review, we provide an overview of strain engineering approaches useful for enhancing the expression of hard-to-produce proteins, including heterologous membrane proteins. PMID:21569582

  14. Clinical translation of controlled protein delivery systems for tissue engineering.

    Science.gov (United States)

    Spiller, Kara L; Vunjak-Novakovic, Gordana

    2015-04-01

    Strategies that utilize controlled release of drugs and proteins for tissue engineering have enormous potential to regenerate damaged organs and tissues. The multiple advantages of controlled release strategies merit overcoming the significant challenges to translation, including high costs and long, difficult regulatory pathways. This review highlights the potential of controlled release of proteins for tissue engineering and regenerative medicine. We specifically discuss treatment modalities that have reached preclinical and clinical trials, with emphasis on controlled release systems for bone tissue engineering, the most advanced application with several products already in clinic. Possible strategies to address translational and regulatory concerns are also discussed.

  15. Integrating protein engineering with process design for biocatalysis

    DEFF Research Database (Denmark)

    Woodley, John M.

    2017-01-01

    important driver. In the early applications of the technology, it was necessary to design processes to match the properties of the biocatalyst. With the advent of protein engineering, organic chemists started to develop and improve enzymes to suit their needs. Likewise in industry, although not widespread......, a new paradigm was already implemented several years ago to engineer enzymes to suit process needs. Today, a new era is entered, where the effectiveness with which such integrated protein and process engineering is achieved becomes critical to implementation. In this paper, the development of a tool...... to improve the effectiveness of this approach is discussed, namely the use of target-setting based on process requirements, to guide the necessary protein engineering....

  16. Protein design in systems metabolic engineering for industrial strain development.

    Science.gov (United States)

    Chen, Zhen; Zeng, An-Ping

    2013-05-01

    Accelerating the process of industrial bacterial host strain development, aimed at increasing productivity, generating new bio-products or utilizing alternative feedstocks, requires the integration of complementary approaches to manipulate cellular metabolism and regulatory networks. Systems metabolic engineering extends the concept of classical metabolic engineering to the systems level by incorporating the techniques used in systems biology and synthetic biology, and offers a framework for the development of the next generation of industrial strains. As one of the most useful tools of systems metabolic engineering, protein design allows us to design and optimize cellular metabolism at a molecular level. Here, we review the current strategies of protein design for engineering cellular synthetic pathways, metabolic control systems and signaling pathways, and highlight the challenges of this subfield within the context of systems metabolic engineering. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Evaluation of mass spectrometric techniques for characterization of engineered proteins

    DEFF Research Database (Denmark)

    Roepstorff, P; Schram, K H; Andersen, Jens S.

    1995-01-01

    Mass spectrometric characterization of engineered proteins has been examined using bovine recombinant Acyl-CoA-Binding Protein (rACBP), [15N]-labeled rACBP, and a number of sequence variants of ACBP produced by site-directed mutagenesis. The mass spectrometric techniques include ESIMS and MALDIMS...

  18. Engineered protein nano-compartments for targeted enzyme localization.

    Directory of Open Access Journals (Sweden)

    Swati Choudhary

    Full Text Available Compartmentalized co-localization of enzymes and their substrates represents an attractive approach for multi-enzymatic synthesis in engineered cells and biocatalysis. Sequestration of enzymes and substrates would greatly increase reaction efficiency while also protecting engineered host cells from potentially toxic reaction intermediates. Several bacteria form protein-based polyhedral microcompartments which sequester functionally related enzymes and regulate their access to substrates and other small metabolites. Such bacterial microcompartments may be engineered into protein-based nano-bioreactors, provided that they can be assembled in a non-native host cell, and that heterologous enzymes and substrates can be targeted into the engineered compartments. Here, we report that recombinant expression of Salmonella enterica ethanolamine utilization (eut bacterial microcompartment shell proteins in E. coli results in the formation of polyhedral protein shells. Purified recombinant shells are morphologically similar to the native Eut microcompartments purified from S. enterica. Surprisingly, recombinant expression of only one of the shell proteins (EutS is sufficient and necessary for creating properly delimited compartments. Co-expression with EutS also facilitates the encapsulation of EGFP fused with a putative Eut shell-targeting signal sequence. We also demonstrate the functional localization of a heterologous enzyme (β-galactosidase targeted to the recombinant shells. Together our results provide proof-of-concept for the engineering of protein nano-compartments for biosynthesis and biocatalysis.

  19. Protein Engineering by Combined Computational and In Vitro Evolution Approaches.

    Science.gov (United States)

    Rosenfeld, Lior; Heyne, Michael; Shifman, Julia M; Papo, Niv

    2016-05-01

    Two alternative strategies are commonly used to study protein-protein interactions (PPIs) and to engineer protein-based inhibitors. In one approach, binders are selected experimentally from combinatorial libraries of protein mutants that are displayed on a cell surface. In the other approach, computational modeling is used to explore an astronomically large number of protein sequences to select a small number of sequences for experimental testing. While both approaches have some limitations, their combination produces superior results in various protein engineering applications. Such applications include the design of novel binders and inhibitors, the enhancement of affinity and specificity, and the mapping of binding epitopes. The combination of these approaches also aids in the understanding of the specificity profiles of various PPIs. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Engineering bioluminescent proteins: expanding their analytical potential.

    Science.gov (United States)

    Rowe, Laura; Dikici, Emre; Daunert, Sylvia

    2009-11-01

    Bioluminescent proteins are used in a plethora of analytical methods, from ultrasensitive assay development to the in vivo imaging of cellular processes. This article reviews the most pertinent current bioluminescent-protein-based technologies and suggests the future direction of this vein of research. (To listen to a podcast about this feature, please go to the Analytical Chemistry multimedia page at pubs.acs.org/page/ancham/audio/index.html .).

  1. Engineering Bioluminescent Proteins: Expanding their Analytical Potential

    OpenAIRE

    Rowe, Laura; Dikici, Emre; Daunert, Sylvia

    2009-01-01

    Bioluminescence has been observed in nature since the dawn of time, but now, scientists are harnessing it for analytical applications. Laura Rowe, Emre Dikici, and Sylvia Daunert of the University of Kentucky describe the origins of bioluminescent proteins and explore their uses in the modern chemistry laboratory. The cover features spectra of bioluminescent light superimposed on an image of jellyfish, which are a common source of bioluminescent proteins. Images courtesy of Emre Dikici and Sh...

  2. An ontology-based search engine for protein-protein interactions.

    Science.gov (United States)

    Park, Byungkyu; Han, Kyungsook

    2010-01-18

    Keyword matching or ID matching is the most common searching method in a large database of protein-protein interactions. They are purely syntactic methods, and retrieve the records in the database that contain a keyword or ID specified in a query. Such syntactic search methods often retrieve too few search results or no results despite many potential matches present in the database. We have developed a new method for representing protein-protein interactions and the Gene Ontology (GO) using modified Gödel numbers. This representation is hidden from users but enables a search engine using the representation to efficiently search protein-protein interactions in a biologically meaningful way. Given a query protein with optional search conditions expressed in one or more GO terms, the search engine finds all the interaction partners of the query protein by unique prime factorization of the modified Gödel numbers representing the query protein and the search conditions. Representing the biological relations of proteins and their GO annotations by modified Gödel numbers makes a search engine efficiently find all protein-protein interactions by prime factorization of the numbers. Keyword matching or ID matching search methods often miss the interactions involving a protein that has no explicit annotations matching the search condition, but our search engine retrieves such interactions as well if they satisfy the search condition with a more specific term in the ontology.

  3. Genetically engineered luminescent proteins in biosensing

    Science.gov (United States)

    Rowe, Laura; Ensor, Mark; Scott, Daniel; Deo, Sapna; Daunert, Sylvia

    2006-02-01

    Luminescent proteins originally isolated from marine or terrestrial organisms have played a key role in the development of several biosensing systems. These proteins have been used in a variety of applications including, immunoassays, binding assays, cell-based sensing, high throughput screening, optical imaging, etc. Among the luminescent proteins isolated, the bioluminescent protein aequorin has been one of the proteins at the forefront in terms of its use in a vast number of biosensing systems. In our laboratory, we have employed aequorin as a label in the development of highly sensitive assays through chemical and genetic modifications from single step analysis of physiologically important molecules in biological fluids. An important aspect of optimizing these assays for clinical use involves understanding the stability of the various aequorin variants that are available. To this end we have designed several stability studies involving three important aequorin mutants, Mutant S, Mutant 5, and Mutant 53. The cysteine free aequorin, Mutant S, has been the most ubiquitously used aequorin variant in our laboratory because of its increased stability and activity as compared to native aequorin. Mutant 5 and Mutant 53 contain a single cyteine residue at position 5 and 53 in the protein, respectively. Because of the presence of a single cysteine residue, Mutant 5 and Mutant 53 both can be site-specifically conjugated. This site specific conjugation capability gives Mutant 5 and Mutant 53 an advantage over native aequorin when developing assays. Additional studies optimizing the expression, purification, and charging of aequorin Mutant S were also performed. A thorough understanding of the efficient expression, purification, and storage of these aequorin mutants will allow for the more practical utilization of these mutants in the development of future biosensing systems.

  4. Opportunities for protein interaction network-guided cellular engineering.

    Science.gov (United States)

    Wright, Phillip C; Jaffe, Stephen; Noirel, Josselin; Zou, Xin

    2013-01-01

    As we move further into the postgenomics age where the mountain of systems biology-generated data keeps growing, as does the number of genomes that have been sequenced, we have the exciting opportunity to understand more deeply the biology of important systems, those that are amenable to genetic manipulation and metabolic engineering. This is, of course, if we can make 'head or tail' of what we have measured and use this for robust predictions. The use of modern mass spectrometry tools has greatly facilitated our understanding of which proteins are present in a particular phenotype, their relative and absolute abundances and their state of modifications. Coupled with modern bioinformatics and systems biology modelling tools, this has the opportunity of not just providing information and understanding but also to provide targets for engineering and suggest new genetic/metabolic designs. Cellular engineering, whether it be via metabolic engineering, synthetic biology or a combination of both approaches, offers exciting potential for biotechnological exploitation in fields as diverse as medicine and energy as well as fine and bulk chemicals production. At the heart of such effective designs, proteins' interactions with other proteins or with DNA will become increasingly important. In this work, we examine the work done until now in protein-protein interactions and how this network knowledge can be used to inform ambitious cellular engineering strategies. Some examples demonstrating small molecules/biofuels and biopharmaceuticals applications are presented. Copyright © 2012 International Union of Biochemistry and Molecular Biology, Inc.

  5. Engineering High Affinity Protein-Protein Interactions Using a High-Throughput Microcapillary Array Platform.

    Science.gov (United States)

    Lim, Sungwon; Chen, Bob; Kariolis, Mihalis S; Dimov, Ivan K; Baer, Thomas M; Cochran, Jennifer R

    2017-02-17

    Affinity maturation of protein-protein interactions requires iterative rounds of protein library generation and high-throughput screening to identify variants that bind with increased affinity to a target of interest. We recently developed a multipurpose protein engineering platform, termed μSCALE (Microcapillary Single Cell Analysis and Laser Extraction). This technology enables high-throughput screening of libraries of millions of cell-expressing protein variants based on their binding properties or functional activity. Here, we demonstrate the first use of the μSCALE platform for affinity maturation of a protein-protein binding interaction. In this proof-of-concept study, we engineered an extracellular domain of the Axl receptor tyrosine kinase to bind tighter to its ligand Gas6. Within 2 weeks, two iterative rounds of library generation and screening resulted in engineered Axl variants with a 50-fold decrease in kinetic dissociation rate, highlighting the use of μSCALE as a new tool for directed evolution.

  6. Maintenance of native-like protein dynamics may not be required for engineering functional proteins.

    Science.gov (United States)

    Gobeil, Sophie M C; Clouthier, Christopher M; Park, Jaeok; Gagné, Donald; Berghuis, Albert M; Doucet, Nicolas; Pelletier, Joelle N

    2014-10-23

    Proteins are dynamic systems, and understanding dynamics is critical for fully understanding protein function. Therefore, the question of whether laboratory engineering has an impact on protein dynamics is of general interest. Here, we demonstrate that two homologous, naturally evolved enzymes with high degrees of structural and functional conservation also exhibit conserved dynamics. Their similar set of slow timescale dynamics is highly restricted, consistent with evolutionary conservation of a functionally important feature. However, we also show that dynamics of a laboratory-engineered chimeric enzyme obtained by recombination of the two homologs exhibits striking difference on the millisecond timescale, despite function and high-resolution crystal structure (1.05 Å) being conserved. The laboratory-engineered chimera is thus functionally tolerant to modified dynamics on the timescale of catalytic turnover. Tolerance to dynamic variation implies that maintenance of native-like protein dynamics may not be required when engineering functional proteins.

  7. Trans-species Engineering of Glycosylated Therapeutic Proteins

    DEFF Research Database (Denmark)

    Yang, Zhang

    understood. Currently, mammalian cells are required for human O-glycosylation. Increasing efforts have been devoted to engineering non-mammalian cells for production of recombinant proteins with “human-like” glycosylation. Substantial success has been achieved with designed N-glycosylation in both lower...... eukaryotes and even prokaryotes. Insect and yeast cells produce O-glycosylation incompatible with use in humans, however recently the yeast Pichia was engineered to perform the first step of human-like O-glycosylation. This review provides an overview of past and current engineering efforts of N...

  8. An Engineered Split Intein for Photoactivated Protein Trans-Splicing.

    Directory of Open Access Journals (Sweden)

    Stanley Wong

    Full Text Available Protein splicing is mediated by inteins that auto-catalytically join two separated protein fragments with a peptide bond. Here we engineered a genetically encoded synthetic photoactivatable intein (named LOVInC, by using the light-sensitive LOV2 domain from Avena sativa as a switch to modulate the splicing activity of the split DnaE intein from Nostoc punctiforme. Periodic blue light illumination of LOVInC induced protein splicing activity in mammalian cells. To demonstrate the broad applicability of LOVInC, synthetic protein systems were engineered for the light-induced reassembly of several target proteins such as fluorescent protein markers, a dominant positive mutant of RhoA, caspase-7, and the genetically encoded Ca2+ indicator GCaMP2. Spatial precision of LOVInC was demonstrated by targeting activity to specific mammalian cells. Thus, LOVInC can serve as a general platform for engineering light-based control for modulating the activity of many different proteins.

  9. Engineering protein folding and translocation improves heterologous protein secretion in Saccharomyces cerevisiae.

    Science.gov (United States)

    Tang, Hongting; Bao, Xiaoming; Shen, Yu; Song, Meihui; Wang, Shenghuan; Wang, Chengqiang; Hou, Jin

    2015-09-01

    Saccharomyces cerevisiae is widely used as a producer of heterologous proteins of medical and industrial interest. Numerous efforts have been made to overcome bottlenecks in protein expression and secretion. However, the effect of engineering protein translocation to heterologous protein secretion has not been studied extensively in S. cerevisiae. In this work, we confirmed that heterologous protein expression in S. cerevisiae induced the unfolded protein response (UPR). To enhance protein folding capacity, the endoplasmic reticulum (ER) chaperone protein BiP and the disulfide isomerase Pdi1p were each over-expressed, and the secretion of three heterologous proteins, β-glucosidase, endoglucanase, and α-amylase, was improved. The impact of engineering key translocation components was also studied. The over-expression of co-translational translocation components Srp14p and Srp54p enhanced the secretion of three heterologous proteins (β-glucosidase, endoglucanase, and α-amylase), but over-expressing the cytosolic chaperone Ssa1p (involved in post-translational translocation) only enhanced the secretion of β-glucosidase. By engineering both co-translational translocation and protein folding, we obtained strains with β-glucosidase, endoglucanase, and α-amylase activities increased by 72%, 60%, and 103% compared to the controls. Our results show that protein translocation may be a limiting factor for heterologous protein production. © 2015 Wiley Periodicals, Inc.

  10. Engineering Lipid Bilayer Membranes for Protein Studies

    Science.gov (United States)

    Khan, Muhammad Shuja; Dosoky, Noura Sayed; Williams, John Dalton

    2013-01-01

    Lipid membranes regulate the flow of nutrients and communication signaling between cells and protect the sub-cellular structures. Recent attempts to fabricate artificial systems using nanostructures that mimic the physiological properties of natural lipid bilayer membranes (LBM) fused with transmembrane proteins have helped demonstrate the importance of temperature, pH, ionic strength, adsorption behavior, conformational reorientation and surface density in cellular membranes which all affect the incorporation of proteins on solid surfaces. Much of this work is performed on artificial templates made of polymer sponges or porous materials based on alumina, mica, and porous silicon (PSi) surfaces. For example, porous silicon materials have high biocompatibility, biodegradability, and photoluminescence, which allow them to be used both as a support structure for lipid bilayers or a template to measure the electrochemical functionality of living cells grown over the surface as in vivo. The variety of these media, coupled with the complex physiological conditions present in living systems, warrant a summary and prospectus detailing which artificial systems provide the most promise for different biological conditions. This study summarizes the use of electrochemical impedance spectroscopy (EIS) data on artificial biological membranes that are closely matched with previously published biological systems using both black lipid membrane and patch clamp techniques. PMID:24185908

  11. Protein-inorganic nanohybrids: A potential symbiosis in tissue engineering.

    Science.gov (United States)

    Freag, May S; Elzoghby, Ahmed O

    2017-10-27

    recently, a great interest has been paid to the development of hybrid protein-inorganic nanoparticles (NPs) for tissue engineering applications to combine the merits of both inorganic and protein nanocarriers. this short review primarily discusses the most important advances in the application of the hybrids of proteins (gelatin, zein, silk fibroin,….etc) with inorganic NPs (calcium phosphate NPs, cadmium QDs, carbon nanotubes,…etc) in tissue engineering. various strategies that have been utilized for the preparation of protein-functionalized inorganic NPs are discussed. Nanocomposite films, electrospun nanofibrous scaffolds, nanostructured colloidal composite gels and nanocomposite lyophilized sponges are among the most common platforms of protein-inorganic nanohybrid formulations used in regenerative medicine. protein-inorganic nanohybrids could serve as promising platforms for different biomedical applications including bone and cartilage tissue regeneration, imaging of engineered tissues, development of antithrombogenic implant biomaterials and anti-bacterial wound dressing as well. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  12. Introduction to current and future protein therapeutics: a protein engineering perspective.

    Science.gov (United States)

    Carter, Paul J

    2011-05-15

    Protein therapeutics and its enabling sister discipline, protein engineering, have emerged since the early 1980s. The first protein therapeutics were recombinant versions of natural proteins. Proteins purposefully modified to increase their clinical potential soon followed with enhancements derived from protein or glycoengineering, Fc fusion or conjugation to polyethylene glycol. Antibody-based drugs subsequently arose as the largest and fastest growing class of protein therapeutics. The rationale for developing better protein therapeutics with enhanced efficacy, greater safety, reduced immunogenicity or improved delivery comes from the convergence of clinical, scientific, technological and commercial drivers that have identified unmet needs and provided strategies to address them. Future protein drugs seem likely to be more extensively engineered to improve their performance, e.g., antibodies and Fc fusion proteins with enhanced effector functions or extended half-life. Two old concepts for improving antibodies, namely antibody-drug conjugates and bispecific antibodies, have advanced to the cusp of clinical success. As for newer protein therapeutic platform technologies, several engineered protein scaffolds are in early clinical development and offer differences and some potential advantages over antibodies. Copyright © 2011 Elsevier Inc. All rights reserved.

  13. The development and application of engineered proteins for bioremediation

    Energy Technology Data Exchange (ETDEWEB)

    Trewhella, J. [ed.

    1995-09-26

    Clean up of the toxic legacy of the Cold War is projected to be the most expensive domestic project the nation has yet undertaken. Remediation of the Department of Energy and Department of Defense toxic waste sites alone are projected to cost {approximately}$1 trillion over a 20-30 year period. New, cost effective technologies are needed to attack this enormous problem. Los Alamos has put together a cross-divisional team of scientist to develop science based bioremediation technology to work toward this goal. In the team we have expertise in: (1) molecular, ecosystem and transport modeling; (2) genetic and protein engineering; (3) microbiology and microbial ecology; (4) structural biology; and (5) bioinorganic chemistry. This document summarizes talks at a workshop of different aspects of bioremediation technology including the following: Introducing novel function into a Heme enzyme: engineering by excavation; cytochrome P-450: ideal systems for bioremediation?; selection and development of bacterial strains for in situ remediation of cholorinated solvents; genetic analysis and preparation of toluene ortho-monooxygenase for field application in remediation of trichloroethylene; microbial ecology and diversity important to bioremediation; engineering haloalkane dehalogenase for bioremediation; enzymes for oxidative biodegradation; indigenous bacteria as hosts for engineered proteins; performance of indigenous bacterial, hosting engineered proteins in microbial communities.

  14. Protein-hydrogel interactions in tissue engineering: mechanisms and applications.

    Science.gov (United States)

    Zustiak, Silviya P; Wei, Yunqian; Leach, Jennie B

    2013-04-01

    Recent advances in our understanding of the sophistication of the cellular microenvironment and the dynamics of tissue remodeling during development, disease, and regeneration have increased our appreciation of the current challenges facing tissue engineering. As this appreciation advances, we are better equipped to approach problems in the biology and therapeutics of even more complex fields, such as stem cells and cancer. To aid in these studies, as well as the established areas of tissue engineering, including cardiovascular, musculoskeletal, and neural applications, biomaterials scientists have developed an extensive array of materials with specifically designed chemical, mechanical, and biological properties. Herein, we highlight an important topic within this area of biomaterials research, protein-hydrogel interactions. Due to inherent advantages of hydrated scaffolds for soft tissue engineering as well as specialized bioactivity of proteins and peptides, this field is well-posed to tackle major needs within emerging areas of tissue engineering. We provide an overview of the major modes of interactions between hydrogels and proteins (e.g., weak forces, covalent binding, affinity binding), examples of applications within growth factor delivery and three-dimensional scaffolds, and finally future directions within the area of hydrogel-protein interactions that will advance our ability to control the cell-biomaterial interface.

  15. Trans-species Engineering of Glycosylated Therapeutic Proteins

    DEFF Research Database (Denmark)

    Yang, Zhang

    eukaryotes and even prokaryotes. Insect and yeast cells produce O-glycosylation incompatible with use in humans, however recently the yeast Pichia was engineered to perform the first step of human-like O-glycosylation. This review provides an overview of past and current engineering efforts of N...... important to address. Whenever glycosylation has been found to be an important PTM for function or bioactivity, human therapeutics have generally been produced in mammalian Chinese hamster ovary (CHO) cell line. Oglycosylation is one of the most complex regulated PTMs of proteins but also one of the least...... understood. Currently, mammalian cells are required for human O-glycosylation. Increasing efforts have been devoted to engineering non-mammalian cells for production of recombinant proteins with “human-like” glycosylation. Substantial success has been achieved with designed N-glycosylation in both lower...

  16. Rewiring cell signalling through chimaeric regulatory protein engineering.

    Science.gov (United States)

    Wang, Baojun; Barahona, Mauricio; Buck, Martin; Schumacher, Jörg

    2013-10-01

    Bacterial cells continuously sense and respond to their environment using their inherent signalling and gene regulatory networks. Cells are equipped with parallel signalling pathways, which can specifically cope with individual input signals, while interconnectivities between pathways lead to an enhanced complexity of regulatory responses that enable sophisticated adaptation. In principle, any cell signalling pathway may be rewired to respond to non-cognate signals by exchanging and recombining their underlying cognate signalling components. In the present article, we review the engineering strategies and use of chimaeric regulatory proteins in cell signalling pathways, especially the TCS (two-component signalling) system in bacteria, to achieve novel customized signalling or regulatory functions. We envisage that engineered chimaeric regulatory proteins can play an important role to aid both forward and reverse engineering of biological systems for many desired applications.

  17. Protein engineering strategies with potential applications for altering clinically relevant cellular pathways at the protein level.

    Science.gov (United States)

    Regan, Lynne; Hinrichsen, Michael R; Oi, Curran

    2016-05-01

    All diseases can be fundamentally viewed as the result of malfunctioning cellular pathways. Protein engineering offers the potential to develop new tools that will allow these dysfunctional pathways to be better understood, in addition to potentially providing new routes to restore proper function. Here we discuss different approaches that can be used to change the intracellular activity of a protein by intervening at the protein level: targeted protein sequestration, protein recruitment, protein degradation, and selective inhibition of binding interfaces. The potential of each of these tools to be developed into effective therapeutic treatments will also be discussed, along with any major barriers that currently block their translation into the clinic.

  18. Engineering Globular Protein Vesicles through Tunable Self-Assembly of Recombinant Fusion Proteins.

    Science.gov (United States)

    Jang, Yeongseon; Choi, Won Tae; Heller, William T; Ke, Zunlong; Wright, Elizabeth R; Champion, Julie A

    2017-09-01

    Vesicles assembled from folded, globular proteins have potential for functions different from traditional lipid or polymeric vesicles. However, they also present challenges in understanding the assembly process and controlling vesicle properties. From detailed investigation of the assembly behavior of recombinant fusion proteins, this work reports a simple strategy to engineer protein vesicles containing functional, globular domains. This is achieved through tunable self-assembly of recombinant globular fusion proteins containing leucine zippers and elastin-like polypeptides. The fusion proteins form complexes in solution via high affinity binding of the zippers, and transition through dynamic coacervates to stable hollow vesicles upon warming. The thermal driving force, which can be tuned by protein concentration or temperature, controls both vesicle size and whether vesicles are single or bi-layered. These results provide critical information to engineer globular protein vesicles via self-assembly with desired size and membrane structure. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Biomineralization of Engineered Spider Silk Protein-Based Composite Materials for Bone Tissue Engineering

    Directory of Open Access Journals (Sweden)

    John G. Hardy

    2016-07-01

    Full Text Available Materials based on biodegradable polyesters, such as poly(butylene terephthalate (PBT or poly(butylene terephthalate-co-poly(alkylene glycol terephthalate (PBTAT, have potential application as pro-regenerative scaffolds for bone tissue engineering. Herein, the preparation of films composed of PBT or PBTAT and an engineered spider silk protein, (eADF4(C16, that displays multiple carboxylic acid moieties capable of binding calcium ions and facilitating their biomineralization with calcium carbonate or calcium phosphate is reported. Human mesenchymal stem cells cultured on films mineralized with calcium phosphate show enhanced levels of alkaline phosphatase activity suggesting that such composites have potential use for bone tissue engineering.

  20. Rational Protein Engineering Guided by Deep Mutational Scanning.

    Science.gov (United States)

    Shin, HyeonSeok; Cho, Byung-Kwan

    2015-09-23

    Sequence-function relationship in a protein is commonly determined by the three-dimensional protein structure followed by various biochemical experiments. However, with the explosive increase in the number of genome sequences, facilitated by recent advances in sequencing technology, the gap between protein sequences available and three-dimensional structures is rapidly widening. A recently developed method termed deep mutational scanning explores the functional phenotype of thousands of mutants via massive sequencing. Coupled with a highly efficient screening system, this approach assesses the phenotypic changes made by the substitution of each amino acid sequence that constitutes a protein. Such an informational resource provides the functional role of each amino acid sequence, thereby providing sufficient rationale for selecting target residues for protein engineering. Here, we discuss the current applications of deep mutational scanning and consider experimental design.

  1. Trans-species Engineering of Glycosylated Therapeutic Proteins

    DEFF Research Database (Denmark)

    Yang, Zhang

    Recombinant expression of therapeutic proteins is one of the major tasks in modern biomedicine. One of the most important factors with respect to therapeutic use in human is posttranslational modifications (PTMs) of the recombinant proteins, of which protein glycosylation is by far the most...... important to address. Whenever glycosylation has been found to be an important PTM for function or bioactivity, human therapeutics have generally been produced in mammalian Chinese hamster ovary (CHO) cell line. Oglycosylation is one of the most complex regulated PTMs of proteins but also one of the least...... understood. Currently, mammalian cells are required for human O-glycosylation. Increasing efforts have been devoted to engineering non-mammalian cells for production of recombinant proteins with “human-like” glycosylation. Substantial success has been achieved with designed N-glycosylation in both lower...

  2. Trans-species Engineering of Glycosylated Therapeutic Proteins

    DEFF Research Database (Denmark)

    Yang, Zhang

    understood. Currently, mammalian cells are required for human O-glycosylation. Increasing efforts have been devoted to engineering non-mammalian cells for production of recombinant proteins with “human-like” glycosylation. Substantial success has been achieved with designed N-glycosylation in both lower......Recombinant expression of therapeutic proteins is one of the major tasks in modern biomedicine. One of the most important factors with respect to therapeutic use in human is posttranslational modifications (PTMs) of the recombinant proteins, of which protein glycosylation is by far the most...... important to address. Whenever glycosylation has been found to be an important PTM for function or bioactivity, human therapeutics have generally been produced in mammalian Chinese hamster ovary (CHO) cell line. Oglycosylation is one of the most complex regulated PTMs of proteins but also one of the least...

  3. Chaperonin of Group I: Oligomeric Spectrum and Biochemical and Biological Implications

    Directory of Open Access Journals (Sweden)

    Silvia Vilasi

    2018-01-01

    Full Text Available Chaperonins play various physiological roles and can also be pathogenic. Elucidation of their structure, e.g., oligomeric status and post-translational modifications (PTM, is necessary to understand their functions and mechanisms of action in health and disease. Group I chaperonins form tetradecamers with two stacked heptameric rings. The tetradecamer is considered the typical functional complex for folding of client polypeptides. However, other forms such as the monomer and oligomers with smaller number of subunits than the classical tetradecamer, also occur in cells. The properties and functions of the monomer and oligomers, and their roles in chaperonin-associated diseases are still incompletely understood. Chaperonin I in eukaryotes occurs in various locations, not just the mitochondrion, which is its canonical place of residence and function. Eukaryotic Chaperonin I, namely Hsp60 (designated HSP60 or HSPD1 in humans has, indeed, been found in the cytosol; the plasma-cell membrane; on the outer surface of cells; in the intercellular space; in biological liquids such as lymph, blood, and cerebrospinal fluid; and in secretions, for instance saliva and urine. Hsp60 has also been found in cell-derived vesicles such as exosomes. The functions of Hsp60 in all these non-canonical locales are still poorly characterized and one of the questions not yet answered is in what form, i.e., monomer or oligomer, is the chaperonin present in these non-canonical locations. In view of the steady increase in interest on chaperonopathies over the last several years, we have studied human HSP60 to determine its role in various diseases, its locations in cells and tissues and migrations in the body, and its post-translational modifications that might have an impact on its location and function. We also carried out experiments to characterize the oligomeric status of extramitochondrial of HSP60 in solution. Here, we provide an overview of our results, focusing on

  4. Integrating protein engineering with process design for biocatalysis.

    Science.gov (United States)

    Woodley, John M

    2018-01-13

    Biocatalysis uses enzymes for chemical synthesis and production, offering selective, safe and sustainable catalysis. While today the majority of applications are in the pharmaceutical sector, new opportunities are arising every day in other industry sectors, where production costs become a more important driver. In the early applications of the technology, it was necessary to design processes to match the properties of the biocatalyst. With the advent of protein engineering, organic chemists started to develop and improve enzymes to suit their needs. Likewise in industry, although not widespread, a new paradigm was already implemented several years ago to engineer enzymes to suit process needs. Today, a new era is entered, where the effectiveness with which such integrated protein and process engineering is achieved becomes critical to implementation. In this paper, the development of a tool to improve the effectiveness of this approach is discussed, namely the use of target-setting based on process requirements, to guide the necessary protein engineering.This article is part of a discussion meeting issue 'Providing sustainable catalytic solutions for a rapidly changing world'. © 2017 The Author(s).

  5. Mycobacterium tuberculosis Chaperonin 10 Is Secreted in the Macrophage Phagosome: Is Secretion Due to Dissociation and Adoption of a Partially Helical Structure at the Membrane?

    Science.gov (United States)

    Fossati, Gianluca; Izzo, Gaetano; Rizzi, Emanuele; Gancia, Emanuela; Modena, Daniela; Moras, Maria Luisa; Niccolai, Neri; Giannozzi, Elena; Spiga, Ottavia; Bono, Letizia; Marone, Piero; Leone, Eugenio; Mangili, Francesca; Harding, Stephen; Errington, Neil; Walters, Christopher; Henderson, Brian; Roberts, Michael M.; Coates, Anthony R. M.; Casetta, Bruno; Mascagni, Paolo

    2003-01-01

    To confirm that Mycobacterium tuberculosis chaperonin 10 (Cpn10) is secreted outside the live bacillus, infected macrophages were examined by electron microscopy. This revealed that the mycobacterial protein accumulates both in the wall of the bacterium and in the matrix of the phagosomes in which ingested mycobacteria survive within infected macrophages. To understand the structural implications underlying this secretion, a structural study of M. tuberculosis Cpn10 was performed under conditions that are generally believed to mimic the membrane environment. It was found that in buffer-organic solvent mixtures, the mycobacterial protein forms two main species, namely, a partially helical monomer that prevails in dilute solutions at room temperature and a dimer that folds into a β-sheet-dominated structure and prevails in either concentrated protein solutions at room temperature or in dilute solutions at low temperature. A partially helical monomer was also found and was completely associated with negatively charged detergents in a micelle-bound state. Remarkably, zwitterionic lipids had no effect on the protein structure. By using N- and C-truncated forms of the protein, the C- and N-terminal sequences were identified as possessing an amphiphilic helical character and as selectively associating with acidic detergent micelles. When the study was extended to other chaperonins, it was found that human Cpn10 is also monomeric and partially helical in dilute organic solvent-buffer mixtures. In contrast, Escherichia coli Cpn10 is mostly dimeric and predominately β-sheet in both dilute and concentrated solutions. Interestingly, human Cpn10 also crosses biological membranes, whereas the E. coli homologue is strictly cytosolic. These results suggest that dissociation to partially helical monomers and interaction with acidic lipids may be two important steps in the mechanism of secretion of M. tuberculosis Cpn10 to the external environment. PMID:12837802

  6. The chaperonin containing TCP1 complex (CCT/TRiC) is involved in mediating sperm-oocyte interaction.

    Science.gov (United States)

    Dun, Matthew D; Smith, Nathan D; Baker, Mark A; Lin, Minjie; Aitken, R John; Nixon, Brett

    2011-10-21

    Sperm-oocyte interactions are among the most remarkable processes in cell biology. These cellular recognition events are initiated by an exquisitely specific adhesion of free-swimming spermatozoa to the zona pellucida, an acellular matrix that surrounds the ovulated oocyte. Decades of research focusing on this interaction have led to the establishment of a widely held paradigm that the zona pellucida receptor is a single molecular entity that is constitutively expressed on the sperm cell surface. In contrast, we have employed the techniques of blue native-polyacrylamide gel electrophoresis, far Western blotting, and proximity ligation to secure the first direct evidence in support of a novel hypothesis that zona binding is mediated by multimeric sperm receptor complex(es). Furthermore, we show that one such multimeric association, comprising the chaperonin-containing TCP1 complex (CCT/TRiC) and a zona-binding protein, zona pellucida-binding protein 2, is present on the surface of capacitated spermatozoa and could account for the zona binding activity of these cells. Collectively, these data provide an important biochemical insight into the molecular basis of sperm-zona pellucida interaction and a plausible explanation for how spermatozoa gain their ability to fertilize.

  7. Linker engineering for fusion protein construction: Improvement and characterization of a GLP-1 fusion protein.

    Science.gov (United States)

    Kong, Yuelin; Tong, Yue; Gao, Mingming; Chen, Chen; Gao, Xiangdong; Yao, Wenbing

    2016-01-01

    Protein engineering has been successfully applied in protein drug discovery. Using this technology, we previously have constructed a fusion protein by linking the globular domain of adiponectin to the C-terminus of a glucagon-like peptide-1 (GLP-1) analog. Herein, to further improve its bioactivity, we reconstructed this fusion protein by introducing linker peptides of different length and flexibility. The reconstructed fusion proteins were overexpressed in Escherichia coli and purified using nickel affinity chromatography. Their agonist activity towards receptors of GLP-1 and adiponectin were assessed in vitro by using luciferase assay and AMP-activated protein kinase (AMPK) immunoblotting, respectively. The effects of the selected fusion protein on glucose and lipid metabolism were evaluated in mice. The fusion protein reconstructed using a linker peptide of AMGPSSGAPGGGGS showed high potency in activating GLP-1 receptor and triggering AMPK phosphorylation via activating the adiponectin receptor. Remarkably, the optimized fusion protein was highly effective in lowering blood glucose and lipids in mice. Collectively, these findings demonstrate that the bioactivity of this GLP-1 fusion protein can be significantly promoted by linker engineering, and indicate that the optimized GLP-1 fusion protein is a promising lead structure for anti-diabetic drug discovery. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Multifactorial level of extremostability of proteins: can they be exploited for protein engineering?

    Science.gov (United States)

    Chakravorty, Debamitra; Khan, Mohd Faheem; Patra, Sanjukta

    2017-05-01

    Research on extremostable proteins has seen immense growth in the past decade owing to their industrial importance. Basic research of attributes related to extreme-stability requires further exploration. Modern mechanistic approaches to engineer such proteins in vitro will have more impact in industrial biotechnology economy. Developing a priori knowledge about the mechanism behind extreme-stability will nurture better understanding of pathways leading to protein molecular evolution and folding. This review is a vivid compilation about all classes of extremostable proteins and the attributes that lead to myriad of adaptations divulged after an extensive study of 6495 articles belonging to extremostable proteins. Along with detailing on the rationale behind extreme-stability of proteins, emphasis has been put on modern approaches that have been utilized to render proteins extremostable by protein engineering. It was understood that each protein shows different approaches to extreme-stability governed by minute differences in their biophysical properties and the milieu in which they exist. Any general rule has not yet been drawn regarding adaptive mechanisms in extreme environments. This review was further instrumental to understand the drawback of the available 14 stabilizing mutation prediction algorithms. Thus, this review lays the foundation to further explore the biophysical pleiotropy of extreme-stable proteins to deduce a global prediction model for predicting the effect of mutations on protein stability.

  9. Protein turnover during in vitro tissue engineering.

    Science.gov (United States)

    Li, Qiyao; Chang, Zhen; Oliveira, Gisele; Xiong, Maiyer; Smith, Lloyd M; Frey, Brian L; Welham, Nathan V

    2016-03-01

    Repopulating acellular biological scaffolds with phenotypically appropriate cells is a promising approach for regenerating functional tissues and organs. Under this tissue engineering paradigm, reseeded cells are expected to remodel the scaffold by active protein synthesis and degradation; however, the rate and extent of this remodeling remain largely unknown. Here, we present a technique to measure dynamic proteome changes during in vitro remodeling of decellularized tissue by reseeded cells, using vocal fold mucosa as the model system. Decellularization and recellularization were optimized, and a stable isotope labeling strategy was developed to differentiate remnant proteins constituting the original scaffold from proteins newly synthesized by reseeded cells. Turnover of matrix and cellular proteins and the effects of cell-scaffold interaction were elucidated. This technique sheds new light on in vitro tissue remodeling and the process of tissue regeneration, and is readily applicable to other tissue and organ systems. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Protein Design for Nanostructural Engineering: Concluding Remarks and Future Directions.

    Science.gov (United States)

    Grove, Tijana Z; Cortajarena, Aitziber L

    2016-01-01

    This final chapter aims to summarize the main conclusions of the book and to point to possible directions for further research in the field of protein design for nanostructural engineering. Even though this research field is still at its infancy, multidisciplinary research efforts in the design of synthetic protein-based nanostructures and functional materials have resulted in significant progress. The chapters in this book cover several selected examples of the most recent advances concerning the use of proteins and peptides as building blocks for the fabrication of architectures and functional nanostructures, assemblies, and materials. Here, we provide a general overview of the strategies that can be employed to prepare functional protein-based nanostructures, and nanostructured materials and devices. Finally, we highlight some of the main aspects to be considered by the research community to set the path for the near future developments.

  11. Trans-species Engineering of Glycosylated Therapeutic Proteins

    DEFF Research Database (Denmark)

    Yang, Zhang

    understood. Currently, mammalian cells are required for human O-glycosylation. Increasing efforts have been devoted to engineering non-mammalian cells for production of recombinant proteins with “human-like” glycosylation. Substantial success has been achieved with designed N-glycosylation in both lower...... eukaryotes and even prokaryotes. Insect and yeast cells produce O-glycosylation incompatible with use in humans, however recently the yeast Pichia was engineered to perform the first step of human-like O-glycosylation. This review provides an overview of past and current engineering efforts of N......- and Oglycosylation and addresses emerging novel promising platforms such as plants. Plants are safe and low-cost production organisms; they do not perform mammalian-type glycosylation by itself and are easily up-scalable. These features should make it possible to custom design and build capacity for human...

  12. Using Resurrected Ancestral Proviral Proteins to Engineer Virus Resistance

    Directory of Open Access Journals (Sweden)

    Asunción Delgado

    2017-05-01

    Full Text Available Proviral factors are host proteins hijacked by viruses for processes essential for virus propagation such as cellular entry and replication. Pathogens and their hosts co-evolve. It follows that replacing a proviral factor with a functional ancestral form of the same protein could prevent viral propagation without fatally compromising organismal fitness. Here, we provide proof of concept of this notion. Thioredoxins serve as general oxidoreductases in all known cells. We report that several laboratory resurrections of Precambrian thioredoxins display substantial levels of functionality within Escherichia coli. Unlike E. coli thioredoxin, however, these ancestral thioredoxins are not efficiently recruited by the bacteriophage T7 for its replisome and therefore prevent phage propagation in E. coli. These results suggest an approach to the engineering of virus resistance. Diseases caused by viruses may have a devastating effect in agriculture. We discuss how the suggested approach could be applied to the engineering of plant virus resistance.

  13. Protein engineering for bioenergy and biomass-based chemicals.

    Science.gov (United States)

    Clarke, Neil D

    2010-08-01

    The conversion of biomass to fuels and chemicals faces substantial challenges if it is to be done on a large scale, in a sustainable manner, and at reasonable cost. Some of these challenges can be addressed through protein engineering technologies, for example by improving biomass-processing enzymes or by manipulating the activities and substrate specificity of enzymes in metabolic pathways. Recent highlights include structure-guided chimera design to improve the properties of cellulases, the engineering of synthetic scaffold proteins to enhance metabolic flux, and the broadening of substrate specificity to co-opt metabolic pathways to the production of long-chain branched alcohols. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  14. Novel blood protein based scaffolds for cardiovascular tissue engineering

    Directory of Open Access Journals (Sweden)

    Kuhn Antonia I.

    2016-09-01

    Full Text Available A major challenge in cardiovascular tissue engineering is the fabrication of scaffolds, which provide appropriate morphological and mechanical properties while avoiding undesirable immune reactions. In this study electrospinning was used to fabricate scaffolds out of blood proteins for cardiovascular tissue engineering. Lyophilised porcine plasma was dissolved in deionised water at a final concentration of 7.5% m/v and blended with 3.7% m/v PEO. Electrospinning resulted in homogeneous fibre morphologies with a mean fibre diameter of 151 nm, which could be adapted to create macroscopic shapes (mats, tubes. Cross-linking with glutaraldehyde vapour improved the long-term stability of protein based scaffolds in comparison to untreated scaffolds, resulting in a mass loss of 41% and 96% after 28 days of incubation in aqueous solution, respectively.

  15. Protein and metabolic engineering for the production of organic acids.

    Science.gov (United States)

    Liu, Jingjing; Li, Jianghua; Shin, Hyun-Dong; Liu, Long; Du, Guocheng; Chen, Jian

    2017-09-01

    Organic acids are natural metabolites of living organisms. They have been widely applied in the food, pharmaceutical, and bio-based materials industries. In recent years, biotechnological routes to organic acids production from renewable raw materials have been regarded as very promising approaches. In this review, we provide an overview of current developments in the production of organic acids using protein and metabolic engineering strategies. The organic acids include propionic acid, pyruvate, itaconic acid, succinic acid, fumaric acid, malic acid and citric acid. We also expect that rapid developments in the fields of systems biology and synthetic biology will accelerate protein and metabolic engineering for microbial organic acid production in the future. Copyright © 2017. Published by Elsevier Ltd.

  16. Metabolic engineering of Escherichia coli to improve recombinant protein production.

    Science.gov (United States)

    Liu, Min; Feng, Xinjun; Ding, Yamei; Zhao, Guang; Liu, Huizhou; Xian, Mo

    2015-12-01

    Escherichia coli is one of the most widely used strains for recombinant protein production. However, obstacles also exist in both academic researches and industrial applications, such as the metabolic burden, the carbon source waste, and the cells' physiological deterioration. This article reviews recent approaches for improving recombinant protein production in metabolic engineering, including workhorse selection, stress factor application, and carbon flux regulation. Selecting a suitable host is the first key point for recombinant protein production. In general, it all depends on characteristics of the strains and the target proteins. It will be triggered cells physiological deterioration when the medium is significantly different from the cell's natural environment. Coexpression of stress factors can help proteins to fold into their native conformation. Carbon flux regulation is a direct approach for redirecting more carbon flux toward the desirable pathways and products. However, some undesirable consequences are usually found in metabolic engineering, such as glucose transport inhibition, cell growth retardation, and useless metabolite accumulation. More efficient regulators and platform cell factories should be explored to meet a variety of production demands.

  17. Soy Protein Scaffold Biomaterials for Tissue Engineering and Regenerative Medicine

    Science.gov (United States)

    Chien, Karen B.

    Developing functional biomaterials using highly processable materials with tailorable physical and bioactive properties is an ongoing challenge in tissue engineering. Soy protein is an abundant, natural resource with potential use for regenerative medicine applications. Preliminary studies show that soy protein can be physically modified and fabricated into various biocompatible constructs. However, optimized soy protein structures for tissue regeneration (i.e. 3D porous scaffolds) have not yet been designed. Furthermore, little work has established the in vivo biocompatibility of implanted soy protein and the benefit of using soy over other proteins including FDA-approved bovine collagen. In this work, freeze-drying and 3D printing fabrication processes were developed using commercially available soy protein to create porous scaffolds that improve cell growth and infiltration compared to other soy biomaterials previously reported. Characterization of scaffold structure, porosity, and mechanical/degradation properties was performed. In addition, the behavior of human mesenchymal stem cells seeded on various designed soy scaffolds was analyzed. Biological characterization of the cell-seeded scaffolds was performed to assess feasibility for use in liver tissue regeneration. The acute and humoral response of soy scaffolds implanted in an in vivo mouse subcutaneous model was also investigated. All fabricated soy scaffolds were modified using thermal, chemical, and enzymatic crosslinking to change properties and cell growth behavior. 3D printing allowed for control of scaffold pore size and geometry. Scaffold structure, porosity, and degradation rate significantly altered the in vivo response. Freeze-dried soy scaffolds had similar biocompatibility as freeze-dried collagen scaffolds of the same protein content. However, the soy scaffolds degraded at a much faster rate, minimizing immunogenicity. Interestingly, subcutaneously implanted soy scaffolds affected blood

  18. Vibrational entropy differences between mesophile and thermophile proteins and their use in protein engineering.

    Science.gov (United States)

    Frappier, Vincent; Najmanovich, Rafael

    2015-04-01

    We recently introduced ENCoM, an elastic network atomic contact model, as the first coarse-grained normal mode analysis method that accounts for the nature of amino acids and can predict the effect of mutations on thermostability based on changes vibrational entropy. In this proof-of-concept article, we use pairs of mesophile and thermophile homolog proteins with identical structures to determine if a measure of vibrational entropy based on normal mode analysis can discriminate thermophile from mesophile proteins. We observe that in around 60% of cases, thermophile proteins are more rigid at equivalent temperatures than their mesophile counterpart and this difference can guide the design of proteins to increase their thermostability through series of mutations. We observe that mutations separating thermophile proteins from their mesophile orthologs contribute independently to a decrease in vibrational entropy and discuss the application and implications of this methodology to protein engineering. © 2014 The Protein Society.

  19. Oxidatively Responsive Chain Extension to Topologically Entangle Engineered Protein Hydrogels

    Science.gov (United States)

    Olsen, Bradley; Tang, Shengchang; Glassman, Matthew; Li, Shuaili; Socrate, Simona

    2014-03-01

    Hydrogels with increased toughness and extensibility have attracted a great deal of interest as mimics for natural tissues in biomedical applications. Artificial protein polymers provide particularly attractive systems for these applications due to their similarity to the chemistry of the natural extracellular matrix. Here, we show that entanglements can be incorporated into physically associating protein gels using simple oxidative chain extension chemistries, producing hydrogels with multiple time and length scales of relaxation. These oxidative chemistries follow the Jacobson-Stockmayer theory and are fully reversible, enabling responsive formation of entanglements within a material. The entangled protein gels demonstrate extensibility up to engineering strains of greater than 3,000%, a toughness of 65,000 J/m⌃3, and significant reductions in creep compliance and increases in elastic recovery. The rheology of the materials is compared to sticky reptation theory as a function of gel concentration, providing insights into the effect of network structure on different modes of molecular relaxation.

  20. Genetically engineered protein in hydrogels tailors stimuli-responsive characteristics

    Science.gov (United States)

    Ehrick, Jason D.; Deo, Sapna K.; Browning, Tyler W.; Bachas, Leonidas G.; Madou, Marc J.; Daunert, Sylvia

    2005-04-01

    Certain proteins undergo a substantial conformational change in response to a given stimulus. This conformational change can manifest in different manners and result in an actuation, that is, catalytic or signalling event, movement, interaction with other proteins, and so on. In all cases, the sensing-actuation process of proteins is initiated by a recognition event that translates into a mechanical action. Thus, proteins are ideal components for designing new nanomaterials that are intelligent and can perform desired mechanical actions in response to target stimuli. A number of approaches have been undertaken to mimic nature's sensing-actuating process. We now report a new hybrid material that integrates genetically engineered proteins within hydrogels capable of producing a stimulus-responsive action mechanism. The mechanical effect is a result of an induced conformational change and binding affinities of the protein in response to a stimulus. The stimuli-responsive hydrogel exhibits three specific swelling stages in response to various ligands offering additional fine-tuned control over a conventional two-stage swelling hydrogel. The newly prepared material was used in the sensing, and subsequent gating and transport of biomolecules across a polymer network, demonstrating its potential application in microfluidics and miniaturized drug-delivery systems.

  1. Glyco-engineering of biotherapeutic proteins in plants.

    Science.gov (United States)

    Ko, Kisung; Ahn, Mi-Hyun; Song, Mira; Choo, Young-Kug; Kim, Hyun Soon; Ko, Kinarm; Joung, Hyouk

    2008-06-30

    Many therapeutic glycoproteins have been successfully generated in plants. Plants have advantages regarding practical and economic concerns, and safety of protein production over other existing systems. However, plants are not ideal expression systems for the production of biopharmaceutical proteins, due to the fact that they are incapable of the authentic human N-glycosylation process. The majority of therapeutic proteins are glycoproteins which harbor N-glycans, which are often essential for their stability, folding, and biological activity. Thus, several glyco-engineering strategies have emerged for the tailor-making of N-glycosylation in plants, including glycoprotein subcellular targeting, the inhibition of plant specific glycosyltranferases, or the addition of human specific glycosyltransferases. This article focuses on plant N-glycosylation structure, glycosylation variation in plant cell, plant expression system of glycoproteins, and impact of glycosylation on immunological function. Furthermore, plant glyco-engineering techniques currently being developed to overcome the limitations of plant expression systems in the production of therapeutic glycoproteins will be discussed in this review.

  2. Engineering self-assembled bioreactors from protein microcompartments

    Energy Technology Data Exchange (ETDEWEB)

    Savage, David [Univ. of California, Berkeley, CA (United States)

    2016-10-12

    The goals of this research are to understand how organisms such as bacteria segregate certain metabolic processes inside of specific structures, or “microcompartments,” in the cell and apply this knowledge to develop novel engineered microcompartments for use in nanotechnology and metabolic engineering. For example, in some bacteria, self-assembling protein microcompartments called carboxysomes encapsulate the enzymes involved in carbon fixation, enabling the cell to utilize carbon dioxide more effectively than if the enzymes were free in the cell. The proposed research will determine how structures such as carboxysomes assemble and function in bacteria and develop a means for creating novel, synthetic microcompartments for optimizing production of specific energy-rich compounds.

  3. Production of biopharmaceutical proteins by yeast: Advances through metabolic engineering

    DEFF Research Database (Denmark)

    Nielsen, Jens

    2013-01-01

    by yeast are human serum albumin, hepatitis vaccines and virus like particles used for vaccination against human papillomavirus. Here is given a brief overview of biopharmaceutical production by yeast and it is discussed how the secretory pathway can be engineered to ensure more efficient protein...... for production of several large volume products. Insulin and insulin analogs are by far the dominating biopharmaceuticals produced by yeast, and this will increase as the global insulin market is expected to grow from USD12B in 2011 to more than USD32B by 2018. Other important biopharmaceuticals produced...

  4. High-Throughput Screening in Protein Engineering : Recent Advances and Future Perspectives

    NARCIS (Netherlands)

    Wójcik, Magdalena; Telzerow, Aline; Quax, Wim J; Boersma, Ykelien L

    2015-01-01

    Over the last three decades, protein engineering has established itself as an important tool for the development of enzymes and (therapeutic) proteins with improved characteristics. New mutagenesis techniques and computational design tools have greatly aided in the advancement of protein

  5. Using Resurrected Ancestral Proviral Proteins to Engineer Virus Resistance.

    Science.gov (United States)

    Delgado, Asunción; Arco, Rocio; Ibarra-Molero, Beatriz; Sanchez-Ruiz, Jose M

    2017-05-09

    Proviral factors are host proteins hijacked by viruses for processes essential for virus propagation such as cellular entry and replication. Pathogens and their hosts co-evolve. It follows that replacing a proviral factor with a functional ancestral form of the same protein could prevent viral propagation without fatally compromising organismal fitness. Here, we provide proof of concept of this notion. Thioredoxins serve as general oxidoreductases in all known cells. We report that several laboratory resurrections of Precambrian thioredoxins display substantial levels of functionality within Escherichia coli. Unlike E. coli thioredoxin, however, these ancestral thioredoxins are not efficiently recruited by the bacteriophage T7 for its replisome and therefore prevent phage propagation in E. coli. These results suggest an approach to the engineering of virus resistance. Diseases caused by viruses may have a devastating effect in agriculture. We discuss how the suggested approach could be applied to the engineering of plant virus resistance. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  6. Protein engineering of CYP105s for their industrial uses.

    Science.gov (United States)

    Yasuda, Kaori; Sugimoto, Hiroshi; Hayashi, Keiko; Takita, Teisuke; Yasukawa, Kiyoshi; Ohta, Miho; Kamakura, Masaki; Ikushiro, Shinichi; Shiro, Yoshitsugu; Sakaki, Toshiyuki

    2018-01-01

    Cytochrome P450 enzymes belonging to the CYP105 family are predominantly found in bacteria belonging to the phylum Actinobacteria and the order Actinomycetales. In this review, we focused on the protein engineering of P450s belonging to the CYP105 family for industrial use. Two Arg substitutions to Ala of CYP105A1 enhanced its vitamin D 3 25- and 1α-hydroxylation activities by 400 and 100-fold, respectively. The coupling efficiency between product formation and NADPH oxidation was largely improved by the R84A mutation. The quintuple mutant Q87W/T115A/H132L/R194W/G294D of CYP105AB3 showed a 20-fold higher activity than the wild-type enzyme. Amino acids at positions 87 and 191 were located at the substrate entrance channel, and that at position 294 was located close to the heme group. Semi-rational engineering of CYP105A3 selected the best performing mutant, T85F/T119S/V194N/N363Y, for producing pravastatin. The T119S and N363Y mutations synergistically had remarkable effects on the interaction between CYP105A3 and putidaredoxin. Although wild-type CYP105AS1 hydroxylated compactin to 6-epi-pravastatin, the quintuple mutant I95T/Q127R/A180V/L236I/A265N converted almost all compactin to pravastatin. Five amino acid substitutions by two rounds of mutagenesis almost completely changed the stereo-selectivity of CYP105AS1. These results strongly suggest that the protein engineering of CYP105 enzymes greatly increase their industrial utility. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Using an Engineered Protein Model to Constrain Protein-Mineral Interactions

    Science.gov (United States)

    Chacon, S. S.; Reardon, P. N.; Washton, N.; Kleber, M.

    2015-12-01

    Exoenzymes are proteins that can catalyze the depolymerization of soil organic matter (SOM). Proteins can also be an important source of organic N for microorganisms, but must be fragmented into small peptides in order to be transported through their membranes. An exoenzyme's affinity to mineral surfaces found in soil affects their capacity to degrade SOM or other proteins. Our goal was to determine the range of modifications on proteins when they interact with a mineral surface. We hypothesized that pedogenic oxides would fragment or promote greater chemical modifications to a protein than phyllosilicates. A well-characterized protein proxy (Gb1, IEP 4.0, 6.2 kDA) was adsorbed onto functionally different mineral surfaces (goethite, montmorillonite, kaolinite and birnesite) at pH 5 and pH 7. We then generated three engineered proxies of Gb1 by inserting either negatively charged, positively charged or aromatic amino acids into the second loop. We used liquid chromatography coupled with a mass spectrometer (LC-MS/MS) and solution-state Heteronuclear Single Quantum Coherence Spectroscopy Nuclear Magnetic Resonance (HSQC NMR) to observe modifications to Gb1 that was allowed to equilibrate during the adsorption process for kaolinite, goethite, birnessite, and montmorillonite. We also used Helium Ion Microscopy (HIM) to determine which surface archetypes Gb1 preferentially adsorbed to as a function of the mineral type. The three engineered proxies were used to determine how variation of the amino acid sequence affects a protein interaction with a mineral surface. Preliminary results in the LC-MS/MS indicate that birnessite hydrolytically fragments Gb1 into polypeptides. Our results suggest that not all mineral surfaces in soil may act as sorbents for EEs and that chemical modification of their structure should also be considered as an explanation for decrease in EE activity. Our results also indicate an abiotic pathway for the turnover of proteins, although its relative

  8. Specific inhibition of GPCR-independent G protein signaling by a rationally engineered protein.

    Science.gov (United States)

    Leyme, Anthony; Marivin, Arthur; Maziarz, Marcin; DiGiacomo, Vincent; Papakonstantinou, Maria P; Patel, Prachi P; Blanco-Canosa, Juan B; Walawalkar, Isha A; Rodriguez-Davila, Gonzalo; Dominguez, Isabel; Garcia-Marcos, Mikel

    2017-11-28

    Activation of heterotrimeric G proteins by cytoplasmic nonreceptor proteins is an alternative to the classical mechanism via G protein-coupled receptors (GPCRs). A subset of nonreceptor G protein activators is characterized by a conserved sequence named the Gα-binding and activating (GBA) motif, which confers guanine nucleotide exchange factor (GEF) activity in vitro and promotes G protein-dependent signaling in cells. GBA proteins have important roles in physiology and disease but remain greatly understudied. This is due, in part, to the lack of efficient tools that specifically disrupt GBA motif function in the context of the large multifunctional proteins in which they are embedded. This hindrance to the study of alternative mechanisms of G protein activation contrasts with the wealth of convenient chemical and genetic tools to manipulate GPCR-dependent activation. Here, we describe the rational design and implementation of a genetically encoded protein that specifically inhibits GBA motifs: GBA inhibitor (GBAi). GBAi was engineered by introducing modifications in Gαi that preclude coupling to every known major binding partner [GPCRs, Gβγ, effectors, guanine nucleotide dissociation inhibitors (GDIs), GTPase-activating proteins (GAPs), or the chaperone/GEF Ric-8A], while favoring high-affinity binding to all known GBA motifs. We demonstrate that GBAi does not interfere with canonical GPCR-G protein signaling but blocks GBA-dependent signaling in cancer cells. Furthermore, by implementing GBAi in vivo, we show that GBA-dependent signaling modulates phenotypes during Xenopus laevis embryonic development. In summary, GBAi is a selective, efficient, and convenient tool to dissect the biological processes controlled by a GPCR-independent mechanism of G protein activation mediated by cytoplasmic factors.

  9. Thermal green protein, an extremely stable, nonaggregating fluorescent protein created by structure-guided surface engineering.

    Science.gov (United States)

    Close, Devin W; Paul, Craig Don; Langan, Patricia S; Wilce, Matthew C J; Traore, Daouda A K; Halfmann, Randal; Rocha, Reginaldo C; Waldo, Geoffery S; Payne, Riley J; Rucker, Joseph B; Prescott, Mark; Bradbury, Andrew R M

    2015-07-01

    In this article, we describe the engineering and X-ray crystal structure of Thermal Green Protein (TGP), an extremely stable, highly soluble, non-aggregating green fluorescent protein. TGP is a soluble variant of the fluorescent protein eCGP123, which despite being highly stable, has proven to be aggregation-prone. The X-ray crystal structure of eCGP123, also determined within the context of this paper, was used to carry out rational surface engineering to improve its solubility, leading to TGP. The approach involved simultaneously eliminating crystal lattice contacts while increasing the overall negative charge of the protein. Despite intentional disruption of lattice contacts and introduction of high entropy glutamate side chains, TGP crystallized readily in a number of different conditions and the X-ray crystal structure of TGP was determined to 1.9 Å resolution. The structural reasons for the enhanced stability of TGP and eCGP123 are discussed. We demonstrate the utility of using TGP as a fusion partner in various assays and significantly, in amyloid assays in which the standard fluorescent protein, EGFP, is undesirable because of aberrant oligomerization. © 2014 Wiley Periodicals, Inc.

  10. Engineering a novel multifunctional green fluorescent protein tag for a wide variety of protein research.

    Directory of Open Access Journals (Sweden)

    Takuya Kobayashi

    Full Text Available BACKGROUND: Genetically encoded tag is a powerful tool for protein research. Various kinds of tags have been developed: fluorescent proteins for live-cell imaging, affinity tags for protein isolation, and epitope tags for immunological detections. One of the major problems concerning the protein tagging is that many constructs with different tags have to be made for different applications, which is time- and resource-consuming. METHODOLOGY/PRINCIPAL FINDINGS: Here we report a novel multifunctional green fluorescent protein (mfGFP tag which was engineered by inserting multiple peptide tags, i.e., octa-histidine (8xHis, streptavidin-binding peptide (SBP, and c-Myc tag, in tandem into a loop of GFP. When fused to various proteins, mfGFP monitored their localization in living cells. Streptavidin agarose column chromatography with the SBP tag successfully isolated the protein complexes in a native form with a high purity. Tandem affinity purification (TAP with 8xHis and SBP tags in mfGFP further purified the protein complexes. mfGFP was clearly detected by c-Myc-specific antibody both in immunofluorescence and immuno-electron microscopy (EM. These findings indicate that mfGFP works well as a multifunctional tag in mammalian cells. The tag insertion was also successful in other fluorescent protein, mCherry. CONCLUSIONS AND SIGNIFICANCE: The multifunctional fluorescent protein tag is a useful tool for a wide variety of protein research, and may have the advantage over other multiple tag systems in its higher expandability and compatibility with existing and future tag technologies.

  11. Engineering

    National Research Council Canada - National Science Library

    Includes papers in the following fields: Aerospace Engineering, Agricultural Engineering, Chemical Engineering, Civil Engineering, Electrical Engineering, Environmental Engineering, Industrial Engineering, Materials Engineering, Mechanical...

  12. Molecular basis of glyphosate resistance: Different approaches through protein engineering

    Science.gov (United States)

    Pollegioni, Loredano; Schonbrunn, Ernst; Siehl, Daniel

    2011-01-01

    Glyphosate (N-phosphonomethyl-glycine) is the most-used herbicide in the world: glyphosate-based formulations exhibit broad-spectrum herbicidal activity with minimal human and environmental toxicity. The extraordinary success of this simple small molecule is mainly due to the high specificity of glyphosate towards the plant enzyme enolpyruvylshikimate-3-phosphate synthase in the shikimate pathway leading to biosynthesis of aromatic amino acids. Starting in 1996, transgenic glyphosate-resistant plants were introduced thus allowing the application of the herbicide to the crop (post-emergence) to remove emerged weeds without crop damage. This review focuses on the evolution of mechanisms of resistance to glyphosate as obtained through natural diversity, the gene shuffling approach to molecular evolution, and a rational, structure-based approach to protein engineering. In addition, we offer rationale for the means by which the modifications made have had their intended effect. PMID:21668647

  13. Process considerations for protein engineering of ω-Transaminase

    DEFF Research Database (Denmark)

    Lima Afonso Neto, Watson; Schwarze, Daniel; Tufvesson, Pär

    Over the past decades, the use of biocatalysis in the chemical and pharmaceutical industry has significantly increased. In parallel and contributing to this trend, many enzymes have been discovered and isolated from different biological sources. This has broadened the scope of biocatalysis...... are important building blocks for the chemical and pharmaceutical industries. On the other hand, there are a number of challenges associated with the use of this enzyme for instance substrate and product inhibition, and a potentially unfavorable equilibrium. In the present work it was investigated how changes...... to a wild type transaminase through protein engineering changed the characteristics of the biocatalyst and the implications this would have on a process. A methodology for characterizing the biocatalyst was developed which was subsequently applied to the wild type and 5 mutants selected. It was seen...

  14. Engineering spatial gradients of signaling proteins using magnetic nanoparticles.

    Science.gov (United States)

    Bonnemay, L; Hostachy, S; Hoffmann, C; Gautier, J; Gueroui, Z

    2013-11-13

    Intracellular biochemical reactions are often localized in space and time, inducing gradients of enzymatic activity that may play decisive roles in determining cell's fate and functions. However, the techniques available to examine such enzymatic gradients of activity remain limited. Here, we propose a new method to engineer a spatial gradient of signaling protein concentration within Xenopus egg extracts using superparamagnetic nanoparticles. We show that, upon the application of a magnetic field, a concentration gradient of nanoparticles with a tunable length extension is established within confined egg extracts. We then conjugate the nanoparticles to RanGTP, a small G-protein controlling microtubule assembly. We found that the generation of an artificial gradient of Ran-nanoparticles modifies the spatial positioning of microtubule assemblies. Furthermore, the spatial control of the level of Ran concentration allows us to correlate the local fold increase in Ran-nanoparticle concentration with the spatial positioning of the microtubule-asters. Our assay provides a bottom-up approach to examine the minimum ingredients generating polarization and symmetry breaking within cells. More generally, these results show how magnetic nanoparticles and magnetogenetic tools can be used to control the spatiotemporal dynamics of signaling pathways.

  15. Epigenetic engineering of ribosomal RNA genes enhances protein production.

    Directory of Open Access Journals (Sweden)

    Raffaella Santoro

    Full Text Available Selection of mammalian high-producer cell lines remains a major challenge for the biopharmaceutical manufacturing industry. Ribosomal RNA (rRNA genes encode the major component of the ribosome but many rRNA gene copies are not transcribed due to epigenetic silencing by the nucleolar remodelling complex (NoRC [6], which may limit the cell's full production capacity. Here we show that the knockdown of TIP5, a subunit of NoRC, decreases the number of silent rRNA genes, upregulates rRNA transcription, enhances ribosome synthesis and increases production of recombinant proteins. However, general enhancement of rRNA transcription rate did not stimulate protein synthesis. Our data demonstrates that the number of transcriptionally competent rRNA genes limits efficient ribosome synthesis. Epigenetic engineering of ribosomal RNA genes offers new possibilities for improving biopharmaceutical manufacturing and provides novel insights into the complex regulatory network which governs the translation machinery in normal cellular processes as well as in pathological conditions like cancer.

  16. Engineering Proteins for Thermostability with iRDP Web Server: e0139486

    National Research Council Canada - National Science Library

    Priyabrata Panigrahi; Manas Sule; Avinash Ghanate; Sureshkumar Ramasamy; C G Suresh

    2015-01-01

    ... been targeted through rational protein engineering. Although a range of factors contributing to thermal stability have been identified and widely researched, the in silico implementation of these as strategies directed towards enhancement of protein...

  17. Molecular diagnostic tools for detection and differentiation of phytoplasmas based on chaperonin-60 reveal differences in host plant infection patterns.

    Directory of Open Access Journals (Sweden)

    Tim J Dumonceaux

    Full Text Available Phytoplasmas ('Candidatus Phytoplasma' spp. are insect-vectored bacteria that infect a wide variety of plants, including many agriculturally important species. The infections can cause devastating yield losses by inducing morphological changes that dramatically alter inflorescence development. Detection of phytoplasma infection typically utilizes sequences located within the 16S-23S rRNA-encoding locus, and these sequences are necessary for strain identification by currently accepted standards for phytoplasma classification. However, these methods can generate PCR products >1400 bp that are less divergent in sequence than protein-encoding genes, limiting strain resolution in certain cases. We describe a method for accessing the chaperonin-60 (cpn60 gene sequence from a diverse array of 'Ca.Phytoplasma' spp. Two degenerate primer sets were designed based on the known sequence diversity of cpn60 from 'Ca.Phytoplasma' spp. and used to amplify cpn60 gene fragments from various reference samples and infected plant tissues. Forty three cpn60 sequences were thereby determined. The cpn60 PCR-gel electrophoresis method was highly sensitive compared to 16S-23S-targeted PCR-gel electrophoresis. The topology of a phylogenetic tree generated using cpn60 sequences was congruent with that reported for 16S rRNA-encoding genes. The cpn60 sequences were used to design a hybridization array using oligonucleotide-coupled fluorescent microspheres, providing rapid diagnosis and typing of phytoplasma infections. The oligonucleotide-coupled fluorescent microsphere assay revealed samples that were infected simultaneously with two subtypes of phytoplasma. These tools were applied to show that two host plants, Brassica napus and Camelina sativa, displayed different phytoplasma infection patterns.

  18. Engineered pendrin protein, an anion transporter and molecular motor.

    Science.gov (United States)

    Tang, Jie; Pecka, Jason L; Tan, Xiaodong; Beisel, Kirk W; He, David Z Z

    2011-09-02

    Pendrin and prestin both belong to a distinct anion transporter family called solute carrier protein 26A, or SLC26A. Pendrin (SLC26A4) is a chloride-iodide transporter that is found at the luminal membrane of follicular cells in the thyroid gland as well as in the endolymphatic duct and sac of the inner ear, whereas prestin (SLC26A5) is expressed in the plasma membrane of cochlear outer hair cells and functions as a unique voltage-dependent motor. We recently identified a motif that is critical for the motor function of prestin. We questioned whether it was possible to create a chimeric pendrin protein with motor capability by integrating this motility motif from prestin. The chimeric pendrin was constructed by substituting residues 160-179 in human pendrin with residues 156-169 from gerbil prestin. Non-linear capacitance and somatic motility, two hallmarks representing prestin function, were measured from chimeric pendrin-transfected human embryonic kidney 293 cells using the voltage clamp technique and photodiode-based displacement measurement system. We showed that this 14-amino acid substitution from prestin was able to confer pendrin with voltage-dependent motor capability despite the amino acid sequence disparity between pendrin and prestin. The molecular mechanism that facilitates motor function appeared to be the same as prestin because the motor activity depended on the concentration of intracellular chloride and was blocked by salicylate treatment. Radioisotope-labeled formate uptake measurements showed that the chimeric pendrin protein retained the capability to transport formate, suggesting that the gain of motor function was not at the expense of its inherent transport capability. Thus, the engineered pendrin was capable of both transporting anions and generating force.

  19. Nonequilibrium synthesis and assembly of hybrid inorganic-protein nanostructures using an engineered DNA binding protein.

    Science.gov (United States)

    Dai, Haixia; Choe, Woo-Seok; Thai, Corrine K; Sarikaya, Mehmet; Traxler, Beth A; Baneyx, François; Schwartz, Daniel T

    2005-11-09

    We show that a protein with no intrinsic inorganic synthesis activity can be endowed with the ability to control the formation of inorganic nanostructures under thermodynamically unfavorable (nonequilibrium) conditions, reproducing a key feature of biological hard-tissue growth and assembly. The nonequilibrium synthesis of Cu(2)O nanoparticles is accomplished using an engineered derivative of the DNA-binding protein TraI in a room-temperature precursor electrolyte. The functional TraI derivative (TraIi1753::CN225) is engineered to possess a cysteine-constrained 12-residue Cu(2)O binding sequence, designated CN225, that is inserted into a permissive site in TraI. When TraIi1753::CN225 is included in the precursor electrolyte, stable Cu(2)O nanoparticles form, even though the concentrations of [Cu(+)] and [OH(-)] are at 5% of the solubility product (K(sp,Cu2O)). Negative control experiments verify that Cu(2)O formation is controlled by inclusion of the CN225 binding sequence. Transmission electron microscopy and electron diffraction reveal a core-shell structure for the nonequilibrium nanoparticles: a 2 nm Cu(2)O core is surrounded by an adsorbed protein shell. Quantitative protein adsorption studies show that the unexpected stability of Cu(2)O is imparted by the nanomolar surface binding affinity of TraIi1753::CN225 for Cu(2)O (K(d) = 1.2 x 10(-)(8) M), which provides favorable interfacial energetics (-45 kJ/mol) for the core-shell configuration. The protein shell retains the DNA-binding traits of TraI, as evidenced by the spontaneous organization of nanoparticles onto circular double-stranded DNA.

  20. Use of a protein engineering strategy to overcome limitations in the production of "Difficult to Express" recombinant proteins.

    Science.gov (United States)

    Hussain, Hirra; Fisher, David I; Abbott, W Mark; Roth, Robert G; Dickson, Alan J

    2017-10-01

    Certain recombinant proteins are deemed "difficult to express" in mammalian expression systems requiring significant cell and/or process engineering to abrogate expression bottlenecks. With increasing demand for the production of recombinant proteins in mammalian cells, low protein yields can have significant consequences for industrial processes. To investigate the molecular mechanisms that restrict expression of recombinant proteins, naturally secreted model proteins were analyzed from the tissue inhibitors of metalloproteinase (TIMP) protein family. In particular, TIMP-2 and TIMP-3 were subjected to detailed study. TIMP proteins share significant sequence homology (∼50% identity and ∼70% similarity in amino acid sequence). However, they show marked differences in secretion in mammalian expression systems despite this extensive sequence homology. Using these two proteins as models, this study characterized the molecular mechanisms responsible for poor recombinant protein production. Our results reveal that both TIMP-2 and TIMP-3 are detectable at mRNA and protein level within the cell but only TIMP-2 is secreted effectively into the extracellular medium. Analysis of protein localization and the nature of intracellular protein suggest TIMP-3 is severely limited in its post-translational processing. To overcome this challenge, modification of the TIMP-3 sequence to include a furin protease-cleavable pro-sequence resulted in secretion of the modified TIMP-3 protein, however, incomplete processing was observed. Based on the TIMP-3 data, the protein engineering approach was optimized and successfully applied in combination with cell engineering, the overexpression of furin, to another member of the TIMP protein family (the poorly expressed TIMP-4). Use of the described protein engineering strategy resulted in successful secretion of poorly (TIMP-4) and non-secreted (TIMP-3) targets, and presents a novel strategy to enhance the production of "difficult" recombinant

  1. Engineering Recombinant Protein Sensors for Quantifying Histone Acetylation.

    Science.gov (United States)

    Sanchez, Oscar F; Mendonca, Agnes; Carneiro, Ana D; Yuan, Chongli

    2017-03-24

    H3K14ac (acetylation of lysine 14 of histone H3) is one of the most important epigentic modifications. Aberrant changes in H3K14ac have been associated with various diseases, including cancers and neurological disorders. Tools that enable detection and quantification of H3K14ac levels in cell extracts and in situ are thus of critical importance to reveal its role in various biological processes. Current detection techniques of specific histone modifications, however, are constrained by tedious sample pretreatments, lack of quantitative accuracy, and reliance on high quality antibodies. To address this issue, we engineered recombinant sensors that are suitable for probing histone acetylation levels using various biological samples. The protein sensor contains recongition domain(s) with sequences derived from the bromodomain of human polybromo-1 (PB1), a natural H3K14ac reader domain. Various sensor designs were tested using nuclear extracts and live cells. The sensor containing dimeric repeats of bromodomain was found most effective in quantifying H3K14ac level in both in vitro and in situ assays. The sensor has a linear detection range of 0.5-50 nM when mixed with nuclear extracts. The sensor colocalizes with H3K14ac antibodies in situ when transfected into human embryonic kidney 293T (HEK293T) cells and is thus capable of providing spatial details of histone modification within the nucleus. Corrected nuclear fluorescence intensity was used to quantify the modification level in situ and found to correlate well with our in vitro assays. Our sensor offers a novel tool to characterize the histone modification level using nuclear extracts and probe histone modification change in live cells.

  2. From peptides to proteins: lessons from my years at the Centre for Protein Engineering.

    Science.gov (United States)

    Friedler, Assaf

    2011-01-01

    The MRC Centre for Protein Engineering (CPE) hosted and trained many scientists over the years. It is a unique research environment that shaped the career of many scientists in all aspects. These include research directions and methodologies, but even more important--issues such as how to approach scientific problems and how to manage a research team. Alan Fersht was the director of the CPE when I joined it as a postdoc in the year 2000. In the current article for the PEDS special CPE issue, I will review how my scientific research and my approach to science developed from the days I arrived to the CPE as a young peptide chemist and throughout the years I spent at the CPE, and how it shaped my current research interests and attitude. I will focus on two major fields: (i) Using peptides to study and modulate the structure and interactions of proteins; (ii) Using quantitative biophysical methods to study proteins and their interactions at the molecular level.

  3. Integrating the protein and metabolic engineering toolkits for next-generation chemical biosynthesis.

    Science.gov (United States)

    Pirie, Christopher M; De Mey, Marjan; Jones Prather, Kristala L; Ajikumar, Parayil Kumaran

    2013-04-19

    Through microbial engineering, biosynthesis has the potential to produce thousands of chemicals used in everyday life. Metabolic engineering and synthetic biology are fields driven by the manipulation of genes, genetic regulatory systems, and enzymatic pathways for developing highly productive microbial strains. Fundamentally, it is the biochemical characteristics of the enzymes themselves that dictate flux through a biosynthetic pathway toward the product of interest. As metabolic engineers target sophisticated secondary metabolites, there has been little recognition of the reduced catalytic activity and increased substrate/product promiscuity of the corresponding enzymes compared to those of central metabolism. Thus, fine-tuning these enzymatic characteristics through protein engineering is paramount for developing high-productivity microbial strains for secondary metabolites. Here, we describe the importance of protein engineering for advancing metabolic engineering of secondary metabolism pathways. This pathway integrated enzyme optimization can enhance the collective toolkit of microbial engineering to shape the future of chemical manufacturing.

  4. DARPins and other repeat protein scaffolds : advances in engineering and applications

    NARCIS (Netherlands)

    Boersma, Ykelien L; Plückthun, Andreas

    2011-01-01

    Antibodies have long been regarded as the only class of binding proteins. With the emergence of protein engineering techniques, new binding proteins based on alternative scaffolds have been designed. Additionally, modern technologies for selection and evolution from libraries are independent of the

  5. Trans-species Engineering of Glycosylated Therapeutic Proteins

    DEFF Research Database (Denmark)

    Yang, Zhang

    eukaryotes and even prokaryotes. Insect and yeast cells produce O-glycosylation incompatible with use in humans, however recently the yeast Pichia was engineered to perform the first step of human-like O-glycosylation. This review provides an overview of past and current engineering efforts of N...

  6. Co-overexpression of bacterial GroESL chaperonins partly overcomes non-productive folding and tetramer assembly of E. coli-expressed human medium-chain acyl-CoA dehydrogenase (MCAD) carrying the prevalent disease-causing K304E mutation

    DEFF Research Database (Denmark)

    Bross, P; Andresen, B S; Winter, V

    1993-01-01

    The influence of co-overexpression of the bacterial chaperonins GroEL and GroES on solubility, tetramer formation and enzyme activity of three variants of heterologously-expressed human medium-chain acyl-CoA dehydrogenase (MCAD) was analysed in order to investigate the molecular mechanism...... and the enzyme activity measured as observed for the wild-type protein. (iii) Solubility of the K304E mutant is in a similar fashion GroESL responsive as the K304Q mutant, but the amount of tetramer observed and the enzyme activity measured do not correlate with the amount of soluble K304E MCAD protein detected...

  7. PIA: An Intuitive Protein Inference Engine with a Web-Based User Interface.

    Science.gov (United States)

    Uszkoreit, Julian; Maerkens, Alexandra; Perez-Riverol, Yasset; Meyer, Helmut E; Marcus, Katrin; Stephan, Christian; Kohlbacher, Oliver; Eisenacher, Martin

    2015-07-02

    Protein inference connects the peptide spectrum matches (PSMs) obtained from database search engines back to proteins, which are typically at the heart of most proteomics studies. Different search engines yield different PSMs and thus different protein lists. Analysis of results from one or multiple search engines is often hampered by different data exchange formats and lack of convenient and intuitive user interfaces. We present PIA, a flexible software suite for combining PSMs from different search engine runs and turning these into consistent results. PIA can be integrated into proteomics data analysis workflows in several ways. A user-friendly graphical user interface can be run either locally or (e.g., for larger core facilities) from a central server. For automated data processing, stand-alone tools are available. PIA implements several established protein inference algorithms and can combine results from different search engines seamlessly. On several benchmark data sets, we show that PIA can identify a larger number of proteins at the same protein FDR when compared to that using inference based on a single search engine. PIA supports the majority of established search engines and data in the mzIdentML standard format. It is implemented in Java and freely available at https://github.com/mpc-bioinformatics/pia.

  8. Bacterial lipid modification of proteins requires appropriate secretory signals even for expression - implications for biogenesis and protein engineering.

    Science.gov (United States)

    Kumar, Subramani; Balamurali, M M; Sankaran, Krishnan

    2014-09-01

    Sec- and Tat-mediated bacterial lipid modification of proteins are important posttranslational processes owing to their vital roles in cellular functions, membrane targeting and biotechnological applications like ELISA, biosensor, adjuvant-free vaccines, liposomal drug delivery etc. However a better understanding of the tight coupling of secretory and lipid modification machineries and the processes associated will help unravel this essential biological event and utilize it for engineering applications. Further, there is a need for a systematic and convincing investigation into membrane targeting, solubilization and ease-of-purification of engineered lipoproteins to facilitate scientists in readily applying this new protein engineering tool. Therefore, in this study, we have investigated systematically recombinant expression, translocation, solubilization and purification of three White Spot Syndrome Viral (WSSV) proteins, ICP11, VP28 and VP281. Our study shows that the lipid modification and secretion processes are tightly coupled to the extent that mismatch between folding kinetics and signal sequence of target proteins could lead to transcriptional-translational uncoupling or aborted translation. The proteins expressed as lipoproteins through Tat-pathway were targeted to the inner membrane achieving considerable enrichment. These His-tagged proteins were then purified to apparent homogeneity in detergent-free form using single-step Immobilized Metal Affinity Chromatography. This study has interesting findings in lipoprotein biogenesis enhancing the scope of this unique post-translational protein engineering tool for obtaining pure detergent-free, membrane or hydrophobic surface-associating diagnostic targets and vaccine candidates for WSSV.

  9. Protein Engineering: Development of a Metal Ion Dependent Switch

    Science.gov (United States)

    2017-05-22

    Lehninger Principles of Biochemistry Current Opinion in Chemical Biology Protein Science PNAS Journal of Molecular Biology Science Direct Royal...Molecular Biology Biochemistry Royal Society of Chemistry Proteins : Structure, Function, and Bioinformatics Journal of Molecular Biology Biophysical...Biophysical Journal Protein Science Journal of Computational Chemistry Current Opinion in Chemical Biology Royal Society of Chemistry

  10. Engineered mammalian cells for production of recombinant proteins

    DEFF Research Database (Denmark)

    2017-01-01

    The present invention relates to mammalian cells modified to provide for improved expression of a recombinant protein of interest. In particular, the invention relates to CHO cells and other host cells in which the expression of one or more endogenous secreted proteins has been disrupted, as well...... as to the preparation, identification and use of such cells in the production of recombinant proteins....

  11. HotSpot Wizard: a web server for identification of hot spots in protein engineering

    National Research Council Canada - National Science Library

    Pavelka, Antonin; Chovancova, Eva; Damborsky, Jiri

    2009-01-01

    HotSpot Wizard is a web server for automatic identification of 'hot spots' for engineering of substrate specificity, activity or enantioselectivity of enzymes and for annotation of protein structures...

  12. Improved Enantioselectivity of Subtilisin Carlsberg towards Secondary Alcohols by Protein Engineering

    DEFF Research Database (Denmark)

    Dorau, Robin; Görbe, Tamas; Svedendahl Humble, Maria

    2017-01-01

    Generally, the catalytic activity of subtilisin Carlsberg (SC) for transacylation reactions with secondary alcohols in organic solvent is low. Enzyme immobilization and protein engineering was performed to improve the enantioselectivity of SC towards secondary alcohols. Possible amino-acid residues...

  13. Structural Insights for Engineering Binding Proteins Based on Non-Antibody Scaffolds

    OpenAIRE

    Gilbreth, Ryan N.; Koide, Shohei

    2012-01-01

    Engineered binding proteins derived from non-antibody scaffolds constitute an increasingly prominent class of reagents in both research and therapeutic applications. The growing number of crystal structures of these “alternative” scaffold-based binding proteins in complex with their targets illustrate the mechanisms of molecular recognition that are common among these systems and those unique to each. This information is useful for critically assessing and improving/expanding engineering stra...

  14. Healing Osteoarthritis: Engineered Proteins Created for Therapeutic Cartilage Regeneration

    Directory of Open Access Journals (Sweden)

    Kevin M. Cherry

    2012-01-01

    Full Text Available Millions of people worldwide are afflicted with painfulosteoarthritis, which is characterized by degradationof articular cartilage found in major joints such as thehip or knee. Symptoms include inflammation, pain,and decreased mobility. Because cartilage has a limitedability to self-heal, researchers have focused efforts onmethods that trigger cartilage regeneration. Our approachis to develop an injectable, protein-based hydrogel withmechanical properties analogous to healthy articularcartilage. The hydrogel provides an environment for cellgrowth and stimulates new tissue formation. We utilizedrecombinant DNA technology to create multifunctional,elastomeric proteins. The recombinant proteins weredesigned with biologically active domains to influence cellbehavior and resilin structural domains that mimic thestiffness of native cartilage. Resilin, a protein found in thewing and leg joints of mosquitoes, provided inspiration forthe mechanical domain in the recombinant protein. Thenew resilin-based protein was expressed in E. coli bacteria.Forming hydrogels requires a large quantity of engineeredprotein, so parameters such as bacterial host, incubationtemperature, expression time, and induction method wereoptimized to increase the protein yield. Using salt toprecipitate the protein and exploiting resilin’s heat stability,27 mg/L of recombinant protein was recovered at 95%purity. The protein expression and purification protocolswere established by analyzing experimental samples onSDS-PAGE gels and by Western blotting. The mechanicalproperties and interactions with stem cells are currentlybeing evaluated to assess the potential of the resilin-basedhydrogel as a treatment for osteoarthritis.

  15. Protein-based materials in load-bearing tissue-engineering applications.

    Science.gov (United States)

    Sayin, Esen; Baran, Erkan Türker; Hasirci, Vasif

    2014-01-01

    Proteins such as collagen and elastin are robust molecules that constitute nanocomponents in the hierarchically organized ultrastructures of bone and tendon as well as in some of the soft tissues that have load-bearing functions. In the present paper, the macromolecular structure and function of the proteins are reviewed and the potential of mammalian and non-mammalian proteins in the engineering of load-bearing tissue substitutes are discussed. Chimeric proteins have become an important structural biomaterial source and their potential in tissue engineering is highlighted. Processing of proteins challenge investigators and in this review rapid prototyping and microfabrication are proposed as methods for obtaining precisely defined custom-built tissue engineered structures with intrinsic microarchitecture.

  16. Protein consensus-based surface engineering (ProCoS): a computer-assisted method for directed protein evolution.

    Science.gov (United States)

    Shivange, Amol V; Hoeffken, Hans Wolfgang; Haefner, Stefan; Schwaneberg, Ulrich

    2016-12-01

    Protein consensus-based surface engineering (ProCoS) is a simple and efficient method for directed protein evolution combining computational analysis and molecular biology tools to engineer protein surfaces. ProCoS is based on the hypothesis that conserved residues originated from a common ancestor and that these residues are crucial for the function of a protein, whereas highly variable regions (situated on the surface of a protein) can be targeted for surface engineering to maximize performance. ProCoS comprises four main steps: (i) identification of conserved and highly variable regions; (ii) protein sequence design by substituting residues in the highly variable regions, and gene synthesis; (iii) in vitro DNA recombination of synthetic genes; and (iv) screening for active variants. ProCoS is a simple method for surface mutagenesis in which multiple sequence alignment is used for selection of surface residues based on a structural model. To demonstrate the technique's utility for directed evolution, the surface of a phytase enzyme from Yersinia mollaretii (Ymphytase) was subjected to ProCoS. Screening just 1050 clones from ProCoS engineering-guided mutant libraries yielded an enzyme with 34 amino acid substitutions. The surface-engineered Ymphytase exhibited 3.8-fold higher pH stability (at pH 2.8 for 3 h) and retained 40% of the enzyme's specific activity (400 U/mg) compared with the wild-type Ymphytase. The pH stability might be attributed to a significantly increased (20 percentage points; from 9% to 29%) number of negatively charged amino acids on the surface of the engineered phytase.

  17. Effects of a mutation in the HSPE1 gene encoding the mitochondrial co-chaperonin HSP10 and its potential association with a neurological and developmental disorder

    Directory of Open Access Journals (Sweden)

    Anne Sigaard Bie

    2016-10-01

    Full Text Available We here report molecular investigations of a missense mutation in the HSPE1 gene encoding the HSP10 subunit of the HSP60/ HSP10 chaperonin complex that assists protein folding in the mitochondrial matrix. The mutation was identified in an infant who came to clinical attention due to infantile spasms at three months of age. Clinical exome sequencing revealed heterozygosity for a HSPE1 NM_002157.2:c.217C>T de novo mutation causing replacement of leucine with phenylalanine at position 73 of the HSP10 protein. This variation has never been observed in public exome sequencing databases or the literature.To evaluate whether the mutation may be disease-associated we investigated its effects by in vitro and ex vivo studies. Our in vitro studies indicated that the purified mutant protein was functional, yet its thermal stability, spontaneous refolding propensity, and resistance to proteolytic treatment were profoundly impaired. Mass spectrometric analysis of patient fibroblasts revealed barely detectable levels of HSP10-p.Leu73Phe protein resulting in an almost 2-fold decrease of the ratio of HSP10 to HSP60 subunits. Amounts of the mitochondrial superoxide dismutase SOD2, a protein whose folding is known to strongly depend on the HSP60/HSP10 complex, were decreased to approximately 20% in patient fibroblasts in spite of unchanged SOD2 transcript levels. As a likely consequence, mitochondrial superoxide levels were increased about 2-fold. Although we cannot exclude other causative or contributing factors, our experimental data support the notion that the HSP10-p.Leu73Phe mutation could be the cause or a strong contributing factor for the disorder in the described patient.

  18. Mycobacterium tuberculosis chaperonin 10 and N-truncated fragments. Their synthesis and purification by the isoelectric focusing technique carried out in solution.

    Science.gov (United States)

    Lucietto, P; Fossati, G; Ball, H L; Giuliani, P; Mascagni, P

    1997-04-01

    The Mycobacterium tuberculosis chaperonin 10 protein and fragments corresponding to sequences 59-99, 51-99 and 26-99 were synthesised by the solid-phase methodology using a double coupling protocol and without the aid of capping agents. After the final acid cleavage using the low TFMSA-high HF protocol the polypeptides were purified by either the ion exchange chromatography/RP-HPLC combination or the isoelectric separation carried out in solution and followed by semi-preparative RP-HPLC. Comparison of the results obtained through the two approaches indicated that in general the isoelectricfocusing/HPLC combination was superior both in terms of recovery of final material and its purity. The advantages found were as follows: (i) Unlike ion exchange chromatography, no tailoring of the separation conditions is required, (ii) Several consecutive focusings can be carried out in progressively narrower pH gradients. This increases the separation resolution without the need of changing other separation parameters, (iii) Very little manipulation is needed, and each focusing requires 3-5h. (iv) Full compatibility with non-ionic denaturants such as 8 M urea. This increases solubility so that using the ROTOFOR instrument described here 50-100 mg crude polypeptide can be processed daily. Thus the isoelectric focusing technique carried out in solution is a valid and inexpensive alternative to ion exchange chromatography.

  19. Engineering therapeutic proteins for cell entry: the natural approach.

    Science.gov (United States)

    Guillard, Sandrine; Minter, Ralph R; Jackson, Ronald H

    2015-03-01

    Owing to the challenges of cell entry, protein-based therapies have so far been restricted to extracellular targets, whereas intracellular targets have been almost exclusively addressed by small molecules. The specificity and potency of proteins would enable them to be effective intracellular drugs, provided that the proteins are delivered efficiently to appropriate intracellular compartments within specific cell types. By mimicking the natural mechanisms of toxins and other natural proteins, new intracellular delivery systems are being developed, the first of which are showing clinical efficacy. This review highlights a range of ingenious approaches designed to adapt natural entry mechanisms to facilitate delivery of proteins and open up a range of validated intracellular targets to modulation by potent and specific therapeutic drugs. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. Engineering of vesicle trafficking improves heterologous protein secretion in Saccharomyces cerevisiae.

    Science.gov (United States)

    Hou, Jin; Tyo, Keith; Liu, Zihe; Petranovic, Dina; Nielsen, Jens

    2012-03-01

    The yeast Saccharomyces cerevisiae is a widely used platform for the production of heterologous proteins of medical or industrial interest. However, heterologous protein productivity is often restricted due to the limitations of the host strain. In the protein secretory pathway, the protein trafficking between different organelles is catalyzed by the soluble NSF (N-ethylmaleimide-sensitive factor) receptor (SNARE) complex and regulated by the Sec1/Munc18 (SM) proteins. In this study, we report that over-expression of the SM protein encoding genes SEC1 and SLY1, improves the protein secretion in S. cerevisiae. Engineering Sec1p, the SM protein that is involved in vesicle trafficking from Golgi to cell membrane, improves the secretion of heterologous proteins human insulin precursor and α-amylase, and also the secretion of an endogenous protein invertase. Enhancing Sly1p, the SM protein regulating the vesicle fusion from endoplasmic reticulum (ER) to Golgi, increases α-amylase production only. Our study demonstrates that strengthening the protein trafficking in ER-to-Golgi and Golgi-to-plasma membrane process is a novel secretory engineering strategy for improving heterologous protein production in S. cerevisiae. Copyright © 2012 Elsevier Inc. All rights reserved.

  1. Modulation of protein stability and aggregation properties by surface charge engineering.

    Science.gov (United States)

    Raghunathan, Govindan; Sokalingam, Sriram; Soundrarajan, Nagasundarapandian; Madan, Bharat; Munussami, Ganapathiraman; Lee, Sun-Gu

    2013-09-01

    An attempt to alter protein surface charges through traditional protein engineering approaches often affects the native protein structure significantly and induces misfolding. This limitation is a major hindrance in modulating protein properties through surface charge variations. In this study, as a strategy to overcome such a limitation, we attempted to co-introduce stabilizing mutations that can neutralize the destabilizing effect of protein surface charge variation. Two sets of rational mutations were designed; one to increase the number of surface charged amino acids and the other to decrease the number of surface charged amino acids by mutating surface polar uncharged amino acids and charged amino acids, respectively. These two sets of mutations were introduced into Green Fluorescent Protein (GFP) together with or without stabilizing mutations. The co-introduction of stabilizing mutations along with mutations for surface charge modification allowed us to obtain functionally active protein variants (s-GFP(+15-17) and s-GFP(+5-6)). When the protein properties such as fluorescent activity, folding rate and kinetic stability were assessed, we found the possibility that the protein stability can be modulated independently of activity and folding by engineering protein surface charges. The aggregation properties of GFP could also be altered through the surface charge engineering.

  2. Engineering periplasmic ligand binding proteins as glucose nanosensors

    Directory of Open Access Journals (Sweden)

    Constance J. Jeffery

    2011-01-01

    Full Text Available Diabetes affects over 100 million people worldwide. Better methods for monitoring blood glucose levels are needed for improving disease management. Several labs have previously made glucose nanosensors by modifying members of the periplasmic ligand binding protein superfamily. This minireview summarizes recent developments in constructing new versions of these proteins that are responsive within the physiological range of blood glucose levels, employ new reporter groups, and/or are more robust. These experiments are important steps in the development of novel proteins that have the characteristics needed for an implantable glucose nanosensor for diabetes management: specificity for glucose, rapid response, sensitivity within the physiological range of glucose concentrations, reproducibility, and robustness.

  3. Allosteric transitions of supramolecular systems explored by network models: application to chaperonin GroEL.

    Directory of Open Access Journals (Sweden)

    Zheng Yang

    2009-04-01

    Full Text Available Identification of pathways involved in the structural transitions of biomolecular systems is often complicated by the transient nature of the conformations visited across energy barriers and the multiplicity of paths accessible in the multidimensional energy landscape. This task becomes even more challenging in exploring molecular systems on the order of megadaltons. Coarse-grained models that lend themselves to analytical solutions appear to be the only possible means of approaching such cases. Motivated by the utility of elastic network models for describing the collective dynamics of biomolecular systems and by the growing theoretical and experimental evidence in support of the intrinsic accessibility of functional substates, we introduce a new method, adaptive anisotropic network model (aANM, for exploring functional transitions. Application to bacterial chaperonin GroEL and comparisons with experimental data, results from action minimization algorithm, and previous simulations support the utility of aANM as a computationally efficient, yet physically plausible, tool for unraveling potential transition pathways sampled by large complexes/assemblies. An important outcome is the assessment of the critical inter-residue interactions formed/broken near the transition state(s, most of which involve conserved residues.

  4. Chaperonin-Inspired pH Protection by Mesoporous Silica SBA-15 on Myoglobin and Lysozyme.

    Science.gov (United States)

    Lynch, Michele M; Liu, Jichuan; Nigra, Michael; Coppens, Marc-Olivier

    2016-09-20

    While enzymes are valuable tools in many fields of biotechnology, they are fragile and must be protected against denaturing conditions such as unfavorable solution pH. Within living organisms, chaperonins help enzymes fold into their native shape and protect them from damage. Inspired by this natural solution, mesoporous silica SBA-15 with different pore diameters is synthesized as a support material for immobilizing and protecting enzymes. In separate experiments, the model enzymes myoglobin and lysozyme are physically adsorbed to SBA-15 and exposed to a range of buffered pH conditions. The immobilized enzymes' biocatalytic activities are quantified and compared to the activities of nonimmobilized enzymes in the same solution conditions. It has been observed that myoglobin immobilized on SBA-15 is protected from acidic denaturation from pH 3.6 to 5.1, exhibiting relative activity of up to 350%. Immobilized lysozyme is protected from unfavorable conditions from pH 6.6 to 7.6, with relative activity of up to 200%. These results indicate that the protective effects conferred to enzymes immobilized by physical adsorption to SBA-15 are driven by the enzymes' electrostatic attraction to the material's surface. The pore diameter of SBA-15 affects the quality of protection given to immobilized enzymes, but the contribution of this effect at different pH values remains unclear.

  5. Structural insights for engineering binding proteins based on non-antibody scaffolds.

    Science.gov (United States)

    Gilbreth, Ryan N; Koide, Shohei

    2012-08-01

    Engineered binding proteins derived from non-antibody scaffolds constitute an increasingly prominent class of reagents in both research and therapeutic applications. The growing number of crystal structures of these 'alternative' scaffold-based binding proteins in complex with their targets illustrate the mechanisms of molecular recognition that are common among these systems and those unique to each. This information is useful for critically assessing and improving/expanding engineering strategies. Furthermore, the structural features of these synthetic proteins produced under tightly controlled, directed evolution deepen our understanding of the underlying principles governing molecular recognition. Copyright © 2012 Elsevier Ltd. All rights reserved.

  6. Protein engineering of cyclodextrin glycosyltransferase from Bacillus circulans strain 251

    NARCIS (Netherlands)

    Penninga, Dirk

    1996-01-01

    An enormous diversity of molecular functions in living organisms is carried out by proteins. Our studies have focussed on the functional analysis of a starch-converting enzyme, cyclodextrin glycosyltransferase (CGTase) from Bacillus circulans strain 251. Zie: Summary

  7. Protein engineering in designing tailored enzymes and microorganisms for biofuels production

    Science.gov (United States)

    Wen, Fei; Nair, Nikhil U; Zhao, Huimin

    2009-01-01

    Summary Lignocellulosic biofuels represent a sustainable, renewable, and the only foreseeable alternative energy source to transportation fossil fuels. However, the recalcitrant nature of lignocellulose poses technical hurdles to an economically viable biorefinery. Low enzymatic hydrolysis efficiency and low productivity, yield, and titer of biofuels are among the top cost contributors. Protein engineering has been used to improve the performances of lignocellulose-degrading enzymes, as well as proteins involved in biofuel synthesis pathways. Unlike its great success seen in other industrial applications, protein engineering has achieved only modest results in improving the lignocellulose-to-biofuels efficiency. This review will discuss the unique challenges that protein engineering faces in the process of converting lignocellulose to biofuels and how they are addressed by recent advances in this field. PMID:19660930

  8. Engineering Protein Self-Assembly: A New Approach for the Design of Octahedral Cages.

    Science.gov (United States)

    Cougnon, Fabien B L

    2016-12-14

    A new symmetry-based approach allowed the self-assembly of an octahedral protein nanostructure. C3 trimeric and C4 tetrameric oligomerization domains can be combined in an engineered protein to direct assembly into a desired object. This work might provide the basis for a more general and flexible strategy to control protein self-assembly. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Review: Advances in Vascular Tissue Engineering Using Protein-Based Biomaterials

    Science.gov (United States)

    Stegemann, Jan P.; Kaszuba, Stephanie N.; Rowe, Shaneen L.

    2008-01-01

    The clinical need for improved blood vessel substitutes, especially in small-diameter applications, drives the field of vascular tissue engineering. The blood vessel has a well-characterized structure and function, but it is a complex tissue, and it has proven difficult to create engineered tissues that are suitable for widespread clinical use. This review is focused on approaches to vascular tissue engineering that use proteins as the primary matrix or “scaffold” material for creating fully biological blood vessel replacements. In particular, this review covers four main approaches to vascular tissue engineering: 1) cell-populated protein hydrogels, 2) cross-linked protein scaffolds, 3) decellularized native tissues, and 4) self-assembled scaffolds. Recent advances in each of these areas are discussed, along with advantages of and drawbacks to these approaches. The first fully biological engineered blood vessels have entered clinical trials, but important challenges remain before engineered vascular tissues will have a wide clinical effect. Cell sourcing and recapitulating the biological and mechanical function of the native blood vessel continue to be important outstanding hurdles. In addition, the path to commercialization for such tissues must be better defined. Continued progress in several complementary approaches to vascular tissue engineering is necessary before blood vessel substitutes can achieve their full potential in improving patient care. PMID:17961004

  10. Engineering and characterization of an enhanced fluorescent protein voltage sensor.

    Directory of Open Access Journals (Sweden)

    Dimitar Dimitrov

    Full Text Available BACKGROUND: Fluorescent proteins have been used to generate a variety of biosensors to optically monitor biological phenomena in living cells. Among this class of genetically encoded biosensors, reporters for membrane potential have been a particular challenge. The use of presently known voltage sensor proteins is limited by incorrect subcellular localization and small or absent voltage responses in mammalian cells. RESULTS: Here we report on a fluorescent protein voltage sensor with superior targeting to the mammalian plasma membrane and high responsiveness to membrane potential signaling in excitable cells. CONCLUSIONS AND SIGNIFICANCE: This biosensor, which we termed VSFP2.1, is likely to lead to new methods of monitoring electrically active cells with cell type specificity, non-invasively and in large numbers, simultaneously.

  11. Artificial Metalloenzymes through Chemical Modification of Engineered Host Proteins

    KAUST Repository

    Zernickel, Anna

    2014-10-01

    With a few exceptions, all organisms are restricted to the 20 canonical amino acids for ribosomal protein biosynthesis. Addition of new amino acids to the genetic code can introduce novel functionalities to proteins, broadening the diversity of biochemical as well as chemical reactions and providing new tools to study protein structure, reactivity, dynamics and protein-protein-interactions. The site directed in vivo incorporation developed by P. G. SCHULTZ and coworkers, using an archeal orthogonal tRNA/aaRS (aminoacyl-tRNA synthase) pair, allows site-specifically insertion of a synthetic unnatural amino acid (UAA) by reprogramming the amber TAG stop codon. A variety of over 80 different UAAs can be introduced by this technique. However by now a very limited number can form kinetically stable bonds to late transition metals. This thesis aims to develop new catalytically active unnatural amino acids or strategies for a posttranslational modification of site-specific amino acids in order to achieve highly enantioselective metallorganic enzyme hybrids (MOEH). As a requirement a stable protein host has to be established, surviving the conditions for incorporation, posttranslational modification and the final catalytic reactions. mTFP* a fluorescent protein was genetically modified by excluding any exposed Cys, His and Met forming a variant mTFP*, which fulfills the required specifications. Posttranslational chemical modification of mTFP* allow the introduction of single site metal chelating moieties. For modification on exposed cysteines different maleiimid containing ligand structures were synthesized. In order to perform copper catalyzed click reactions, suitable unnatural amino acids (para-azido-(L)-phenylalanine, para-ethynyl-(L)-phenylalanine) were synthesized and a non-cytotoxic protocol was established. The triazole ring formed during this reaction may contribute as a moderate σ-donor/π-acceptor ligand to the metal binding site. Since the cell limits the

  12. Engineering of protein folding and secretion-strategies to overcome bottlenecks for efficient production of recombinant proteins.

    Science.gov (United States)

    Delic, Marizela; Göngrich, Rebecca; Mattanovich, Diethard; Gasser, Brigitte

    2014-07-20

    Recombinant protein production has developed into a huge market with enormous positive implications for human health and for the future direction of a biobased economy. Limitations in the economic and technical feasibility of production processes are often related to bottlenecks of in vivo protein folding. Based on cell biological knowledge, some major bottlenecks have been overcome by the overexpression of molecular chaperones and other folding related proteins, or by the deletion of deleterious pathways that may lead to misfolding, mistargeting, or degradation. While important success could be achieved by this strategy, the list of reported unsuccessful cases is disappointingly long and obviously dependent on the recombinant protein to be produced. Singular engineering of protein folding steps may not lead to desired results if the pathway suffers from several limitations. In particular, the connection between folding quality control and proteolytic degradation needs further attention. Based on recent understanding that multiple steps in the folding and secretion pathways limit productivity, synergistic combinations of the cell engineering approaches mentioned earlier need to be explored. In addition, systems biology-based whole cell analysis that also takes energy and redox metabolism into consideration will broaden the knowledge base for future rational engineering strategies.

  13. Fibrous proteins: At the crossroads of genetic engineering and biotechnological applications.

    Science.gov (United States)

    Yigit, Sezin; Dinjaski, Nina; Kaplan, David L

    2016-05-01

    Fibrous proteins, such as silk, elastin and collagen are finding broad impact in biomaterial systems for a range of biomedical and industrial applications. Some of the key advantages of biosynthetic fibrous proteins compared to synthetic polymers include the tailorability of sequence, protein size, degradation pattern, and mechanical properties. Recombinant DNA production and precise control over genetic sequence of these proteins allows expansion and fine tuning of material properties to meet the needs for specific applications. We review current approaches in the design, cloning, and expression of fibrous proteins, with a focus on strategies utilized to meet the challenges of repetitive fibrous protein production. We discuss recent advances in understanding the fundamental basis of structure-function relationships and the designs that foster fibrous protein self-assembly towards predictable architectures and properties for a range of applications. We highlight the potential of functionalization through genetic engineering to design fibrous protein systems for biotechnological and biomedical applications. © 2015 Wiley Periodicals, Inc.

  14. A discussion of molecular biology methods for protein engineering

    CSIR Research Space (South Africa)

    Zawaira, A

    2011-09-01

    Full Text Available A number of molecular biology techniques are available to generate variants from a particular start gene for eventual protein expression. The authors discuss the basic principles of these methods in a repertoire that may be used to achieve...

  15. Lipase-catalyzed process for biodiesel production: protein engineering and lipase production.

    Science.gov (United States)

    Hwang, Hyun Tae; Qi, Feng; Yuan, Chongli; Zhao, Xuebing; Ramkrishna, Doraiswami; Liu, Dehua; Varma, Arvind

    2014-04-01

    Biodiesel is an environment-friendly and renewable fuel produced by transesterification of various feedstocks. Although the lipase-catalyzed biodiesel production has many advantages over the conventional alkali catalyzed process, its industrial applications have been limited by high-cost and low-stability of lipase enzymes. This review provides a general overview of the recent advances in lipase engineering, including both protein modification and production. Recent advances in biotechnology such as in protein engineering, recombinant methods and metabolic engineering have been employed but are yet to impact lipase engineering for cost-effective production of biodiesel. A summary of the current challenges and perspectives for potential solutions are also provided. © 2013 Wiley Periodicals, Inc.

  16. The composition, structure and stability of a group II chaperonin are temperature regulated in a hyperthermophilic archaeon.

    Science.gov (United States)

    Kagawa, Hiromi K; Yaoi, Takuro; Brocchieri, Luciano; McMillan, R Andrew; Alton, Thomas; Trent, Jonathan D

    2003-04-01

    The hyperthermoacidophilic archaeon Sulfolobus shibatae contains group II chaperonins, known as rosettasomes, which are two nine-membered rings composed of three different 60 kDa subunits (TF55 alpha, beta and gamma). We sequenced the gene for the gamma subunit and studied the temperature-dependent changes in alpha, beta and gamma expression, their association into rosettasomes and their phylogenetic relationships. Alpha and beta gene expression was increased by heat shock (30 min, 86 degrees C) and decreased by cold shock (30 min, 60 degrees C). Gamma expression was undetectable at heat shock temperatures and low at normal temperatures (75-79 degrees C), but induced by cold shock. Polyacrylamide gel electrophoresis indicated that in vitro alpha and beta subunits form homo-oligomeric rosettasomes, and mixtures of alpha, beta and gamma form hetero-oligomeric rosettasomes. Transmission electron microscopy revealed that beta homo-oligomeric rosettasomes and all hetero-oligomeric rosettasomes associate into filaments. In vivo rosettasomes were hetero-oligomeric with an average subunit ratio of 1alpha:1beta:0.1gamma in cultures grown at 75 degrees C, a ratio of 1alpha:3beta:1gamma in cultures grown at 60 degrees C and a ratio of 2alpha:3beta:0gamma after 86 degrees C heat shock. Using differential scanning calorimetry, we determined denaturation temperatures (Tm) for alpha, beta and gamma subunits of 95.7 degrees C, 96.7 degrees C and 80.5 degrees C, respectively, and observed that rosettasomes containing gamma were relatively less stable than those with alpha and/or beta only. We propose that, in vivo, the rosettasome structure is determined by the relative abundance of subunits and not by a fixed geometry. Furthermore, phylogenetic analyses indicate that archaeal chaperonin subunits underwent multiple duplication events within species (paralogy). The independent evolution of these paralogues raises the possibility that chaperonins have functionally diversified between

  17. Genetic engineering: from a clone to a protein

    Directory of Open Access Journals (Sweden)

    Ljubijankić Goran

    2002-01-01

    Full Text Available October 15, 1980, was a date when the highest increase of the share price of one company was recorded on the world most powerful New York change - rates of shares of the company Genetech jumped by more than 2.5 times in only 20 minutes. This event was a grand entrance of a new technology into the global economy and it irreversibly established a brand new status of biology within the development of civilization. Genetech is one of the first companies within the field of molecular biotechnology that accomplished such an enormous commercial success no more than seven years upon the last discoveries in the series of scientific findings that had provided formulation of a new technology designated as genetic engineering or a recombinant DNA technology. Today, 20 years later, it can be rightfully claimed that expectations of the new technology are fulfilled: even if the qualitative shift it provided to fundamental research is disregarded, its direct commercial effects are very convincing - the total value of sold products produced by this technology exceeded the sum of 60 billions dollars in 2000. Let us cite market parameters of only some products manufactured by the application of genetic engineering: annual global consumption of recombinant human insulin amounts to approximately 4.6 tons only in the industrialized part of the world, while the annual sale of recombinant human erythropoietin, interferon's and a human growth hormone amount to 2, i.e. 1.5 billions dollars, respectively.

  18. Engineering a pH responsive pore forming protein

    Science.gov (United States)

    Kisovec, Matic; Rezelj, Saša; Knap, Primož; Cajnko, Miša Mojca; Caserman, Simon; Flašker, Ajda; Žnidaršič, Nada; Repič, Matej; Mavri, Janez; Ruan, Yi; Scheuring, Simon; Podobnik, Marjetka; Anderluh, Gregor

    2017-02-01

    Listeriolysin O (LLO) is a cytolysin capable of forming pores in cholesterol-rich lipid membranes of host cells. It is conveniently suited for engineering a pH-governed responsiveness, due to a pH sensor identified in its structure that was shown before to affect its stability. Here we introduced a new level of control of its hemolytic activity by making a variant with hemolytic activity that was pH-dependent. Based on detailed structural analysis coupled with molecular dynamics and mutational analysis, we found that the bulky side chain of Tyr406 allosterically affects the pH sensor. Molecular dynamics simulation further suggested which other amino acid residues may also allosterically influence the pH-sensor. LLO was engineered to the point where it can, in a pH-regulated manner, perforate artificial and cellular membranes. The single mutant Tyr406Ala bound to membranes and oligomerized similarly to the wild-type LLO, however, the final membrane insertion step was pH-affected by the introduced mutation. We show that the mutant toxin can be activated at the surface of artificial membranes or living cells by a single wash with slightly acidic pH buffer. Y406A mutant has a high potential in development of novel nanobiotechnological applications such as controlled release of substances or as a sensor of environmental pH.

  19. Gene composer: database software for protein construct design, codon engineering, and gene synthesis.

    Science.gov (United States)

    Lorimer, Don; Raymond, Amy; Walchli, John; Mixon, Mark; Barrow, Adrienne; Wallace, Ellen; Grice, Rena; Burgin, Alex; Stewart, Lance

    2009-04-21

    To improve efficiency in high throughput protein structure determination, we have developed a database software package, Gene Composer, which facilitates the information-rich design of protein constructs and their codon engineered synthetic gene sequences. With its modular workflow design and numerous graphical user interfaces, Gene Composer enables researchers to perform all common bio-informatics steps used in modern structure guided protein engineering and synthetic gene engineering. An interactive Alignment Viewer allows the researcher to simultaneously visualize sequence conservation in the context of known protein secondary structure, ligand contacts, water contacts, crystal contacts, B-factors, solvent accessible area, residue property type and several other useful property views. The Construct Design Module enables the facile design of novel protein constructs with altered N- and C-termini, internal insertions or deletions, point mutations, and desired affinity tags. The modifications can be combined and permuted into multiple protein constructs, and then virtually cloned in silico into defined expression vectors. The Gene Design Module uses a protein-to-gene algorithm that automates the back-translation of a protein amino acid sequence into a codon engineered nucleic acid gene sequence according to a selected codon usage table with minimal codon usage threshold, defined G:C% content, and desired sequence features achieved through synonymous codon selection that is optimized for the intended expression system. The gene-to-oligo algorithm of the Gene Design Module plans out all of the required overlapping oligonucleotides and mutagenic primers needed to synthesize the desired gene constructs by PCR, and for physically cloning them into selected vectors by the most popular subcloning strategies. We present a complete description of Gene Composer functionality, and an efficient PCR-based synthetic gene assembly procedure with mis-match specific endonuclease

  20. Gene Composer: database software for protein construct design, codon engineering, and gene synthesis

    Directory of Open Access Journals (Sweden)

    Mixon Mark

    2009-04-01

    Full Text Available Abstract Background To improve efficiency in high throughput protein structure determination, we have developed a database software package, Gene Composer, which facilitates the information-rich design of protein constructs and their codon engineered synthetic gene sequences. With its modular workflow design and numerous graphical user interfaces, Gene Composer enables researchers to perform all common bio-informatics steps used in modern structure guided protein engineering and synthetic gene engineering. Results An interactive Alignment Viewer allows the researcher to simultaneously visualize sequence conservation in the context of known protein secondary structure, ligand contacts, water contacts, crystal contacts, B-factors, solvent accessible area, residue property type and several other useful property views. The Construct Design Module enables the facile design of novel protein constructs with altered N- and C-termini, internal insertions or deletions, point mutations, and desired affinity tags. The modifications can be combined and permuted into multiple protein constructs, and then virtually cloned in silico into defined expression vectors. The Gene Design Module uses a protein-to-gene algorithm that automates the back-translation of a protein amino acid sequence into a codon engineered nucleic acid gene sequence according to a selected codon usage table with minimal codon usage threshold, defined G:C% content, and desired sequence features achieved through synonymous codon selection that is optimized for the intended expression system. The gene-to-oligo algorithm of the Gene Design Module plans out all of the required overlapping oligonucleotides and mutagenic primers needed to synthesize the desired gene constructs by PCR, and for physically cloning them into selected vectors by the most popular subcloning strategies. Conclusion We present a complete description of Gene Composer functionality, and an efficient PCR-based synthetic gene

  1. Using Evolution to Guide Protein Engineering: The Devil IS in the Details.

    Science.gov (United States)

    Swint-Kruse, Liskin

    2016-07-12

    For decades, protein engineers have endeavored to reengineer existing proteins for novel applications. Overall, protein folds and gross functions can be readily transferred from one protein to another by transplanting large blocks of sequence (i.e., domain recombination). However, predictably fine-tuning function (e.g., by adjusting ligand affinity, specificity, catalysis, and/or allosteric regulation) remains a challenge. One approach has been to use the sequences of protein families to identify amino acid positions that change during the evolution of functional variation. The rationale is that these nonconserved positions could be mutated to predictably fine-tune function. Evolutionary approaches to protein design have had some success, but the engineered proteins seldom replicate the functional performances of natural proteins. This Biophysical Perspective reviews several complexities that have been revealed by evolutionary and experimental studies of protein function. These include 1) challenges in defining computational and biological thresholds that define important amino acids; 2) the co-occurrence of many different patterns of amino acid changes in evolutionary data; 3) difficulties in mapping the patterns of amino acid changes to discrete functional parameters; 4) the nonconventional mutational outcomes that occur for a particular group of functionally important, nonconserved positions; 5) epistasis (nonadditivity) among multiple mutations; and 6) the fact that a large fraction of a protein's amino acids contribute to its overall function. To overcome these challenges, new goals are identified for future studies. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  2. Bacterial autoinducer-2 detection via an engineered quorum sensing protein.

    Science.gov (United States)

    Raut, Nilesh; Joel, Smita; Pasini, Patrizia; Daunert, Sylvia

    2015-03-03

    Autoinducer-2 (AI-2) is a Quorum Sensing (QS) molecule utilized by bacteria in interspecies communication. More recently, it is identified to be vital in regulating QS pathways in a number of human and foodborne pathogens. Methods to detect AI-2 in a rapid and highly sensitive manner can help in the early detection of bacterial infections. Herein, we describe a rapid, selective, and highly sensitive protein based biosensing system employing the Fluorescence Resonance Energy Transfer (FRET) between a protein fusion LuxP-EGFP and 7-diethylamino-3-[N-(2-maleimidoethyl)carbamoyl]coumarin (MDCC). The developed biosensing system, which can detect AI-2 at subnanomolar levels, was successfully applied to detect AI-2 in clinical samples such as saliva and blood serum.

  3. Protein Fibrillar Hydrogels for three-Dimensional Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Hui Yan

    2009-01-01

    Full Text Available Protein self-assembly into highly ordered fibrillar aggregates has attracted increasing attention over recent years, due primarily to its association with disease states such as Alzheimer's. More recently, however, research has focused on understanding the generic behavior of protein self-assembly where fibrillation is typically induced under harsh conditions of low pH and/or high temperature. Moreover the inherent properties of these fibrils, including their nanoscale dimension, environmental responsiveness, and biological compatibility, are attracting substantial interest for exploiting these fibrils for the creation of new materials. Here we will show how protein fibrils can be formed under physiological conditions and their subsequent gelation driven using the ionic strength of cell culture media while simultaneously incorporating cells homogeneously throughout the gel network. The fibrillar and elastic nature of the gel have been confirmed using cryo-transmission electron microscopy and oscillatory rheology, respectively; while cell culture work shows that our hydrogels promote cell spreading, attachment, and proliferation in three dimensions.

  4. Mitogen-activated protein kinase-regulated AZI1 – an attractive candidate for genetic engineering

    Science.gov (United States)

    Pitzschke, Andrea; Datta, Sneha; Persak, Helene

    2014-01-01

    Mitogen-activated protein kinases and their targets have been in the limelight of plant stress research. Signaling pathways mediating the responses to multiple stresses deserve particular attention. In a recent study, we reported AZI1, a member of the lipid transfer protein family, to play a role in MPK3-mediated responses to salt stress in Arabidopsis thaliana. MPK3 controls AZI1 at the transcriptional and posttranslational level. The AZI1 protein has several properties that make it very attractive for genetic engineering. A model of multi-level control of AZI1 by MPK3 is proposed, and strategies toward optimizing AZI1 protein properties are briefly discussed. PMID:24518841

  5. Mitogen-activated protein kinase-regulated AZI1 - an attractive candidate for genetic engineering.

    Science.gov (United States)

    Pitzschke, Andrea; Datta, Sneha; Persak, Helene

    2014-01-01

    Mitogen-activated protein kinases and their targets have been in the limelight of plant stress research. Signaling pathways mediating the responses to multiple stresses deserve particular attention. In a recent study, we reported AZI1, a member of the lipid transfer protein family, to play a role in MPK3-mediated responses to salt stress in Arabidopsis thaliana. MPK3 controls AZI1 at the transcriptional and posttranslational level. The AZI1 protein has several properties that make it very attractive for genetic engineering. A model of multi-level control of AZI1 by MPK3 is proposed, and strategies toward optimizing AZI1 protein properties are briefly discussed.

  6. Formation of the chaperonin complex studied by 2D NMR spectroscopy

    Science.gov (United States)

    Takenaka, Toshio; Nakamura, Takashi; Yanaka, Saeko; Yagi-Utsumi, Maho; Chandak, Mahesh S.; Takahashi, Kazunobu; Paul, Subhankar; Makabe, Koki; Arai, Munehito; Kato, Koichi

    2017-01-01

    We studied the interaction between GroES and a single-ring mutant (SR1) of GroEL by the NMR titration of 15N-labeled GroES with SR1 at three different temperatures (20, 25 and 30°C) in the presence of 3 mM ADP in 100 mM KCl and 10 mM MgCl2 at pH 7.5. We used SR1 instead of wild-type double-ring GroEL to precisely control the stoichiometry of the GroES binding to be 1:1 ([SR1]:[GroES]). Native heptameric GroES was very flexible, showing well resolved cross peaks of the residues in a mobile loop segment (residue 17–34) and at the top of a roof hairpin (Asn51) in the heteronuclear single quantum coherence spectra. The binding of SR1 to GroES caused the cross peaks to disappear simultaneously, and hence it occurred in a single-step cooperative manner with significant immobilization of the whole GroES structure. The binding was thus entropic with a positive entropy change (219 J/mol/K) and a positive enthalpy change (35 kJ/mol), and the binding constant was estimated at 1.9×105 M−1 at 25°C. The NMR titration in 3 mM ATP also indicated that the binding constant between GroES and SR1 increased more than tenfold as compared with the binding constant in 3 mM ADP. These results will be discussed in relation to the structure and mechanisms of the chaperonin GroEL/GroES complex. PMID:29059240

  7. Formation of the chaperonin complex studied by 2D NMR spectroscopy.

    Directory of Open Access Journals (Sweden)

    Toshio Takenaka

    Full Text Available We studied the interaction between GroES and a single-ring mutant (SR1 of GroEL by the NMR titration of 15N-labeled GroES with SR1 at three different temperatures (20, 25 and 30°C in the presence of 3 mM ADP in 100 mM KCl and 10 mM MgCl2 at pH 7.5. We used SR1 instead of wild-type double-ring GroEL to precisely control the stoichiometry of the GroES binding to be 1:1 ([SR1]:[GroES]. Native heptameric GroES was very flexible, showing well resolved cross peaks of the residues in a mobile loop segment (residue 17-34 and at the top of a roof hairpin (Asn51 in the heteronuclear single quantum coherence spectra. The binding of SR1 to GroES caused the cross peaks to disappear simultaneously, and hence it occurred in a single-step cooperative manner with significant immobilization of the whole GroES structure. The binding was thus entropic with a positive entropy change (219 J/mol/K and a positive enthalpy change (35 kJ/mol, and the binding constant was estimated at 1.9×105 M-1 at 25°C. The NMR titration in 3 mM ATP also indicated that the binding constant between GroES and SR1 increased more than tenfold as compared with the binding constant in 3 mM ADP. These results will be discussed in relation to the structure and mechanisms of the chaperonin GroEL/GroES complex.

  8. Engineering a periplasmic binding protein for amino acid sensors with improved binding properties.

    Science.gov (United States)

    Ko, Wooseok; Kim, Sanggil; Lee, Hyun Soo

    2017-10-25

    Periplasmic binding proteins (PBPs) are members of a widely distributed protein superfamily found in bacteria and archaea, and are involved in the cellular uptake of solutes. In this report, a leucine-binding PBP was engineered to detect l-Leu based on a fluorescence resonance energy transfer (FRET) change upon ligand binding. A fluorescent unnatural amino acid, l-(7-hydroxycoumarin-4-yl)ethylglycine (CouA), was genetically incorporated into the protein as a FRET donor, and a yellow fluorescent protein (YFP) was fused with its N-terminus as a FRET acceptor. When CouA was incorporated into position 178, the sensor protein showed a 2.5-fold increase in the FRET ratio. Protein engineering significantly improved its substrate specificity, showing minimal changes in the FRET ratio with the other 19 natural amino acids and d-Leu. Further modification increased the sensitivity of the sensor protein (14-fold) towards l-Leu, and it recognized l-Met as well with moderate binding affinity. Selected mutant sensors were used to measure concentrations of l-Leu in a biological sample (fetal bovine serum) and to determine the optical purity of Leu and Met. This FRET-based sensor design strategy allowed us to easily manipulate the natural receptor to improve its binding affinity and specificity and to recognize other natural molecules, which are not recognized by the wild-type receptor. The design strategy can be applied to other natural receptors, enabling engineering receptors that sense biochemically interesting molecules.

  9. Chitosan based nanoparticles as a sustained protein release carrier for tissue engineering applications

    Science.gov (United States)

    Hou, Yaping; Hu, Junli; Park, Hyejin; Lee, Min

    2012-01-01

    Chitosan/tripolyphosphate/chondroitin sulfate (Chi/TPP/CS) nanoparticles were prepared by an ionic gelation method to obtain a controlled release of proteins. Using Nel-like molecule-1 (Nell-1), a novel osteogenic protein, as a model protein, it was demonstrated that adjusting the composition of the particles modulated the protein association and release kinetics of incorporated proteins. Increasing the amounts of chitosan crosslinking agents, TPP and CS, in the particles achieved sustained protein release. An increase in crosslinking density decreased degradation rates of the particles. Furthermore, the bioactivity of the protein was preserved during the encapsulating procedure into the particles. To demonstrate the feasibility of Chi/TPP/CS nanoparticles as sustained release carriers for tissue engineering scaffold applications, protein-loaded nanoparticles were successfully incorporated into collagen hydrogels or prefabricated porous poly (lactide-co-glycolide) (PLGA) scaffolds without obstructing the integrity of the hydrogels or porous structure of the scaffolds. Thus, we expect that these particles have a potential for efficient protein carriers in tissue engineering applications, and will be further evaluated in vivo. PMID:22275184

  10. Chitosan-based nanoparticles as a sustained protein release carrier for tissue engineering applications.

    Science.gov (United States)

    Hou, Yaping; Hu, Junli; Park, Hyejin; Lee, Min

    2012-04-01

    Chitosan/tripolyphosphate/chondroitin sulfate (Chi/TPP/CS) nanoparticles were prepared by an ionic gelation method to obtain a controlled release of proteins. Using Nel-like molecule-1 (Nell-1), a novel osteogenic protein, as a model protein, it was demonstrated that adjusting the composition of the particles modulated the protein association and release kinetics of incorporated proteins. Increasing the amounts of Chi crosslinking agents, TPP and CS, in the particles achieved sustained protein release. An increase in crosslinking density decreased degradation rates of the particles. Furthermore, the bioactivity of the protein was preserved during the encapsulating procedure into the particles. To demonstrate the feasibility of Chi/TPP/CS nanoparticles as sustained release carriers for tissue engineering scaffold applications, protein-loaded nanoparticles were successfully incorporated into collagen hydrogels or prefabricated porous poly(lactide-co-glycolide) (PLGA) scaffolds without obstructing the integrity of the hydrogels or porous structure of the scaffolds. Thus, we expect that these particles have a potential for efficient protein carriers in tissue engineering applications, and will be further evaluated in vivo. Copyright © 2012 Wiley Periodicals, Inc.

  11. Efficient expression of SRK intracellular domain by a modeling-based protein engineering.

    Science.gov (United States)

    Murase, Kohji; Hirano, Yoshinori; Takayama, Seiji; Hakoshima, Toshio

    2017-03-01

    S-locus protein kinase (SRK) is a receptor kinase that plays a critical role in self-recognition in the Brassicaceae self-incompatibility (SI) response. SRK is activated by binding of its ligand S-locus protein 11 (SP11) and subsequently induced phosphorylation of the intracellular kinase domain. However, a detailed activation mechanism of SRK is still largely unknown because of the difficulty in stably expressing SRK recombinant proteins. Here, we performed modeling-based protein engineering of the SRK kinase domain for stable expression in Escherichia coli. The engineered SRK intracellular domain was expressed about 54-fold higher production than wild type SRK, without loss of the kinase activity, suggesting it could be useful for further biochemical and structural studies. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Controlling Topological Entanglement in Engineered Protein Hydrogels with a Variety of Thiol Coupling Chemistries

    Directory of Open Access Journals (Sweden)

    Shengchang eTang

    2014-05-01

    Full Text Available Topological entanglements between polymer chains are achieved in associating protein hydrogels through the synthesis of high molecular weight proteins via chain extension using a variety of thiol coupling chemistries, including disulfide formation, thiol-maleimide, thiol-bromomaleimide and thiol-ene. Coupling of cysteines via disulfide formation results in the most pronounced entanglement effect in hydrogels, while other chemistries provide versatile means of changing the extent of entanglement, achieving faster chain extension, and providing a facile method of controlling the network hierarchy and incorporating stimuli responsivities. The addition of trifunctional coupling agents causes incomplete crosslinking and introduces branching architecture to the protein molecules. The high-frequency plateau modulus and the entanglement plateau modulus can be tuned by changing the ratio of difunctional chain extender to the trifunctional branching unit. Therefore, these chain extension reactions show promise in delicately controlling the relaxation and mechanical properties of engineered protein hydrogels in ways that complement their design through genetic engineering.

  13. High-Level Production of Heterologous Protein by Engineered Yeasts Grown in Cottage Cheese Whey

    Science.gov (United States)

    Maullu, Carlo; Lampis, Giorgio; Desogus, Alessandra; Ingianni, Angela; Rossolini, Gian Maria; Pompei, Raffaello

    1999-01-01

    Cottage cheese whey is a cheese industry by-product still rich in proteins and lactose. Its recycling is seldom cost-effective. In this work we show that the lactose-utilizing yeast Kluyveromyces lactis, engineered for production of recombinant human lysozyme, can be grown in cottage cheese whey, resulting in high-level production of the heterologous protein (125 μg/ml). PMID:10347071

  14. High-Level Production of Heterologous Protein by Engineered Yeasts Grown in Cottage Cheese Whey

    OpenAIRE

    Maullu, Carlo; Lampis, Giorgio; Desogus, Alessandra; Ingianni, Angela; Rossolini, Gian Maria; Pompei, Raffaello

    1999-01-01

    Cottage cheese whey is a cheese industry by-product still rich in proteins and lactose. Its recycling is seldom cost-effective. In this work we show that the lactose-utilizing yeast Kluyveromyces lactis, engineered for production of recombinant human lysozyme, can be grown in cottage cheese whey, resulting in high-level production of the heterologous protein (125 μg/ml).

  15. Protein Corona Influences Cell-Biomaterial Interactions in Nanostructured Tissue Engineering Scaffolds.

    Science.gov (United States)

    Serpooshan, Vahid; Mahmoudi, Morteza; Zhao, Mingming; Wei, Ke; Sivanesan, Senthilkumar; Motamedchaboki, Khatereh; Malkovskiy, Andrey V; Gladstone, Andrew B; Cohen, Jeffrey E; Yang, Phillip C; Rajadas, Jayakumar; Bernstein, Daniel; Woo, Y Joseph; Ruiz-Lozano, Pilar

    2015-07-22

    Biomaterials are extensively used to restore damaged tissues, in the forms of implants (e.g. tissue engineered scaffolds) or biomedical devices (e.g. pacemakers). Once in contact with the physiological environment, nanostructured biomaterials undergo modifications as a result of endogenous proteins binding to their surface. The formation of this macromolecular coating complex, known as 'protein corona', onto the surface of nanoparticles and its effect on cell-particle interactions are currently under intense investigation. In striking contrast, protein corona constructs within nanostructured porous tissue engineering scaffolds remain poorly characterized. As organismal systems are highly dynamic, it is conceivable that the formation of distinct protein corona on implanted scaffolds might itself modulate cell-extracellular matrix interactions. Here, we report that corona complexes formed onto the fibrils of engineered collagen scaffolds display specific, distinct, and reproducible compositions that are a signature of the tissue microenvironment as well as being indicative of the subject's health condition. Protein corona formed on collagen matrices modulated cellular secretome in a context-specific manner ex-vivo , demonstrating their role in regulating scaffold-cellular interactions. Together, these findings underscore the importance of custom-designing personalized nanostructured biomaterials, according to the biological milieu and disease state. We propose the use of protein corona as in situ biosensor of temporal and local biomarkers.

  16. Protein Corona Influences Cell–Biomaterial Interactions in Nanostructured Tissue Engineering Scaffolds

    Science.gov (United States)

    Zhao, Mingming; Wei, Ke; Sivanesan, Senthilkumar; Motamedchaboki, Khatereh; Malkovskiy, Andrey V.; Gladstone, Andrew B.; Cohen, Jeffrey E.; Yang, Phillip C.; Rajadas, Jayakumar; Bernstein, Daniel; Woo, Y. Joseph; Ruiz-Lozano, Pilar

    2016-01-01

    Biomaterials are extensively used to restore damaged tissues, in the forms of implants (e.g. tissue engineered scaffolds) or biomedical devices (e.g. pacemakers). Once in contact with the physiological environment, nanostructured biomaterials undergo modifications as a result of endogenous proteins binding to their surface. The formation of this macromolecular coating complex, known as ‘protein corona’, onto the surface of nanoparticles and its effect on cell-particle interactions are currently under intense investigation. In striking contrast, protein corona constructs within nanostructured porous tissue engineering scaffolds remain poorly characterized. As organismal systems are highly dynamic, it is conceivable that the formation of distinct protein corona on implanted scaffolds might itself modulate cell-extracellular matrix interactions. Here, we report that corona complexes formed onto the fibrils of engineered collagen scaffolds display specific, distinct, and reproducible compositions that are a signature of the tissue microenvironment as well as being indicative of the subject's health condition. Protein corona formed on collagen matrices modulated cellular secretome in a context-specific manner ex-vivo, demonstrating their role in regulating scaffold-cellular interactions. Together, these findings underscore the importance of custom-designing personalized nanostructured biomaterials, according to the biological milieu and disease state. We propose the use of protein corona as in situ biosensor of temporal and local biomarkers. PMID:27516731

  17. Exosome engineering for efficient intracellular delivery of soluble proteins using optically reversible protein-protein interaction module.

    Science.gov (United States)

    Yim, Nambin; Ryu, Seung-Wook; Choi, Kyungsun; Lee, Kwang Ryeol; Lee, Seunghee; Choi, Hojun; Kim, Jeongjin; Shaker, Mohammed R; Sun, Woong; Park, Ji-Ho; Kim, Daesoo; Heo, Won Do; Choi, Chulhee

    2016-07-22

    Nanoparticle-mediated delivery of functional macromolecules is a promising method for treating a variety of human diseases. Among nanoparticles, cell-derived exosomes have recently been highlighted as a new therapeutic strategy for the in vivo delivery of nucleotides and chemical drugs. Here we describe a new tool for intracellular delivery of target proteins, named 'exosomes for protein loading via optically reversible protein-protein interactions' (EXPLORs). By integrating a reversible protein-protein interaction module controlled by blue light with the endogenous process of exosome biogenesis, we are able to successfully load cargo proteins into newly generated exosomes. Treatment with protein-loaded EXPLORs is shown to significantly increase intracellular levels of cargo proteins and their function in recipient cells in vitro and in vivo. These results clearly indicate the potential of EXPLORs as a mechanism for the efficient intracellular transfer of protein-based therapeutics into recipient cells and tissues.

  18. Structure- and sequence-analysis inspired engineering of proteins for enhanced thermostability

    NARCIS (Netherlands)

    Wijma, Hein J.; Floor, Robert J.; Janssen, Dick B.

    Protein engineering strategies for increasing stability can be improved by replacing random mutagenesis and high-throughput screening by approaches that include bioinformatics and computational design. Mutations can be focused on regions in the structure that are most flexible and involved in the

  19. Developing Progranulin-Loaded Protein Engineered Hydrogel for Post-Traumatic Osteoarthritis

    Science.gov (United States)

    2017-09-01

    at 4 °C and 4000 rpm for 25 minutes. The cells were re-suspended in lysis buffer (50 mM sodium phosphate dibasic pH 8, 6 M urea, and 20 mM...which may prove to be unique in assemblies from other protein engineered systems in the biomaterials field. What was the impact on other

  20. Development of Genome Engineering Tools from Plant-Specific PPR Proteins Using Animal Cultured Cells.

    Science.gov (United States)

    Kobayashi, Takehito; Yagi, Yusuke; Nakamura, Takahiro

    2016-01-01

    The pentatricopeptide repeat (PPR) motif is a sequence-specific RNA/DNA-binding module. Elucidation of the RNA/DNA recognition mechanism has enabled engineering of PPR motifs as new RNA/DNA manipulation tools in living cells, including for genome editing. However, the biochemical characteristics of PPR proteins remain unknown, mostly due to the instability and/or unfolding propensities of PPR proteins in heterologous expression systems such as bacteria and yeast. To overcome this issue, we constructed reporter systems using animal cultured cells. The cell-based system has highly attractive features for PPR engineering: robust eukaryotic gene expression; availability of various vectors, reagents, and antibodies; highly efficient DNA delivery ratio (>80 %); and rapid, high-throughput data production. In this chapter, we introduce an example of such reporter systems: a PPR-based sequence-specific translational activation system. The cell-based reporter system can be applied to characterize plant genes of interested and to PPR engineering.

  1. Pathway and protein engineering approaches to produce novel and commodity small molecules.

    Science.gov (United States)

    Bhan, Namita; Xu, Peng; Koffas, Mattheos A G

    2013-12-01

    Nature has provided us the basis of designing the most integrated and efficacious production platforms. Cell factories via their millions of years of evolution have nearly perfected each of their production systems. We have been trying to imitate, utilize and tweak this system to our advantage by using slightly overlapping and greatly interdependent approaches such as metabolic engineering and systems biology to make nature work for us in an efficient and robust way, without producing toxic waste and/or unnecessary side products. Systems biology, metabolic engineering and 'omics' technologies have paved the way for protein and pathway engineering. To this end we will talk about the recent advances in production of novel pharmaceutical and commodity small molecules by designing novel proteins and pathways. Copyright © 2013 Elsevier Ltd. All rights reserved.

  2. Engineering Archeal Surrogate Systems for the Development of Protein-Protein Interaction Inhibitors against Human RAD51.

    Science.gov (United States)

    Moschetti, Tommaso; Sharpe, Timothy; Fischer, Gerhard; Marsh, May E; Ng, Hong Kin; Morgan, Matthew; Scott, Duncan E; Blundell, Tom L; R Venkitaraman, Ashok; Skidmore, John; Abell, Chris; Hyvönen, Marko

    2016-11-20

    Protein-protein interactions (PPIs) are increasingly important targets for drug discovery. Efficient fragment-based drug discovery approaches to tackle PPIs are often stymied by difficulties in the production of stable, unliganded target proteins. Here, we report an approach that exploits protein engineering to "humanise" thermophilic archeal surrogate proteins as targets for small-molecule inhibitor discovery and to exemplify this approach in the development of inhibitors against the PPI between the recombinase RAD51 and tumour suppressor BRCA2. As human RAD51 has proved impossible to produce in a form that is compatible with the requirements of fragment-based drug discovery, we have developed a surrogate protein system using RadA from Pyrococcus furiosus. Using a monomerised RadA as our starting point, we have adopted two parallel and mutually instructive approaches to mimic the human enzyme: firstly by mutating RadA to increase sequence identity with RAD51 in the BRC repeat binding sites, and secondly by generating a chimeric archaeal human protein. Both approaches generate proteins that interact with a fourth BRC repeat with affinity and stoichiometry comparable to human RAD51. Stepwise humanisation has also allowed us to elucidate the determinants of RAD51 binding to BRC repeats and the contributions of key interacting residues to this interaction. These surrogate proteins have enabled the development of biochemical and biophysical assays in our ongoing fragment-based small-molecule inhibitor programme and they have allowed us to determine hundreds of liganded structures in support of our structure-guided design process, demonstrating the feasibility and advantages of using archeal surrogates to overcome difficulties in handling human proteins. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  3. Engineering an FMN-based iLOV protein for the detection of arsenic ions.

    Science.gov (United States)

    Ravikumar, Yuvaraj; Nadarajan, Saravanan Prabhu; Lee, Chong-Soon; Yun, Hyungdon

    2017-05-15

    Over the past few decades, genetically encoded fluorescent proteins have been widely used as efficient probes to explore and investigate the roles of metal ions in biological processes. The discovery of small FMN-based fluorescent proteins, such as iLOV and FbFP, has enabled researchers to exploit these fluorescent reporter proteins for metal-sensing applications. In this study, we report the inherent binding properties of iLOV towards arsenic ions. The fluorescence quenching of iLOV was linearly related to the concentration of arsenic ions, and engineered proteins showed better sensitivity than the wild-type protein. Engineering key residues around the chromophore converted the iLOV protein into a highly sensitive sensor for As3+ ions. iLOVN468S exhibited an improved binding affinity with a dissociation constant of 1.5 μM. Furthermore, the circular dichroism spectra indicated that the fluorescence quenching mechanism might be related to arsenic-protein complex formation. Thus, the reagentless sensing of arsenic can potentially be exploited to determine intracellular or environmental arsenic using a genetically encoded biosensing approach. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Effects of protein engineering and rational mutagenesis on crystal lattice of single chain antibody fragments.

    Science.gov (United States)

    Kalyoncu, Sibel; Hyun, Jeongmin; Pai, Jennifer C; Johnson, Jennifer L; Entzminger, Kevin; Jain, Avni; Heaner, David P; Morales, Ivan A; Truskett, Thomas M; Maynard, Jennifer A; Lieberman, Raquel L

    2014-09-01

    Protein crystallization is dependent upon, and sensitive to, the intermolecular contacts that assist in ordering proteins into a three-dimensional lattice. Here we used protein engineering and mutagenesis to affect the crystallization of single chain antibody fragments (scFvs) that recognize the EE epitope (EYMPME) with high affinity. These hypercrystallizable scFvs are under development to assist difficult proteins, such as membrane proteins, in forming crystals, by acting as crystallization chaperones. Guided by analyses of intermolecular crystal lattice contacts, two second-generation anti-EE scFvs were produced, which bind to proteins with installed EE tags. Surprisingly, although noncomplementarity determining region (CDR) lattice residues from the parent scFv framework remained unchanged through the processes of protein engineering and rational design, crystal lattices of the derivative scFvs differ. Comparison of energy calculations and the experimentally-determined lattice interactions for this basis set provides insight into the complexity of the forces driving crystal lattice choice and demonstrates the availability of multiple well-ordered surface features in our scFvs capable of forming versatile crystal contacts. © 2014 Wiley Periodicals, Inc.

  5. Cyclotides as grafting frameworks for protein engineering and drug design applications.

    Science.gov (United States)

    Poth, Aaron G; Chan, Lai Y; Craik, David J

    2013-09-01

    Cyclotides are a family of naturally occurring backbone-cyclized macrocyclic mini-proteins from plants that have a knotted trio of intramolecular disulfide bonds. Their structural features imbue cyclotides with extraordinary stability against degradation at elevated temperatures or in the presence of proteolytic enzymes. The plasticity of their intracysteine loop sequences is exemplified by the more than 250 natural cyclotides sequenced to date, and this tolerance to sequence variation, along with their diverse bioactivities, underpins the suitability of the cyclic cystine knot motif as a valuable drug design scaffold and research tool for protein engineering studies. Here, we review the recent literature on applications of cyclotides for the stabilization of peptide epitopes and related protein engineering studies. Possible future directions in this field are also described. Copyright © 2013 Wiley Periodicals, Inc.

  6. Solid-state NMR and EPR Spectroscopy of Mn2+ -Substituted ATP-Fueled Protein Engines.

    Science.gov (United States)

    Wiegand, Thomas; Lacabanne, Denis; Keller, Katharina; Cadalbert, Riccardo; Lecoq, Lauriane; Yulikov, Maxim; Terradot, Laurent; Jeschke, Gunnar; Meier, Beat H; Böckmann, Anja

    2017-03-13

    Paramagnetic metal ions deliver structural information both in EPR and solid-state NMR experiments, offering a profitable synergetic approach to study bio-macromolecules. We demonstrate the spectral consequences of Mg2+ / Mn2+ substitution and the resulting information contents for two different ATP:Mg2+ -fueled protein engines, a DnaB helicase from Helicobacter pylori active in the bacterial replisome, and the ABC transporter BmrA, a bacterial efflux pump. We show that, while EPR spectra report on metal binding and provide information on the geometry of the metal centers in the proteins, paramagnetic relaxation enhancements identified in the NMR spectra can be used to localize residues at the binding site. Protein engines are ubiquitous and the methods described herein should be applicable in a broad context. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Labeling Cell Surface GPIs and GPI-Anchored Proteins through Metabolic Engineering with Artificial Inositol Derivatives.

    Science.gov (United States)

    Lu, Lili; Gao, Jian; Guo, Zhongwu

    2015-08-10

    Glycosylphosphatidylinositol (GPI) anchoring of proteins to the cell surface is important for various biological processes, but GPI-anchored proteins are difficult to study. An effective strategy was developed for the metabolic engineering of cell-surface GPIs and GPI-anchored proteins by using inositol derivatives carrying an azido group. The azide-labeled GPIs and GPI-anchored proteins were then tagged with biotin on live cells through a click reaction, which allows further elaboration with streptavidin-conjugated dyes or other molecules. The strategy can be used to label GPI-anchored proteins with various tags for biological studies. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Exploring the past and the future of protein evolution with ancestral sequence reconstruction: the 'retro' approach to protein engineering.

    Science.gov (United States)

    Gumulya, Yosephine; Gillam, Elizabeth M J

    2017-01-01

    A central goal in molecular evolution is to understand the ways in which genes and proteins evolve in response to changing environments. In the absence of intact DNA from fossils, ancestral sequence reconstruction (ASR) can be used to infer the evolutionary precursors of extant proteins. To date, ancestral proteins belonging to eubacteria, archaea, yeast and vertebrates have been inferred that have been hypothesized to date from between several million to over 3 billion years ago. ASR has yielded insights into the early history of life on Earth and the evolution of proteins and macromolecular complexes. Recently, however, ASR has developed from a tool for testing hypotheses about protein evolution to a useful means for designing novel proteins. The strength of this approach lies in the ability to infer ancestral sequences encoding proteins that have desirable properties compared with contemporary forms, particularly thermostability and broad substrate range, making them good starting points for laboratory evolution. Developments in technologies for DNA sequencing and synthesis and computational phylogenetic analysis have led to an escalation in the number of ancient proteins resurrected in the last decade and greatly facilitated the use of ASR in the burgeoning field of synthetic biology. However, the primary challenge of ASR remains in accurately inferring ancestral states, despite the uncertainty arising from evolutionary models, incomplete sequences and limited phylogenetic trees. This review will focus, firstly, on the use of ASR to uncover links between sequence and phenotype and, secondly, on the practical application of ASR in protein engineering. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  9. ECM protein nanofibers and nanostructures engineered using surface-initiated assembly.

    Science.gov (United States)

    Szymanski, John M; Jallerat, Quentin; Feinberg, Adam W

    2014-04-17

    The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer

  10. Orthogonal dual-modification of proteins for the engineering of multivalent protein scaffolds

    Directory of Open Access Journals (Sweden)

    Michaela Mühlberg

    2015-05-01

    Full Text Available To add new tools to the repertoire of protein-based multivalent scaffold design, we have developed a novel dual-labeling strategy for proteins that combines residue-specific incorporation of unnatural amino acids with chemical oxidative aldehyde formation at the N-terminus of a protein. Our approach relies on the selective introduction of two different functional moieties in a protein by mutually orthogonal copper-catalyzed azide–alkyne cycloaddition (CuAAC and oxime ligation. This method was applied to the conjugation of biotin and β-linked galactose residues to yield an enzymatically active thermophilic lipase, which revealed specific binding to Erythrina cristagalli lectin by SPR binding studies.

  11. PIPE: a protein-protein interaction prediction engine based on the re-occurring short polypeptide sequences between known interacting protein pairs

    Directory of Open Access Journals (Sweden)

    Greenblatt Jack

    2006-07-01

    Full Text Available Abstract Background Identification of protein interaction networks has received considerable attention in the post-genomic era. The currently available biochemical approaches used to detect protein-protein interactions are all time and labour intensive. Consequently there is a growing need for the development of computational tools that are capable of effectively identifying such interactions. Results Here we explain the development and implementation of a novel Protein-Protein Interaction Prediction Engine termed PIPE. This tool is capable of predicting protein-protein interactions for any target pair of the yeast Saccharomyces cerevisiae proteins from their primary structure and without the need for any additional information or predictions about the proteins. PIPE showed a sensitivity of 61% for detecting any yeast protein interaction with 89% specificity and an overall accuracy of 75%. This rate of success is comparable to those associated with the most commonly used biochemical techniques. Using PIPE, we identified a novel interaction between YGL227W (vid30 and YMR135C (gid8 yeast proteins. This lead us to the identification of a novel yeast complex that here we term vid30 complex (vid30c. The observed interaction was confirmed by tandem affinity purification (TAP tag, verifying the ability of PIPE to predict novel protein-protein interactions. We then used PIPE analysis to investigate the internal architecture of vid30c. It appeared from PIPE analysis that vid30c may consist of a core and a secondary component. Generation of yeast gene deletion strains combined with TAP tagging analysis indicated that the deletion of a member of the core component interfered with the formation of vid30c, however, deletion of a member of the secondary component had little effect (if any on the formation of vid30c. Also, PIPE can be used to analyse yeast proteins for which TAP tagging fails, thereby allowing us to predict protein interactions that are not

  12. A Real-Time All-Atom Structural Search Engine for Proteins

    OpenAIRE

    Gonzalez, Gabriel; Hannigan, Brett; DeGrado, William F.

    2014-01-01

    Protein designers use a wide variety of software tools for de novo design, yet their repertoire still lacks a fast and interactive all-atom search engine. To solve this, we have built the Suns program: a real-time, atomic search engine integrated into the PyMOL molecular visualization system. Users build atomic-level structural search queries within PyMOL and receive a stream of search results aligned to their query within a few seconds. This instant feedback cycle enables a new “designabilit...

  13. A real-time all-atom structural search engine for proteins.

    OpenAIRE

    Gabriel Gonzalez; Brett Hannigan; DeGrado, William F.

    2014-01-01

    Protein designers use a wide variety of software tools for de novo design, yet their repertoire still lacks a fast and interactive all-atom search engine. To solve this, we have built the Suns program: a real-time, atomic search engine integrated into the PyMOL molecular visualization system. Users build atomic-level structural search queries within PyMOL and receive a stream of search results aligned to their query within a few seconds. This instant feedback cycle enables a new "designabilit...

  14. Cytocompatible and water-stable camelina protein films for tissue engineering.

    Science.gov (United States)

    Zhao, Yi; Jiang, Qiuran; Xu, Helan; Reddy, Narendra; Xu, Lan; Yang, Yiqi

    2014-05-01

    In this research, films with compressive strength and aqueous stability were developed from camelina protein (CP) for tissue engineering. Protein based scaffolds have poor mechanical properties and aqueous stability and generally require chemical or physical modifications to make them applicable for medical applications. However, these modifications such as crosslinking could reduce biocompatibility and/or degradability of the scaffolds. Using proteins that are inherently water-stable could avoid modifications and provide scaffolds with the desired properties. CP with a high degree of disulfide cross-linkage has the potential to provide water-stable biomaterials, but it is difficult to dissolve CP and develop scaffolds. In this study, a new method of dissolving highly cross-linked proteins that results in limited hydrolysis and preserves the protein backbone was developed to produce water-stable films from CP without any modification. Only 12 % weight loss of camelina films was observed after 7 days in phosphate buffer saline (PBS) at 37°C. NIH 3T3 fibroblasts could attach and proliferate better on camelina films than on citric acid cross-linked collagen films. Therefore, CP films have the potential to be used for tissue engineering, and this extraction-dissolution method can be used for developing biomedical materials from various water-stable plant proteins. Copyright © 2013 Wiley Periodicals, Inc.

  15. Engineering Synthetic Proteins to Generate Ca2+Signals in Mammalian Cells.

    Science.gov (United States)

    Qudrat, Anam; Truong, Kevin

    2017-03-17

    The versatility of Ca 2+ signals allows it to regulate diverse cellular processes such as migration, apoptosis, motility and exocytosis. In some receptors (e.g., VEGFR2), Ca 2+ signals are generated upon binding their ligand(s) (e.g., VEGF-A). Here, we employed a design strategy to engineer proteins that generate a Ca 2+ signal upon binding various extracellular stimuli by creating fusions of protein domains that oligomerize to the transmembrane domain and the cytoplasmic tail of the VEGFR2. To test the strategy, we created chimeric proteins that generate Ca 2+ signals upon stimulation with various extracellular stimuli (e.g., rapamycin, EDTA or extracellular free Ca 2+ ). By coupling these chimeric proteins that generate Ca 2+ signals with proteins that respond to Ca 2+ signals, we rewired, for example, dynamic cellular blebbing to increases in extracellular free Ca 2+ . Thus, using this design strategy, it is possible to engineer proteins to generate a Ca 2+ signal to rewire a wide range of extracellular stimuli to a wide range of Ca 2+ -activated processes.

  16. Enzymatic Cross-Linking of Resilin-Based Proteins for Vascular Tissue Engineering Applications.

    Science.gov (United States)

    Kim, Yeji; Gill, Emily E; Liu, Julie C

    2016-08-08

    Protein-based biomaterials have received significant attention for tissue engineering applications. For example, resilin-based protein gels have been produced with different cross-linking chemistries for applications in cartilage, cardiovascular, and vocal fold engineering. In this study, we investigate an alternative cross-linking chemistry to form resilin-based protein hydrogels and demonstrate the versatility of the gels for investigating cell response to matrix stiffness. Specifically, transglutaminase was used to cross-link proteins and resulted in gel surfaces more suitable for long-term cell attachment compared to those formed by a Mannich-type condensation reaction. Since matrix stiffness is an important determinant in modulating cell response, we first tuned matrix stiffness by varying total protein concentration. Next, we observed that matrix stiffness modulated cell spreading and endothelial differentiation of human mesenchymal stem cells. In particular, our results show that cells differentiated on our matrices, which have a stiffness similar to subendothelial layers, had statistically equivalent endothelial function compared to cells differentiated on hard glass surfaces. Thus, our protein-based matrix system is a promising tool to provide substrates favorable for long-term cell attachment and better mimics the native subendothelial environment compared to conventional hard culture substrates.

  17. From Protein Engineering to Immobilization: Promising Strategies for the Upgrade of Industrial Enzymes

    Directory of Open Access Journals (Sweden)

    Raushan Kumar Singh

    2013-01-01

    Full Text Available Enzymes found in nature have been exploited in industry due to their inherent catalytic properties in complex chemical processes under mild experimental and environmental conditions. The desired industrial goal is often difficult to achieve using the native form of the enzyme. Recent developments in protein engineering have revolutionized the development of commercially available enzymes into better industrial catalysts. Protein engineering aims at modifying the sequence of a protein, and hence its structure, to create enzymes with improved functional properties such as stability, specific activity, inhibition by reaction products, and selectivity towards non-natural substrates. Soluble enzymes are often immobilized onto solid insoluble supports to be reused in continuous processes and to facilitate the economical recovery of the enzyme after the reaction without any significant loss to its biochemical properties. Immobilization confers considerable stability towards temperature variations and organic solvents. Multipoint and multisubunit covalent attachments of enzymes on appropriately functionalized supports via linkers provide rigidity to the immobilized enzyme structure, ultimately resulting in improved enzyme stability. Protein engineering and immobilization techniques are sequential and compatible approaches for the improvement of enzyme properties. The present review highlights and summarizes various studies that have aimed to improve the biochemical properties of industrially significant enzymes.

  18. A real-time all-atom structural search engine for proteins.

    Science.gov (United States)

    Gonzalez, Gabriel; Hannigan, Brett; DeGrado, William F

    2014-07-01

    Protein designers use a wide variety of software tools for de novo design, yet their repertoire still lacks a fast and interactive all-atom search engine. To solve this, we have built the Suns program: a real-time, atomic search engine integrated into the PyMOL molecular visualization system. Users build atomic-level structural search queries within PyMOL and receive a stream of search results aligned to their query within a few seconds. This instant feedback cycle enables a new "designability"-inspired approach to protein design where the designer searches for and interactively incorporates native-like fragments from proven protein structures. We demonstrate the use of Suns to interactively build protein motifs, tertiary interactions, and to identify scaffolds compatible with hot-spot residues. The official web site and installer are located at http://www.degradolab.org/suns/ and the source code is hosted at https://github.com/godotgildor/Suns (PyMOL plugin, BSD license), https://github.com/Gabriel439/suns-cmd (command line client, BSD license), and https://github.com/Gabriel439/suns-search (search engine server, GPLv2 license).

  19. From Protein Engineering to Immobilization: Promising Strategies for the Upgrade of Industrial Enzymes

    Science.gov (United States)

    Singh, Raushan Kumar; Tiwari, Manish Kumar; Singh, Ranjitha; Lee, Jung-Kul

    2013-01-01

    Enzymes found in nature have been exploited in industry due to their inherent catalytic properties in complex chemical processes under mild experimental and environmental conditions. The desired industrial goal is often difficult to achieve using the native form of the enzyme. Recent developments in protein engineering have revolutionized the development of commercially available enzymes into better industrial catalysts. Protein engineering aims at modifying the sequence of a protein, and hence its structure, to create enzymes with improved functional properties such as stability, specific activity, inhibition by reaction products, and selectivity towards non-natural substrates. Soluble enzymes are often immobilized onto solid insoluble supports to be reused in continuous processes and to facilitate the economical recovery of the enzyme after the reaction without any significant loss to its biochemical properties. Immobilization confers considerable stability towards temperature variations and organic solvents. Multipoint and multisubunit covalent attachments of enzymes on appropriately functionalized supports via linkers provide rigidity to the immobilized enzyme structure, ultimately resulting in improved enzyme stability. Protein engineering and immobilization techniques are sequential and compatible approaches for the improvement of enzyme properties. The present review highlights and summarizes various studies that have aimed to improve the biochemical properties of industrially significant enzymes. PMID:23306150

  20. From protein engineering to immobilization: promising strategies for the upgrade of industrial enzymes.

    Science.gov (United States)

    Singh, Raushan Kumar; Tiwari, Manish Kumar; Singh, Ranjitha; Lee, Jung-Kul

    2013-01-10

    Enzymes found in nature have been exploited in industry due to their inherent catalytic properties in complex chemical processes under mild experimental and environmental conditions. The desired industrial goal is often difficult to achieve using the native form of the enzyme. Recent developments in protein engineering have revolutionized the development of commercially available enzymes into better industrial catalysts. Protein engineering aims at modifying the sequence of a protein, and hence its structure, to create enzymes with improved functional properties such as stability, specific activity, inhibition by reaction products, and selectivity towards non-natural substrates. Soluble enzymes are often immobilized onto solid insoluble supports to be reused in continuous processes and to facilitate the economical recovery of the enzyme after the reaction without any significant loss to its biochemical properties. Immobilization confers considerable stability towards temperature variations and organic solvents. Multipoint and multisubunit covalent attachments of enzymes on appropriately functionalized supports via linkers provide rigidity to the immobilized enzyme structure, ultimately resulting in improved enzyme stability. Protein engineering and immobilization techniques are sequential and compatible approaches for the improvement of enzyme properties. The present review highlights and summarizes various studies that have aimed to improve the biochemical properties of industrially significant enzymes.

  1. Engineered Bacterial Metal-binding Proteins for Nanoscale Self-assembly and heavy Metal Tolerance

    Science.gov (United States)

    Hall Sedlak, Ruth Amanda

    Implementing biological principles in material synthesis and assembly is one way to expand our abilities to efficiently assemble nanoscale materials and devices. Specifically, recent advances in identifying peptides that bind inorganic materials with high affinity and specificity has spurred investigation of protein models for nanoscale inorganic assembly. This dissertation presents the results of my studies of several E. coli proteins engineered to bind inorganic materials through simple peptide motifs. I demonstrate that these proteins modulate the self-assembly of DNA-based nanostructures and can introduce heavy metal tolerance into metal-sensitive bacteria. Chapter 2 explores use of the engineered F plasmid DNA relaxase/helicase TraI for the self-assembly of complex DNA-protein-gold nanostructures. The full-length protein is engineered with a gold binding motif at an internal permissive site (TraI369GBP1-7x), while a truncated version of TraI is engineered with the same gold binding motif at the C-terminus (TraI361GBP1-7x). Both constructs bind gold nanoparticles while maintaining their DNA binding activity, and transmission electron microscopy reveals TraI369GBP1-7x utilizes its non-specific DNA binding activity to decorate single-stranded and double-stranded DNA with gold nanoparticles. The self assembly principles demonstrated in this work will be fundamental to constructing higher ordered hybrid nanostructures through DNA-protein-nanoparticle interactions. Chapter 3 studies the effects of expressing inorganic binding peptides within cells. I identified a silver binding peptide that, when fused to the periplasmic maltose binding protein, protects E. coli from silver toxicity in batch culture and reduces silver ions to silver nanoparticles within the bacterial periplasm. Engineered metal-ion tolerant microorganisms such as this E. coli could potentially be used in applications ranging from remediation to interrogation of biomolecule-metal interactions in vivo

  2. Cellular engineering for therapeutic protein production: product quality, host modification, and process improvement.

    Science.gov (United States)

    Wells, Evan; Robinson, Anne Skaja

    2017-01-01

    Recombinant proteins offer many therapeutic advantages unavailable in traditional small molecule drugs, but the need for cellular versus chemical synthesis complicates production. Avenues for producing therapeutic biologics are continuously expanding, and developments in biochemistry, cell biology, and bioengineering fuel new discoveries that promise safer, more efficient, and cheaper drugs for consumers. Numerous approaches to express recombinant proteins exist, but Escherichia coli, Saccharomyces cerevisiae, and mammalian systems (e.g. Chinese hamster ovary cells, CHO) are the most widely utilized. Improvements to production in these hosts have focused on novel expression cassettes, cell line modifications, engineering secretion pathways, and media design. Here, we describe recent developments for improving protein production in E. coli, S. cerevisiae, and CHO systems and compare recent advancements to previous knowledge in the field. With the expanding importance and prevalence of protein therapeutics, these improvements will serve as the framework for future discoveries. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. A directed approach for engineering conditional protein stability using biologically silent small molecules.

    Science.gov (United States)

    Maynard-Smith, Lystranne A; Chen, Ling-Chun; Banaszynski, Laura A; Ooi, A G Lisa; Wandless, Thomas J

    2007-08-24

    The ability to regulate the function of specific proteins using cell-permeable molecules can be a powerful method for interrogating biological systems. To bring this type of "chemical genetic" control to a wide range of proteins, we recently developed an experimental system in which the stability of a small protein domain expressed in mammalian cells depends on the presence of a high affinity ligand. This ligand-dependent stability is conferred to any fused partner protein. The FK506- and rapamycin-binding protein (FKBP12) has been the subject of extensive biophysical analyses, including both kinetic and thermodynamic studies of the wild-type protein as well as dozens of mutants. The goal of this study was to determine if the thermodynamic stabilities (DeltaDeltaG(U-F)) of various amino acid substitutions within a given protein are predictive for engineering additional ligand-dependent destabilizing domains. We used FKBP12 as a model system and found that in vitro thermodynamic stability correlates weakly with intracellular degradation rates of the mutants and that the ability of a given mutation to destabilize the protein is context-dependent. We evaluated several new FKBP12 ligands for their ability to stabilize these mutants and found that a cell-permeable molecule called Shield-1 is the most effective stabilizing ligand. We then performed an unbiased microarray analysis of NIH3T3 cells treated with various concentrations of Shield-1. These studies show that Shield-1 does not elicit appreciable cellular responses.

  4. Self-Assembly of Protein Monolayers Engineered for Improved Monoclonal Immunoglobulin G Binding

    Directory of Open Access Journals (Sweden)

    Jeremy H. Lakey

    2011-08-01

    Full Text Available Bacterial outer membrane proteins, along with a filling lipid molecule can be modified to form stable self-assembled monolayers on gold. The transmembrane domain of Escherichia coli outer membrane protein A has been engineered to create a scaffold protein to which functional motifs can be fused. In earlier work we described the assembly and structure of an antibody-binding array where the Z domain of Staphylococcus aureus protein A was fused to the scaffold protein. Whilst the binding of rabbit polyclonal immunoglobulin G (IgG to the array is very strong, mouse monoclonal IgG dissociates from the array easily. This is a problem since many immunodiagnostic tests rely upon the use of mouse monoclonal antibodies. Here we describe a strategy to develop an antibody-binding array that will bind mouse monoclonal IgG with lowered dissociation from the array. A novel protein consisting of the scaffold protein fused to two pairs of Z domains separated by a long flexible linker was manufactured. Using surface plasmon resonance the self-assembly of the new protein on gold and the improved binding of mouse monoclonal IgG were demonstrated.

  5. ProFET: Feature engineering captures high-level protein functions.

    Science.gov (United States)

    Ofer, Dan; Linial, Michal

    2015-11-01

    The amount of sequenced genomes and proteins is growing at an unprecedented pace. Unfortunately, manual curation and functional knowledge lag behind. Homologous inference often fails at labeling proteins with diverse functions and broad classes. Thus, identifying high-level protein functionality remains challenging. We hypothesize that a universal feature engineering approach can yield classification of high-level functions and unified properties when combined with machine learning approaches, without requiring external databases or alignment. In this study, we present a novel bioinformatics toolkit called ProFET (Protein Feature Engineering Toolkit). ProFET extracts hundreds of features covering the elementary biophysical and sequence derived attributes. Most features capture statistically informative patterns. In addition, different representations of sequences and the amino acids alphabet provide a compact, compressed set of features. The results from ProFET were incorporated in data analysis pipelines, implemented in python and adapted for multi-genome scale analysis. ProFET was applied on 17 established and novel protein benchmark datasets involving classification for a variety of binary and multi-class tasks. The results show state of the art performance. The extracted features' show excellent biological interpretability. The success of ProFET applies to a wide range of high-level functions such as subcellular localization, structural classes and proteins with unique functional properties (e.g. neuropeptide precursors, thermophilic and nucleic acid binding). ProFET allows easy, universal discovery of new target proteins, as well as understanding the features underlying different high-level protein functions. ProFET source code and the datasets used are freely available at https://github.com/ddofer/ProFET. michall@cc.huji.ac.il Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights

  6. Engineering Iron Responses in Mammalian Cells by Signal-Induced Protein Proximity.

    Science.gov (United States)

    Zeng, Guihua; Li, Huanqiu; Wei, Yongyi; Xuan, Weimin; Zhang, Roushu; Breden, Larisa E; Wang, Wei; Liang, Fu-Sen

    2017-06-16

    A new synthetic biology engineering strategy integrating chemical reactivity sensing and small molecule induced protein dimerization has been developed to generate artificial Fe(2+) signaling circuitry to control tailored cellular events in mammalian cells. The dual function probe ABA-FE18 (Fe(2+)-sensing and protein dimerization) derived from ABA was developed and used to control gene activation, signal transduction, and cytoskeletal remodeling in response to Fe(2+). This technology was utilized to design signal circuitry incorporating "AND" and "OR" biologic gates that enables mammalian cells to translate different combinations of Fe(2+) and H2O2 signals into predefined biological outputs.

  7. Chlorella species as hosts for genetic engineering and expression of heterologous proteins: Progress, challenge and perspective.

    Science.gov (United States)

    Yang, Bo; Liu, Jin; Jiang, Yue; Chen, Feng

    2016-10-01

    The species of Chlorella represent a highly specialized group of green microalgae that can produce high levels of protein. Many Chlorella strains can grow rapidly and achieve high cell density under controlled conditions and are thus considered to be promising protein sources. Many advances in the genetic engineering of Chlorella have occurred in recent years, with significant developments in successful expression of heterologous proteins for various applications. Nevertheless, a lot of obstacles remain to be addressed, and a sophisticated and stable Chlorella expression system has yet to emerge. This review provides a brief summary of current knowledge on Chlorella and an overview of recent progress in the genetic engineering of Chlorella, and highlights the advances in the development of a genetic toolbox of Chlorella for heterologous protein expression. Research directions to further exploit the Chlorella expression system with respect to both challenges and perspectives are also discussed. This paper serves as a comprehensive literature review for the Chlorella community and will provide valuable insights into future exploration of Chlorella as a promising host for heterologous protein expression. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Tissue-engineered cells producing complex recombinant proteins inhibit ovarian cancer in vivo

    OpenAIRE

    Stephen, Antonia E.; Masiakos, Peter T.; Segev, Dorry L.; Vacanti, Joseph P.; Donahoe, Patricia K.; MacLaughlin, David T.

    2001-01-01

    Techniques of tissue engineering and cell and molecular biology were used to create a biodegradable scaffold for transfected cells to produce complex proteins. Mullerian Inhibiting Substance (MIS) causes regression of Mullerian ducts in the mammalian embryo. MIS also causes regression in vitro of ovarian tumor cell lines and primary cells from ovarian carcinomas, which derive from Mullerian structures. In a strategy to circumvent the complicated purification protoc...

  9. Tissue-engineered human bioartificial muscles expressing a foreign recombinant protein for gene therapy

    Science.gov (United States)

    Powell, C.; Shansky, J.; Del Tatto, M.; Forman, D. E.; Hennessey, J.; Sullivan, K.; Zielinski, B. A.; Vandenburgh, H. H.

    1999-01-01

    Murine skeletal muscle cells transduced with foreign genes and tissue engineered in vitro into bioartificial muscles (BAMs) are capable of long-term delivery of soluble growth factors when implanted into syngeneic mice (Vandenburgh et al., 1996b). With the goal of developing a therapeutic cell-based protein delivery system for humans, similar genetic tissue-engineering techniques were designed for human skeletal muscle stem cells. Stem cell myoblasts were isolated, cloned, and expanded in vitro from biopsied healthy adult (mean age, 42 +/- 2 years), and elderly congestive heart failure patient (mean age, 76 +/- 1 years) skeletal muscle. Total cell yield varied widely between biopsies (50 to 672 per 100 mg of tissue, N = 10), but was not significantly different between the two patient groups. Percent myoblasts per biopsy (73 +/- 6%), number of myoblast doublings prior to senescence in vitro (37 +/- 2), and myoblast doubling time (27 +/- 1 hr) were also not significantly different between the two patient groups. Fusion kinetics of the myoblasts were similar for the two groups after 20-22 doublings (74 +/- 2% myoblast fusion) when the biopsy samples had been expanded to 1 to 2 billion muscle cells, a number acceptable for human gene therapy use. The myoblasts from the two groups could be equally transduced ex vivo with replication-deficient retroviral expression vectors to secrete 0.5 to 2 microg of a foreign protein (recombinant human growth hormone, rhGH)/10(6) cells/day, and tissue engineered into human BAMs containing parallel arrays of differentiated, postmitotic myofibers. This work suggests that autologous human skeletal myoblasts from a potential patient population can be isolated, genetically modified to secrete foreign proteins, and tissue engineered into implantable living protein secretory devices for therapeutic use.

  10. Interferences of Silica Nanoparticles in Green Fluorescent Protein Folding Processes.

    Science.gov (United States)

    Klein, Géraldine; Devineau, Stéphanie; Aude, Jean Christophe; Boulard, Yves; Pasquier, Hélène; Labarre, Jean; Pin, Serge; Renault, Jean Philippe

    2016-01-12

    We investigated the relationship between unfolded proteins, silica nanoparticles and chaperonin to determine whether unfolded proteins could stick to silica surfaces and how this process could impair heat shock protein activity. The HSP60 catalyzed green fluorescent protein (GFP) folding was used as a model system. The adsorption isotherms and adsorption kinetics of denatured GFP were measured, showing that denaturation increases GFP affinity for silica surfaces. This affinity is maintained even if the surfaces are covered by a protein corona and allows silica NPs to interfere directly with GFP folding by trapping it in its unstructured state. We determined also the adsorption isotherms of HSP60 and its chaperonin activity once adsorbed, showing that SiO2 NP can interfere also indirectly with protein folding through chaperonin trapping and inhibition. This inhibition is specifically efficient when NPs are covered first with a layer of unfolded proteins. These results highlight for the first time the antichaperonin activity of silica NPs and ask new questions about the toxicity of such misfolded proteins/nanoparticles assembly toward cells.

  11. A combinatorial histidine scanning library approach to engineer highly pH-dependent protein switches.

    Science.gov (United States)

    Murtaugh, Megan L; Fanning, Sean W; Sharma, Tressa M; Terry, Alexandra M; Horn, James R

    2011-09-01

    There is growing interest in the development of protein switches, which are proteins whose function, such as binding a target molecule, can be modulated through environmental triggers. Efforts to engineer highly pH sensitive protein-protein interactions typically rely on the rational introduction of ionizable groups in the protein interface. Such experiments are typically time intensive and often sacrifice the protein's affinity at the permissive pH. The underlying thermodynamics of proton-linkage dictate that the presence of multiple ionizable groups, which undergo a pK(a) change on protein binding, are necessary to result in highly pH-dependent binding. To test this hypothesis, a novel combinatorial histidine library was developed where every possible combination of histidine and wild-type residue is sampled throughout the interface of a model anti-RNase A single domain VHH antibody. Antibodies were coselected for high-affinity binding and pH-sensitivity using an in vitro, dual-function selection strategy. The resulting antibodies retained near wild-type affinity yet became highly sensitive to small decreases in pH, drastically decreasing their binding affinity, due to the incorporation of multiple histidine groups. Several trends were observed, such as histidine "hot-spots," which will help enhance the development of pH switch proteins as well as increase our understanding of the role of ionizable residues in protein interfaces. Overall, the combinatorial approach is rapid, general, and robust and should be capable of producing highly pH-sensitive protein affinity reagents for a number of different applications. Copyright © 2011 The Protein Society.

  12. Functional characterization of the α- and β-subunits of a group II chaperonin from Aeropyrum pernix K1.

    Science.gov (United States)

    Lee, Jin-Woo; Kim, Se Won; Kim, Jeong-Hwan; Jeon, Sung-Jong; Kwon, Hyun-Ju; Kim, Byung-Woo; Nam, Soo-Wan

    2013-06-28

    We isolated and functionally characterized the α- and β- subunits (ApCpnA and ApCpnB) of a chaperonin from Aeropyrum pernix K1. The constructed vectors pET3d- ApCpnA and pET21a-ApCpnB were transformed into E. coli Rosetta (DE3), BL21 (DE3), or CodonPlus (DE3) cells. The expression of ApCpnA (60.7 kDa) and ApCpnB (61.2 kDa) was confirmed by SDS-PAGE analysis. Recombinant ApCpnA and ApCpnB were purified by heat-shock treatment and anion-exchange chromatography. ApCpnA and ApCpnB were able to hydrolyze not only ATP, but also CTP, GTP, and UTP, albeit with different efficacies. Purified ApCpnA and ApCpnB showed the highest ATPase, CTPase, UTPase, and GTPase activities at 80°C. Furthermore, the addition of ApCpnA and ApCpnB effectively protected citrate synthase (CS) and alcohol dehydrogenase (ADH) from thermal aggregation and inactivation at 43°C and 50°C, respectively. In particular, the addition of ATP or CTP to ApCpnA and ApCpnB resulted in the most effective prevention of thermal aggregation and inactivation of CS and ADH. The ATPase activity of the two chaperonin subunits was dependent on the salt concentration. Among the ions we examined, potassium ions were the most effective at enhancing the ATP hydrolysis activity of ApCpnA and ApCpnB.

  13. Engineering split intein DnaE from Nostoc punctiforme for rapid protein purification.

    Science.gov (United States)

    Ramirez, Miguel; Valdes, Najla; Guan, Dongli; Chen, Zhilei

    2013-03-01

    We report the engineering of a DnaE intein able to catalyze rapid C-terminal cleavage in the absence of N-terminal cleavage. A single mutation in DnaE intein from Nostoc punctiforme PCC73102 (NpuDnaE), Asp118Gly, was introduced based on sequence alignment with a previously engineered C-terminal cleaving intein mini-MtuRecA. This mutation was able to both suppress N-terminal cleavage and significantly elevate C-terminal cleavage efficiency. Molecular modeling suggests that in NpuDnaE Asp118 forms a hydrogen bond with the penultimate Asn, preventing its spontaneous cyclization prior to N-terminal cleavage. Mutation of Asp118 to Gly essentially abolishes this restriction leading to subsequent C-terminal cleavage in the absence of N-terminal cleavage. The Gly118 NpuDnaE mutant exhibits rapid thio-dependent C-terminal cleavage kinetics with 80% completion within 3 h at room temperature. We used this newly engineered intein to develop both column-free and chromatography-based protein purification methods utilizing the elastin-like-polypeptide and chitin-binding protein as removable purification tags, respectively. We demonstrate rapid target protein purification to electrophoretic purity at yields up to 84 mg per liter of Escherichia coli culture.

  14. Overview on zein protein: a promising pharmaceutical excipient in drug delivery systems and tissue engineering.

    Science.gov (United States)

    Labib, Gihan

    2018-01-01

    Natural pharmaceutical excipients have been applied extensively in the past decades owing to their safety and biocompatibility. Zein, a natural protein of plant origin offers great benefit over other synthetic polymers used in controlled drug and biomedical delivery systems. It was used in a variety of medical fields including pharmaceutical and biomedical drug targeting, vaccine, tissue engineering, and gene delivery. Being biodegradable and biocompatible, the current review focuses on the history and the medical application of zein as an attractive still promising biopolymer. Areas covered: The current review gives a broadscope on zein as a still promising protein excipient in different fields. Zein- based drug and biomedical delivery systems are discussed with special focus on current and potential application in controlled drug delivery systems, and tissue engineering. Expert opinion: Zein as a protein of natural origin can still be considered a promising polymer in the field of drug delivery systems as well as in tissue engineering. Although different researchers spotted light on zein application in different industrial fields extensively, the feasibility of its use in the field of drug delivery replenished by investigators in recent years has not yet been fully approached.

  15. Heart research advances using database search engines, Human Protein Atlas and the Sydney Heart Bank.

    Science.gov (United States)

    Li, Amy; Estigoy, Colleen; Raftery, Mark; Cameron, Darryl; Odeberg, Jacob; Pontén, Fredrik; Lal, Sean; Dos Remedios, Cristobal G

    2013-10-01

    This Methodological Review is intended as a guide for research students who may have just discovered a human "novel" cardiac protein, but it may also help hard-pressed reviewers of journal submissions on a "novel" protein reported in an animal model of human heart failure. Whether you are an expert or not, you may know little or nothing about this particular protein of interest. In this review we provide a strategic guide on how to proceed. We ask: How do you discover what has been published (even in an abstract or research report) about this protein? Everyone knows how to undertake literature searches using PubMed and Medline but these are usually encyclopaedic, often producing long lists of papers, most of which are either irrelevant or only vaguely relevant to your query. Relatively few will be aware of more advanced search engines such as Google Scholar and even fewer will know about Quertle. Next, we provide a strategy for discovering if your "novel" protein is expressed in the normal, healthy human heart, and if it is, we show you how to investigate its subcellular location. This can usually be achieved by visiting the website "Human Protein Atlas" without doing a single experiment. Finally, we provide a pathway to discovering if your protein of interest changes its expression level with heart failure/disease or with ageing. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

  16. Arming Technology in Yeast—Novel Strategy for Whole-cell Biocatalyst and Protein Engineering

    Directory of Open Access Journals (Sweden)

    Mitsuyoshi Ueda

    2013-09-01

    Full Text Available Cell surface display of proteins/peptides, in contrast to the conventional intracellular expression, has many attractive features. This arming technology is especially effective when yeasts are used as a host, because eukaryotic modifications that are often required for functional use can be added to the surface-displayed proteins/peptides. A part of various cell wall or plasma membrane proteins can be genetically fused to the proteins/peptides of interest to be displayed. This technology, leading to the generation of so-called “arming technology”, can be employed for basic and applied research purposes. In this article, we describe various strategies for the construction of arming yeasts, and outline the diverse applications of this technology to industrial processes such as biofuel and chemical productions, pollutant removal, and health-related processes, including oral vaccines. In addition, arming technology is suitable for protein engineering and directed evolution through high-throughput screening that is made possible by the feature that proteins/peptides displayed on cell surface can be directly analyzed using intact cells without concentration and purification. Actually, novel proteins/peptides with improved or developed functions have been created, and development of diagnostic/therapeutic antibodies are likely to benefit from this powerful approach.

  17. Modular Integrated Secretory System Engineering in Pichia pastoris To Enhance G-Protein Coupled Receptor Expression.

    Science.gov (United States)

    Claes, Katrien; Vandewalle, Kristof; Laukens, Bram; Laeremans, Toon; Vosters, Olivier; Langer, Ingrid; Parmentier, Marc; Steyaert, Jan; Callewaert, Nico

    2016-10-21

    Membrane protein research is still hampered by the generally very low levels at which these proteins are naturally expressed, necessitating heterologous expression. Protein degradation, folding problems, and undesired post-translational modifications often occur, together resulting in low expression levels of heterogeneous protein products that are unsuitable for structural studies. We here demonstrate how the integration of multiple engineering modules in Pichia pastoris can be used to increase both the quality and the quantity of overexpressed integral membrane proteins, with the human CXCR4 G-protein coupled receptor as an example. The combination of reduced proteolysis, enhanced ER folding capacity, GlycoDelete-based N-Glycan trimming, and nanobody-based fold stabilization improved the expression of this GPCR in P. pastoris from a low expression level of a heterogeneously glycosylated, proteolyzed product to substantial quantities (2-3 mg/L shake flask culture) of a nonproteolyzed, homogeneously glycosylated proteoform. We expect that this set of tools will contribute to successful expression of more membrane proteins in a quantity and quality suitable for functional and structural studies.

  18. Engineered, highly reactive substrates of microbial transglutaminase enable protein labeling within various secondary structure elements.

    Science.gov (United States)

    Rachel, Natalie M; Quaglia, Daniela; Lévesque, Éric; Charette, André B; Pelletier, Joelle N

    2017-11-01

    Microbial transglutaminase (MTG) is a practical tool to enzymatically form isopeptide bonds between peptide or protein substrates. This natural approach to crosslinking the side-chains of reactive glutamine and lysine residues is solidly rooted in food and textile processing. More recently, MTG's tolerance for various primary amines in lieu of lysine have revealed its potential for site-specific protein labeling with aminated compounds, including fluorophores. Importantly, MTG can label glutamines at accessible positions in the body of a target protein, setting it apart from most labeling enzymes that react exclusively at protein termini. To expand its applicability as a labeling tool, we engineered the B1 domain of Protein G (GB1) to probe the selectivity and enhance the reactivity of MTG toward its glutamine substrate. We built a GB1 library where each variant contained a single glutamine at positions covering all secondary structure elements. The most reactive and selective variants displayed a >100-fold increase in incorporation of a recently developed aminated benzo[a]imidazo[2,1,5-cd]indolizine-type fluorophore, relative to native GB1. None of the variants were destabilized. Our results demonstrate that MTG can react readily with glutamines in α-helical, β-sheet, and unstructured loop elements and does not favor one type of secondary structure. Introducing point mutations within MTG's active site further increased reactivity toward the most reactive substrate variant, I6Q-GB1, enhancing MTG's capacity to fluorescently label an engineered, highly reactive glutamine substrate. This work demonstrates that MTG-reactive glutamines can be readily introduced into a protein domain for fluorescent labeling. © 2017 The Protein Society.

  19. Design and engineering of a man-made diffusive electron-transport protein.

    Science.gov (United States)

    Fry, Bryan A; Solomon, Lee A; Leslie Dutton, P; Moser, Christopher C

    2016-05-01

    Maquettes are man-made cofactor-binding oxidoreductases designed from first principles with minimal reference to natural protein sequences. Here we focus on water-soluble maquettes designed and engineered to perform diffusive electron transport of the kind typically carried out by cytochromes, ferredoxins and flavodoxins and other small proteins in photosynthetic and respiratory energy conversion and oxido-reductive metabolism. Our designs were tested by analysis of electron transfer between heme maquettes and the well-known natural electron transporter, cytochrome c. Electron-transfer kinetics were measured from seconds to milliseconds by stopped-flow, while sub-millisecond resolution was achieved through laser photolysis of the carbon monoxide maquette heme complex. These measurements demonstrate electron transfer from the maquette to cytochrome c, reproducing the timescales and charge complementarity modulation observed in natural systems. The ionic strength dependence of inter-protein electron transfer from 9.7×10(6) M(-1) s(-1) to 1.2×10(9) M(-1) s(-1) follows a simple Debye-Hückel model for attraction between +8 net charged oxidized cytochrome c and -19 net charged heme maquette, with no indication of significant protein dipole moment steering. Successfully recreating essential components of energy conversion and downstream metabolism in man-made proteins holds promise for in vivo clinical intervention and for the production of fuel or other industrial products. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Translation system engineering in Escherichia coli enhances non-canonical amino acid incorporation into proteins.

    Science.gov (United States)

    Gan, Rui; Perez, Jessica G; Carlson, Erik D; Ntai, Ioanna; Isaacs, Farren J; Kelleher, Neil L; Jewett, Michael C

    2017-05-01

    The ability to site-specifically incorporate non-canonical amino acids (ncAAs) into proteins has made possible the study of protein structure and function in fundamentally new ways, as well as the bio synthesis of unnatural polymers. However, the task of site-specifically incorporating multiple ncAAs into proteins with high purity and yield continues to present a challenge. At the heart of this challenge lies the lower efficiency of engineered orthogonal translation system components compared to their natural counterparts (e.g., translation elements that specifically use a ncAA and do not interact with the cell's natural translation apparatus). Here, we show that evolving and tuning expression levels of multiple components of an engineered translation system together as a whole enhances ncAA incorporation efficiency. Specifically, we increase protein yield when incorporating multiple p-azido-phenylalanine(pAzF) residues into proteins by (i) evolving the Methanocaldococcus jannaschii p-azido-phenylalanyl-tRNA synthetase anti-codon binding domain, (ii) evolving the elongation factor Tu amino acid-binding pocket, and (iii) tuning the expression of evolved translation machinery components in a single vector. Use of the evolved translation machinery in a genomically recoded organism lacking release factor one enabled enhanced multi-site ncAA incorporation into proteins. We anticipate that our approach to orthogonal translation system development will accelerate and expand our ability to site-specifically incorporate multiple ncAAs into proteins and biopolymers, advancing new horizons for synthetic and chemical biotechnology. Biotechnol. Bioeng. 2017;114: 1074-1086. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  1. Engineering and optimization of an allosteric biosensor protein for peroxisome proliferator-activated receptor γ ligands

    Science.gov (United States)

    Li, Jingjing; Gierach, Izabela; Gillies, Alison; Warden, Charles D.; Wood, David W.

    2011-01-01

    The peroxisome proliferator-activated receptor gamma (PPARγ or PPARG) belongs to the nuclear receptor superfamily, and is a potential drug target for a variety of diseases. In this work, we constructed a series of bacterial biosensors for the identification of functional PPARγ ligands. These sensors entail modified Escherichia coli cells carrying a four-domain fusion protein, comprised of the PPARγ ligand binding domain (LBD), an engineered mini-intein domain, the E. coli maltose binding protein (MBD), and a thymidylate synthase (TS) reporter enzyme. E. coli cells expressing this protein exhibit hormone ligand-dependent growth phenotypes. Unlike our published estrogen (ER) and thyroid receptor (TR) biosensors, the canonical PPARγ biosensor cells displayed pronounced growth in the absence of ligand. They were able to distinguish agonists and antagonists, however, even in the absence of agonist. To improve ligand sensitivity of this sensor, we attempted to engineer and optimize linker peptides flanking the PPARγ LBD insertion point. Truncation of the original linkers led to decreased basal growth and significantly enhanced ligand sensitivity of the PPARγ sensor, while substitution of the native linkers with optimized G4S (Gly-Gly-Gly-Gly-Ser) linkers further increased the sensitivity. Our studies demonstrate that the properties of linkers, especially the C-terminal linker, greatly influence the efficiency and fidelity of the allosteric signal induced by ligand binding. Our work also suggests an approach to increase allosteric behavior in this multidomain sensor protein, without modification of the functional LBD. PMID:21893405

  2. Analyzing the Folding and Binding Steps of an Intrinsically Disordered Protein by Protein Engineering.

    Science.gov (United States)

    Bonetti, Daniela; Troilo, Francesca; Toto, Angelo; Brunori, Maurizio; Longhi, Sonia; Gianni, Stefano

    2017-07-25

    Intrinsically disordered proteins (IDPs) are functionally active despite lacking a well-defined three-dimensional structure. Such proteins often undergo a disorder-to-order transition, or induced folding, when binding to their specific physiological partner. Because of cooperativity, the folding and binding steps typically appear as a single event, and therefore, induced folding is extremely difficult to characterize experimentally. In this perspective, the interaction between the disordered C-terminal domain of the measles virus nucleoprotein NTAIL and the folded X domain of the viral phosphoprotein (XD) is particularly interesting because the inherent complexity of the observed kinetics allows characterization of the binding and folding steps individually. Here we present a detailed structural description of the folding and binding events occurring in the recognition between NTAIL and XD. This result was achieved by measuring the effect of single-amino acid substitutions in NTAIL on the reaction mechanism. Analysis of the experimental data allowed us (i) to identify the key residues involved in the initial recognition between the two molecules and (ii) to depict the general features of the folding pathway of NTAIL. Furthermore, an analysis of the changes in stability obtained for the whole set of variants highlights how the sequence of this IDP has not been selected during evolution to fold efficiently. This feature might be a consequence of the weakly funneled nature of the energy landscape of IDPs in their unbound state and represents a plausible explanation of their highly dynamic nature even in the bound state, typically defined as "fuzziness".

  3. Integrating Protein Engineering and Bioorthogonal Click Conjugation for Extracellular Vesicle Modulation and Intracellular Delivery.

    Science.gov (United States)

    Wang, Ming; Altinoglu, Sarah; Takeda, Yuji S; Xu, Qiaobing

    2015-01-01

    Exosomes are small, cell-secreted vesicles that transfer proteins and genetic information between cells. This intercellular transmission regulates many physiological and pathological processes. Therefore, exosomes have emerged as novel biomarkers for disease diagnosis and as nanocarriers for drug delivery. Here, we report an easy-to-adapt and highly versatile methodology to modulate exosome composition and conjugate exosomes for intracellular delivery. Our strategy combines the metabolic labeling of newly synthesized proteins or glycan/glycoproteins of exosome-secreting cells with active azides and bioorthogonal click conjugation to modify and functionalize the exosomes. The azide-integrated can be conjugated to a variety of small molecules and proteins and can efficiently deliver conjugates into cells. The metabolic engineering of exosomes diversifies the chemistry of exosomes and expands the functions that can be introduced into exosomes, providing novel, powerful tools to study the roles of exosomes in biology and expand the biomedical potential of exosomes.

  4. Genetic engineering modification and fermentation optimization for extracellular production of recombinant proteins using Escherichia coli.

    Science.gov (United States)

    Zhou, Yuling; Lu, Zhenghui; Wang, Xiang; Selvaraj, Jonathan Nimal; Zhang, Guimin

    2018-02-01

    As a common expression host, Escherichia coli has received more and more attention due to the recently developed secretory expression system, which offers advantages like reduced downstream bioprocesses and improved product quality. These advantages, coupled with high-density fermentation technology, make it a preferred system for large-scale production of many proteins utilized in industry and agriculture at a reduced process cost. To improve the secretion efficiency of target proteins, various strategies, including signal peptide optimization, periplasmic leakage, and chaperones co-expression have been developed. In addition, the optimization of the fermentation conditions such as temperature, inducer, and medium were also taken into account for the extracellular production in the high-density fermentation to reduce the cost of production. Here, these strategies ranging from genetic engineering to fermentation optimization were summarized for the future guidance of extracellular production of recombinant proteins using E. coli.

  5. Specific Internalisation of Gold Nanoparticles into Engineered Porous Protein Cages via Affinity Binding.

    Directory of Open Access Journals (Sweden)

    David Paramelle

    Full Text Available Porous protein cages are supramolecular protein self-assemblies presenting pores that allow the access of surrounding molecules and ions into their core in order to store and transport them in biological environments. Protein cages' pores are attractive channels for the internalisation of inorganic nanoparticles and an alternative for the preparation of hybrid bioinspired nanoparticles. However, strategies based on nanoparticle transport through the pores are largely unexplored, due to the difficulty of tailoring nanoparticles that have diameters commensurate with the pores size and simultaneously displaying specific affinity to the cages' core and low non-specific binding to the cages' outer surface. We evaluated the specific internalisation of single small gold nanoparticles, 3.9 nm in diameter, into porous protein cages via affinity binding. The E2 protein cage derived from the Geobacillus stearothermophilus presents 12 pores, 6 nm in diameter, and an empty core of 13 nm in diameter. We engineered the E2 protein by site-directed mutagenesis with oligohistidine sequences exposing them into the cage's core. Dynamic light scattering and electron microscopy analysis show that the structures of E2 protein cages mutated with bis- or penta-histidine sequences are well conserved. The surface of the gold nanoparticles was passivated with a self-assembled monolayer made of a mixture of short peptidols and thiolated alkane ethylene glycol ligands. Such monolayers are found to provide thin coatings preventing non-specific binding to proteins. Further functionalisation of the peptide coated gold nanoparticles with Ni2+ nitrilotriacetic moieties enabled the specific binding to oligohistidine tagged cages. The internalisation via affinity binding was evaluated by electron microscopy analysis. From the various mutations tested, only the penta-histidine mutated E2 protein cage showed repeatable and stable internalisation. The present work overcomes the

  6. Protein adsorption steers blood contact activation on engineered cobalt chromium alloy oxide layers.

    Science.gov (United States)

    Milleret, Vincent; Buzzi, Stefano; Gehrig, Peter; Ziogas, Algirdas; Grossmann, Jonas; Schilcher, Katrin; Zinkernagel, Annelies S; Zucker, Arik; Ehrbar, Martin

    2015-09-01

    Biomaterials upon implantation are immediately covered by blood proteins which direct the subsequent blood activation. These early events determine the following cascade of biological reactions and consequently the long-term success of implants. The ability to modulate surface properties of biomaterials is therefore of considerable clinical significance. Goal of this study was an in-depth understanding of the biological response to cobalt chromium stent alloys with engineered surface oxide layers, which showed altered body reactions in vivo. We analyzed in vitro the biological events following initial blood contact on engineered cobalt chromium surfaces featuring said oxide layers. Surface-specific blood reactions were confirmed by scanning electron microscopy and the adsorbed protein layers were characterized by mass spectrometry. This powerful proteomics tool allowed the identification and quantification of over hundred surface-adhering proteins. Proteins associated with the coagulation cascade, platelet adhesion and neutrophil function correlated with the various blood surface activations observed. Furthermore, results of pre-coated surfaces with defined fibrinogen-albumin mixtures suggest that neutrophil adhesion was controlled by fibrinogen orientation and conformation rather than quantity. This study highlights the importance of controlling the biological response in the complex protein-implant surface interactions and the potential of the surface modifications to improve the clinical performance of medical implants. The blood contact activation of CoCr alloys is determined by their surface oxide layer properties. Modifications of the oxide layer affected the total amount of adsorbed proteins and the composition of the adsorbed protein layer. Additionally fibrinogen coatings mediated the surface-dependent neutrophil adhesion in a concentration-independent manner, indicating the influence of conformation and/or orientation of the adsorbed protein. Despite the

  7. A fluorescent cassette-based strategy for engineering multiple domain fusion proteins

    Directory of Open Access Journals (Sweden)

    Khorchid Ahmad

    2003-07-01

    Full Text Available Abstract Background The engineering of fusion proteins has become increasingly important and most recently has formed the basis of many biosensors, protein purification systems, and classes of new drugs. Currently, most fusion proteins consist of three or fewer domains, however, more sophisticated designs could easily involve three or more domains. Using traditional subcloning strategies, this requires micromanagement of restriction enzymes sites that results in complex workaround solutions, if any at all. Results Therefore, to aid in the efficient construction of fusion proteins involving multiple domains, we have created a new expression vector that allows us to rapidly generate a library of cassettes. Cassettes have a standard vector structure based on four specific restriction endonuclease sites and using a subtle property of blunt or compatible cohesive end restriction enzymes, they can be fused in any order and number of times. Furthermore, the insertion of PCR products into our expression vector or the recombination of cassettes can be dramatically simplified by screening for the presence or absence of fluorescence. Conclusions Finally, the utility of this new strategy was demonstrated by the creation of basic cassettes for protein targeting to subcellular organelles and for protein purification using multiple affinity tags.

  8. Identifying new therapeutic targets via modulation of protein corona formation by engineered nanoparticles.

    Directory of Open Access Journals (Sweden)

    Rochelle R Arvizo

    Full Text Available We introduce a promising methodology to identify new therapeutic targets in cancer. Proteins bind to nanoparticles to form a protein corona. We modulate this corona by using surface-engineered nanoparticles, and identify protein composition to provide insight into disease development.Using a family of structurally homologous nanoparticles we have investigated the changes in the protein corona around surface-functionalized gold nanoparticles (AuNPs from normal and malignant ovarian cell lysates. Proteomics analysis using mass spectrometry identified hepatoma-derived growth factor (HDGF that is found exclusively on positively charged AuNPs ((+AuNPs after incubation with the lysates. We confirmed expression of HDGF in various ovarian cancer cells and validated binding selectivity to (+AuNPs by Western blot analysis. Silencing of HDGF by siRNA resulted s inhibition in proliferation of ovarian cancer cells.We investigated the modulation of protein corona around surface-functionalized gold nanoparticles as a promising approach to identify new therapeutic targets. The potential of our method for identifying therapeutic targets was demonstrated through silencing of HDGF by siRNA, which inhibited proliferation of ovarian cancer cells. This integrated proteomics, bioinformatics, and nanotechnology strategy demonstrates that protein corona identification can be used to discover novel therapeutic targets in cancer.

  9. Identifying New Therapeutic Targets via Modulation of Protein Corona Formation by Engineered Nanoparticles

    Science.gov (United States)

    Arvizo, Rochelle R.; Giri, Karuna; Moyano, Daniel; Miranda, Oscar R.; Madden, Benjamin; McCormick, Daniel J.; Bhattacharya, Resham; Rotello, Vincent M.; Kocher, Jean-Pierre; Mukherjee, Priyabrata

    2012-01-01

    Background We introduce a promising methodology to identify new therapeutic targets in cancer. Proteins bind to nanoparticles to form a protein corona. We modulate this corona by using surface-engineered nanoparticles, and identify protein composition to provide insight into disease development. Methods/Principal Findings Using a family of structurally homologous nanoparticles we have investigated the changes in the protein corona around surface-functionalized gold nanoparticles (AuNPs) from normal and malignant ovarian cell lysates. Proteomics analysis using mass spectrometry identified hepatoma-derived growth factor (HDGF) that is found exclusively on positively charged AuNPs (+AuNPs) after incubation with the lysates. We confirmed expression of HDGF in various ovarian cancer cells and validated binding selectivity to +AuNPs by Western blot analysis. Silencing of HDGF by siRNA resulted s inhibition in proliferation of ovarian cancer cells. Conclusion We investigated the modulation of protein corona around surface-functionalized gold nanoparticles as a promising approach to identify new therapeutic targets. The potential of our method for identifying therapeutic targets was demonstrated through silencing of HDGF by siRNA, which inhibited proliferation of ovarian cancer cells. This integrated proteomics, bioinformatics, and nanotechnology strategy demonstrates that protein corona identification can be used to discover novel therapeutic targets in cancer. PMID:22442705

  10. HotSpot Wizard 2.0: automated design of site-specific mutations and smart libraries in protein engineering

    National Research Council Canada - National Science Library

    Bendl, Jaroslav; Stourac, Jan; Sebestova, Eva; Vavra, Ondrej; Musil, Milos; Brezovsky, Jan; Damborsky, Jiri

    2016-01-01

    HotSpot Wizard 2.0 is a web server for automated identification of hot spots and design of smart libraries for engineering proteins' stability, catalytic activity, substrate specificity and enantioselectivity...

  11. Quantification of Extracellular Matrix Proteins from a Rat Lung Scaffold to Provide a Molecular Readout for Tissue Engineering*

    Science.gov (United States)

    Hill, Ryan C.; Calle, Elizabeth A.; Dzieciatkowska, Monika; Niklason, Laura E.; Hansen, Kirk C.

    2015-01-01

    The use of extracellular matrix (ECM)1 scaffolds, derived from decellularized tissues for engineered organ generation, holds enormous potential in the field of regenerative medicine. To support organ engineering efforts, we developed a targeted proteomics method to extract and quantify extracellular matrix components from tissues. Our method provides more complete and accurate protein characterization than traditional approaches. This is accomplished through the analysis of both the chaotrope-soluble and -insoluble protein fractions and using recombinantly generated stable isotope labeled peptides for endogenous protein quantification. Using this approach, we have generated 74 peptides, representing 56 proteins to quantify protein in native (nondecellularized) and decellularized lung matrices. We have focused on proteins of the ECM and additional intracellular proteins that are challenging to remove during the decellularization procedure. Results indicate that the acellular lung scaffold is predominantly composed of structural collagens, with the majority of these proteins found in the insoluble ECM, a fraction that is often discarded using widely accepted proteomic methods. The decellularization procedure removes over 98% of intracellular proteins evaluated and retains, to varying degrees, proteoglycans and glycoproteins of the ECM. Accurate characterization of ECM proteins from tissue samples will help advance organ engineering efforts by generating a molecular readout that can be correlated with functional outcome to drive the next generation of engineered organs. PMID:25660013

  12. Quantification of extracellular matrix proteins from a rat lung scaffold to provide a molecular readout for tissue engineering.

    Science.gov (United States)

    Hill, Ryan C; Calle, Elizabeth A; Dzieciatkowska, Monika; Niklason, Laura E; Hansen, Kirk C

    2015-04-01

    The use of extracellular matrix (ECM) scaffolds, derived from decellularized tissues for engineered organ generation, holds enormous potential in the field of regenerative medicine. To support organ engineering efforts, we developed a targeted proteomics method to extract and quantify extracellular matrix components from tissues. Our method provides more complete and accurate protein characterization than traditional approaches. This is accomplished through the analysis of both the chaotrope-soluble and -insoluble protein fractions and using recombinantly generated stable isotope labeled peptides for endogenous protein quantification. Using this approach, we have generated 74 peptides, representing 56 proteins to quantify protein in native (nondecellularized) and decellularized lung matrices. We have focused on proteins of the ECM and additional intracellular proteins that are challenging to remove during the decellularization procedure. Results indicate that the acellular lung scaffold is predominantly composed of structural collagens, with the majority of these proteins found in the insoluble ECM, a fraction that is often discarded using widely accepted proteomic methods. The decellularization procedure removes over 98% of intracellular proteins evaluated and retains, to varying degrees, proteoglycans and glycoproteins of the ECM. Accurate characterization of ECM proteins from tissue samples will help advance organ engineering efforts by generating a molecular readout that can be correlated with functional outcome to drive the next generation of engineered organs. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Exploring the mechanisms used by promiscuous chaperones to assist protein folding in the cell

    Science.gov (United States)

    Jewett, Andrew I.

    There are two popular theories to explain how molecular chaperones boost the yield of folded protein in the cell: According to the Anfinsen cage model, (ACM) chaperonins protect denatured proteins from aggregation. A competing theory, the iterative annealing model (IAM) claims that ATP regulated chaperone binding and release accelerates folding by freeing proteins from long-lived kinetic traps. We present experimental and kinetic evidence to argue that the IAM is not a complete picture of how the GroEL/ES chaperonin works. Surprisingly some substrate proteins experience folding rate enhancements without undergoing multiple rounds of ATP-induced binding and release from the chaperonin. An explanation of this data requires going beyond the ACM and IAM models. Our work uses molecular dynamics simulations to investigate the folding of a highly frustrated protein within a chaperonin cavity. The chaperonin interior is modeled by a sphere with variable degree of attraction to the protein inside. We demonstrate that this cavity, similar to the weakly hydrophobic interior of the GroEL cavity upon complexion with ATP and GroES, is sufficient to accelerate the folding of a frustrated protein by more than an order of magnitude. Our simulations uncover a novel form of the IAM in which the substrate exhibits spontaneous binding and release from the wall of the chaperonin cage. This mimics the behavior observed in the standard IAM, with the difference that thermal fluctuations, rather than ATP, allow the substrate to unbind from the chaperone. An growing number of smaller cageless chaperones have been discovered that can assist protein folding without the consumption of ATP, including artificial "minichaperones" (fragments of larger chaperones). It is tempting to speculate that the same thermally-driven IAM mechanism could play a role with these chaperones as well. We performed additional simulations of protein folding outside the sphere. We find that in order to accelerate

  14. In-depth analysis of protein inference algorithms using multiple search engines and well-defined metrics.

    Science.gov (United States)

    Audain, Enrique; Uszkoreit, Julian; Sachsenberg, Timo; Pfeuffer, Julianus; Liang, Xiao; Hermjakob, Henning; Sanchez, Aniel; Eisenacher, Martin; Reinert, Knut; Tabb, David L; Kohlbacher, Oliver; Perez-Riverol, Yasset

    2017-01-06

    In mass spectrometry-based shotgun proteomics, protein identifications are usually the desired result. However, most of the analytical methods are based on the identification of reliable peptides and not the direct identification of intact proteins. Thus, assembling peptides identified from tandem mass spectra into a list of proteins, referred to as protein inference, is a critical step in proteomics research. Currently, different protein inference algorithms and tools are available for the proteomics community. Here, we evaluated five software tools for protein inference (PIA, ProteinProphet, Fido, ProteinLP, MSBayesPro) using three popular database search engines: Mascot, X!Tandem, and MS-GF+. All the algorithms were evaluated using a highly customizable KNIME workflow using four different public datasets with varying complexities (different sample preparation, species and analytical instruments). We defined a set of quality control metrics to evaluate the performance of each combination of search engines, protein inference algorithm, and parameters on each dataset. We show that the results for complex samples vary not only regarding the actual numbers of reported protein groups but also concerning the actual composition of groups. Furthermore, the robustness of reported proteins when using databases of differing complexities is strongly dependant on the applied inference algorithm. Finally, merging the identifications of multiple search engines does not necessarily increase the number of reported proteins, but does increase the number of peptides per protein and thus can generally be recommended. Protein inference is one of the major challenges in MS-based proteomics nowadays. Currently, there are a vast number of protein inference algorithms and implementations available for the proteomics community. Protein assembly impacts in the final results of the research, the quantitation values and the final claims in the research manuscript. Even though protein

  15. Handheld highly selective plasmonic chem/biosensor using engineered binding proteins for extreme conformational changes

    Science.gov (United States)

    Kosciolek, Derek J.; Sonar, Ajay; Lepak, Lori A.; Schnatz, Peter; Bendoym, Igor; Brown, Mia C.; Koder, Ronald L.; Crouse, David T.

    2017-08-01

    In this project we develop a handheld, portable, highly selective and sensitive chem/biosensor that has potential applications in both airborne and water-based environmental sensing. The device relies on a plasmonic chip of subwavelength-scale periodic gold rods engineered to resonate in the near infrared. The chip is functionalized with a novel class of proteins that exhibit large conformational changes upon binding to a specific target analyte. The subsequent change in local refractive index near the surface of the gold is one to two orders of magnitude greater than current conventional methods, which produces a readily measurable 5 to 10 percent difference in light transmission. This allows us to forgo traditional, bulky tabletop setups in favor of a compact form factor. Using commercially available optics to construct a transmission-based optical train, measured changes in bulk refractive index are presented here. While synthesis of binding protein efforts are focused on heme as analyte for proof of concept validation, the functionalized protein can be engineered to pair with a wide variety of analytes with minimal alterations to the plasmonic chip or device design. Such flexibility allows for this device to potentially meet the needs of first responders and health care professionals in a multitude of scenarios.

  16. Recoding aminoacyl-tRNA synthetases for synthetic biology by rational protein-RNA engineering.

    Science.gov (United States)

    Hadd, Andrew; Perona, John J

    2014-12-19

    We have taken a rational approach to redesigning the amino acid binding and aminoacyl-tRNA pairing specificities of bacterial glutaminyl-tRNA synthetase. The four-stage engineering incorporates generalizable design principles and improves the pairing efficiency of noncognate glutamate with tRNA(Gln) by over 10(5)-fold compared to the wild-type enzyme. Better optimized designs of the protein-RNA complex include substantial reengineering of the globular core region of the tRNA, demonstrating a role for specific tRNA nucleotides in specifying the identity of the genetically encoded amino acid. Principles emerging from this engineering effort open new prospects for combining rational and genetic selection approaches to design novel aminoacyl-tRNA synthetases that ligate noncanonical amino acids onto tRNAs. This will facilitate reconstruction of the cellular translation apparatus for applications in synthetic biology.

  17. A divide and conquer approach to determine the Pareto frontier for optimization of protein engineering experiments

    Science.gov (United States)

    He, Lu; Friedman, Alan M.; Bailey-Kellogg, Chris

    2016-01-01

    In developing improved protein variants by site-directed mutagenesis or recombination, there are often competing objectives that must be considered in designing an experiment (selecting mutations or breakpoints): stability vs. novelty, affinity vs. specificity, activity vs. immunogenicity, and so forth. Pareto optimal experimental designs make the best trade-offs between competing objectives. Such designs are not “dominated”; i.e., no other design is better than a Pareto optimal design for one objective without being worse for another objective. Our goal is to produce all the Pareto optimal designs (the Pareto frontier), in order to characterize the trade-offs and suggest designs most worth considering, but to avoid explicitly considering the large number of dominated designs. To do so, we develop a divide-and-conquer algorithm, PEPFR (Protein Engineering Pareto FRontier), that hierarchically subdivides the objective space, employing appropriate dynamic programming or integer programming methods to optimize designs in different regions. This divide-and-conquer approach is efficient in that the number of divisions (and thus calls to the optimizer) is directly proportional to the number of Pareto optimal designs. We demonstrate PEPFR with three protein engineering case studies: site-directed recombination for stability and diversity via dynamic programming, site-directed mutagenesis of interacting proteins for affinity and specificity via integer programming, and site-directed mutagenesis of a therapeutic protein for activity and immunogenicity via integer programming. We show that PEPFR is able to effectively produce all the Pareto optimal designs, discovering many more designs than previous methods. The characterization of the Pareto frontier provides additional insights into the local stability of design choices as well as global trends leading to trade-offs between competing criteria. PMID:22180081

  18. Fluoro amino acids: a rarity in nature, yet a prospect for protein engineering.

    Science.gov (United States)

    Odar, Corinna; Winkler, Margit; Wiltschi, Birgit

    2015-03-01

    Fluoro amino acids are highly valuable compounds constantly gaining relevance in diverse fields of the biosciences as well as in the pharmaceutical industry. The value of these compounds can be attributed to the properties of the extremely electronegative fluorine atom. This atom forms a highly polarized bond of extraordinary strength with carbon. The formation of the fluorine-carbon bond is challenging: its chemical synthesis demands harsh reaction conditions and to date only one class of enzyme has been found capable of introducing the fluoride ion into an organic compound. Most of these fluorinating enzymes participate in the biosynthesis of 4-fluoro-L-threonine, the only fluoro amino acid of natural origin discovered so far. Despite their scarcity in nature, fluoro amino acids are valuable tools to fluorinate proteins. The fluoro protein variants often show improved stability and folding as well as altered activity and fluorescence characteristics. This review details the biosynthesis of 4-fluoro-L-threonine with a special focus on the fluorinating enzymes. Moreover, we elaborate on the application of fluoro amino acids as building blocks for fluorinated protein variants. Insight into different techniques to incorporate fluoro amino acids into proteins is also provided. We highlight prospects and the current relevance of fluoro amino acids as a tool to engineer proteins with novel traits. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Disulfide by Design 2.0: a web-based tool for disulfide engineering in proteins.

    Science.gov (United States)

    Craig, Douglas B; Dombkowski, Alan A

    2013-12-01

    Disulfide engineering is an important biotechnological tool that has advanced a wide range of research. The introduction of novel disulfide bonds into proteins has been used extensively to improve protein stability, modify functional characteristics, and to assist in the study of protein dynamics. Successful use of this technology is greatly enhanced by software that can predict pairs of residues that will likely form a disulfide bond if mutated to cysteines. We had previously developed and distributed software for this purpose: Disulfide by Design (DbD). The original DbD program has been widely used; however, it has a number of limitations including a Windows platform dependency. Here, we introduce Disulfide by Design 2.0 (DbD2), a web-based, platform-independent application that significantly extends functionality, visualization, and analysis capabilities beyond the original program. Among the enhancements to the software is the ability to analyze the B-factor of protein regions involved in predicted disulfide bonds. Importantly, this feature facilitates the identification of potential disulfides that are not only likely to form but are also expected to provide improved thermal stability to the protein. DbD2 provides platform-independent access and significantly extends the original functionality of DbD. A web server hosting DbD2 is provided at http://cptweb.cpt.wayne.edu/DbD2/.

  20. Interaction of Tissue Engineering Substrates with Serum Proteins and Its Influence on Human Primary Endothelial Cells.

    Science.gov (United States)

    Mohan, Tamilselvan; Niegelhell, Katrin; Nagaraj, Chandran; Reishofer, David; Spirk, Stefan; Olschewski, Andrea; Stana Kleinschek, Karin; Kargl, Rupert

    2017-02-13

    Polymer-based biomaterials particularly polycaprolactone (PCL) are one of the most promising substrates for tissue engineering. The surface chemistry of these materials plays a major role since it governs protein adsorption, cell adhesion, viability, degradation, and biocompatibility in the first place. This study correlates the interaction of the most abundant serum proteins (albumin, immunoglobulins, fibrinogen) with the surface properties of PCL and its influence on the morphology and metabolic activity of primary human arterial endothelial cells that are seeded on the materials. Prior to that, thin films of PCL are manufactured by spin-coating and characterized in detail. A quartz crystal microbalance with dissipation (QCM-D), a multiparameter surface plasmon resonance spectroscopy instrument (MP-SPR), wettability data, and atomic force microscopy are combined to elucidate the pH-dependent protein adsorption on the PCL substrates. Primary endothelial cells are cultured on the protein modified polymer, and conclusions are drawn on the significant impact of type and form of proteins coatings on cell morphology and metabolic activity.

  1. Combining Protein and Strain Engineering for the Production of Glyco-Engineered Horseradish Peroxidase C1A in Pichia pastoris.

    Science.gov (United States)

    Capone, Simona; Ćorajević, Lejla; Bonifert, Günther; Murth, Patrick; Maresch, Daniel; Altmann, Friedrich; Herwig, Christoph; Spadiut, Oliver

    2015-09-24

    Horseradish peroxidase (HRP), conjugated to antibodies and lectins, is widely used in medical diagnostics. Since recombinant production of the enzyme is difficult, HRP isolated from plant is used for these applications. Production in the yeast Pichia pastoris (P. pastoris), the most promising recombinant production platform to date, causes hyperglycosylation of HRP, which in turn complicates conjugation to antibodies and lectins. In this study we combined protein and strain engineering to obtain an active and stable HRP variant with reduced surface glycosylation. We combined four mutations, each being beneficial for either catalytic activity or thermal stability, and expressed this enzyme variant as well as the unmutated wildtype enzyme in both a P. pastoris benchmark strain and a strain where the native α-1,6-mannosyltransferase (OCH1) was knocked out. Considering productivity in the bioreactor as well as enzyme activity and thermal stability, the mutated HRP variant produced in the P. pastoris benchmark strain turned out to be interesting for medical diagnostics. This variant shows considerable catalytic activity and thermal stability and is less glycosylated, which might allow more controlled and efficient conjugation to antibodies and lectins.

  2. Engineered Escherichia coli silver-binding periplasmic protein that promotes silver tolerance.

    Science.gov (United States)

    Sedlak, Ruth Hall; Hnilova, Marketa; Grosh, Carolynn; Fong, Hanson; Baneyx, Francois; Schwartz, Dan; Sarikaya, Mehmet; Tamerler, Candan; Traxler, Beth

    2012-04-01

    Silver toxicity is a problem that microorganisms face in medical and environmental settings. Through exposure to silver compounds, some bacteria have adapted to growth in high concentrations of silver ions. Such adapted microbes may be dangerous as pathogens but, alternatively, could be potentially useful in nanomaterial-manufacturing applications. While naturally adapted isolates typically utilize efflux pumps to achieve metal resistance, we have engineered a silver-tolerant Escherichia coli strain by the use of a simple silver-binding peptide motif. A silver-binding peptide, AgBP2, was identified from a combinatorial display library and fused to the C terminus of the E. coli maltose-binding protein (MBP) to yield a silver-binding protein exhibiting nanomolar affinity for the metal. Growth experiments performed in the presence of silver nitrate showed that cells secreting MBP-AgBP2 into the periplasm exhibited silver tolerance in a batch culture, while those expressing a cytoplasmic version of the fusion protein or MBP alone did not. Transmission electron microscopy analysis of silver-tolerant cells revealed the presence of electron-dense silver nanoparticles. This is the first report of a specifically engineered metal-binding peptide exhibiting a strong in vivo phenotype, pointing toward a novel ability to manipulate bacterial interactions with heavy metals by the use of short and simple peptide motifs. Engineered metal-ion-tolerant microorganisms such as this E. coli strain could potentially be used in applications ranging from remediation to interrogation of biomolecule-metal interactions in vivo.

  3. Mutational breeding and genetic engineering in the development of high grain protein content.

    Science.gov (United States)

    Wenefrida, Ida; Utomo, Herry S; Linscombe, Steve D

    2013-12-04

    Cereals are the most important crops in the world for both human consumption and animal feed. Improving their nutritional values, such as high protein content, will have significant implications, from establishing healthy lifestyles to helping remediate malnutrition problems worldwide. Besides providing a source of carbohydrate, grain is also a natural source of dietary fiber, vitamins, minerals, specific oils, and other disease-fighting phytocompounds. Even though cereal grains contain relatively little protein compared to legume seeds, they provide protein for the nutrition of humans and livestock that is about 3 times that of legumes. Most cereal seeds lack a few essential amino acids; therefore, they have imbalanced amino acid profiles. Lysine (Lys), threonine (Thr), methionine (Met), and tryptophan (Trp) are among the most critical and are a limiting factor in many grain crops for human nutrition. Tremendous research has been put into the efforts to improve these essential amino acids. Development of high protein content can be outlined in four different approaches through manipulating seed protein bodies, modulating certain biosynthetic pathways to overproduce essential and limiting amino acids, increasing nitrogen relocation to the grain through the introduction of transgenes, and exploiting new genetic variance. Various technologies have been employed to improve protein content including conventional and mutational breeding, genetic engineering, marker-assisted selection, and genomic analysis. Each approach involves a combination of these technologies. Advancements in nutrigenomics and nutrigenetics continue to improve public knowledge at a rapid pace on the importance of specific aspects of food nutrition for optimum fitness and health. An understanding of the molecular basis for human health and genetic predisposition to certain diseases through human genomes enables individuals to personalize their nutritional requirements. It is critically important

  4. Engineering the supply chain for protein production/secretion in yeasts and mammalian cells.

    Science.gov (United States)

    Klein, Tobias; Niklas, Jens; Heinzle, Elmar

    2015-03-01

    Metabolic bottlenecks play an increasing role in yeasts and mammalian cells applied for high-performance production of proteins, particularly of pharmaceutical ones that require complex posttranslational modifications. We review the present status and developments focusing on the rational metabolic engineering of such cells to optimize the supply chain for building blocks and energy. Methods comprise selection of beneficial genetic modifications, rational design of media and feeding strategies. Design of better producer cells based on whole genome-wide metabolic network analysis becomes increasingly possible. High-resolution methods of metabolic flux analysis for the complex networks in these compartmented cells are increasingly available. We discuss phenomena that are common to both types of organisms but also those that are different with respect to the supply chain for the production and secretion of pharmaceutical proteins.

  5. Immobilized WNT Proteins Act as a Stem Cell Niche for Tissue Engineering.

    Science.gov (United States)

    Lowndes, Molly; Rotherham, Michael; Price, Joshua C; El Haj, Alicia J; Habib, Shukry J

    2016-07-12

    The timing, location, and level of WNT signaling are highly regulated during embryonic development and for the maintenance of adult tissues. Consequently the ability to provide a defined and directed source of WNT proteins is crucial to fully understand its role in tissue development and to mimic its activity in vitro. Here we describe a one-step immobilization technique to covalently bind WNT3A proteins as a basal surface with easy storage and long-lasting activity. We show that this platform is able to maintain adult and embryonic stem cells while also being adaptable for 3D systems. Therefore, this platform could be used for recapitulating specific stem cell niches with the goal of improving tissue engineering. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  6. Artificial TALE as a Convenient Protein Platform for Engineering Broad-Spectrum Resistance to Begomoviruses

    Directory of Open Access Journals (Sweden)

    Xiaofei Cheng

    2015-08-01

    Full Text Available Transcription activator–like effectors (TALEs are a class of sequence-specific DNA-binding proteins that utilize a simple and predictable modality to recognize target DNA. This unique characteristic allows for the rapid assembly of artificial TALEs, with high DNA binding specificity, to any target DNA sequences for the creation of customizable sequence-specific nucleases used in genome engineering. Here, we report the use of an artificial TALE protein as a convenient platform for designing broad-spectrum resistance to begomoviruses, one of the most destructive plant virus groups, which cause tremendous losses worldwide. We showed that artificial TALEs, which were assembled based on conserved sequence motifs within begomovirus genomes, could confer partial resistance in transgenic Nicotiana benthamiana to all three begomoviruses tested. Furthermore, the resistance was maintained even in the presence of their betasatellite. These results shed new light on the development of broad-spectrum resistance against DNA viruses, such as begomoviruses.

  7. Genetically engineered hybrid proteins from Parietaria judaica pollen for allergen-specific immunotherapy.

    Science.gov (United States)

    González-Rioja, Roberto; Ibarrola, Ignacio; Arilla, M Carmen; Ferrer, Angel; Mir, Amparo; Andreu, Carmen; Martínez, Alberto; Asturias, Juan A

    2007-09-01

    Despite the use of conventional allergen-specific immunotherapy in clinical practice, more defined, efficient, and safer allergy vaccines are required. The aim of the study was to obtain hypoallergenic molecules by deleting B-cell epitopes, which could potentially be applied to Parietaria judaica pollen allergy treatment. Three hybrid molecules (Q1, Q2, and Q3) derived from fragments of the 2 major P judaica pollen allergens, Par j 1 and Par j 2, were engineered by means of PCR. Hybrid structures were compared with their natural components by means of circular dichroism, and their biologic activities were compared by using T-cell proliferation assays. Their IgE-binding activity was determined with Western blotting, skin prick tests, and enzyme allergosorbent and ELISA inhibition tests. The hybrid proteins, especially Q2 and Q3, revealed significantly reduced IgE reactivity compared with the natural allergens, as well as with the whole P judaica extract. Furthermore, in vivo skin prick tests showed that the hybrid proteins had a significantly lower potency to induce cutaneous reactions than the whole P judaica extract. Two (Q1 and Q2) of the 3 hybrid proteins induced a comparable T-cell proliferation response as that produced by the whole extract and natural allergens. Considering its reduced anaphylactogenic potential, together with its conserved T-cell reactivity, the engineered Q2 protein could be used in safe and shortened schedules of allergen-specific immunotherapy against P judaica pollen allergy. Recombinant hybrid Q2 is able to induce T-cell proliferation, thus evidencing a potential therapeutic effect. Its reduced IgE-binding capacity envisages an excellent safety profile.

  8. Engineering hybrid polymer-protein super-aligned nanofibers via rotary jet spinning.

    Science.gov (United States)

    Badrossamay, Mohammad R; Balachandran, Kartik; Capulli, Andrew K; Golecki, Holly M; Agarwal, Ashutosh; Goss, Josue A; Kim, Hansu; Shin, Kwanwoo; Parker, Kevin Kit

    2014-03-01

    Cellular microenvironments are important in coaxing cells to behave collectively as functional, structured tissues. Important cues in this microenvironment are the chemical, mechanical and spatial arrangement of the supporting matrix in the extracellular space. In engineered tissues, synthetic scaffolding provides many of these microenvironmental cues. Key requirements are that synthetic scaffolds should recapitulate the native three-dimensional (3D) hierarchical fibrillar structure, possess biomimetic surface properties and demonstrate mechanical integrity, and in some tissues, anisotropy. Electrospinning is a popular technique used to fabricate anisotropic nanofiber scaffolds. However, it suffers from relatively low production rates and poor control of fiber alignment without substantial modifications to the fiber collector mechanism. Additionally, many biomaterials are not amenable for fabrication via high-voltage electrospinning methods. Hence, we reasoned that we could utilize rotary jet spinning (RJS) to fabricate highly aligned hybrid protein-polymer with tunable chemical and physical properties. In this study, we engineered highly aligned nanofiber constructs with robust fiber alignment from blends of the proteins collagen and gelatin, and the polymer poly-ε-caprolactone via RJS and electrospinning. RJS-spun fibers retain greater protein content on the surface and are also fabricated at a higher production rate compared to those fabricated via electrospinning. We measured increased fiber diameter and viscosity, and decreasing fiber alignment as protein content increased in RJS hybrid fibers. RJS nanofiber constructs also demonstrate highly anisotropic mechanical properties mimicking several biological tissue types. We demonstrate the bio-functionality of RJS scaffold fibers by testing their ability to support cell growth and maturation with a variety of cell types. Our highly anisotropic RJS fibers are therefore able to support cellular alignment

  9. Engineering a recyclable elastin-like polypeptide capturing scaffold for non-chromatographic protein purification.

    Science.gov (United States)

    Liu, Fang; Chen, Wilfred

    2013-01-01

    Previously, we reported a non-chromatographic protein purification method exploiting the highly specific interaction between the dockerin and cohesin domains from Clostridium thermocellum and the reversible aggregation property of elastin-like polypeptide (ELP) to provide fast and cost-effective protein purification. However, the bound dockerin-intein tag cannot be completely dissociated from the ELP-cohesin capturing scaffold due to the high binding affinity, resulting in a single-use approach. In order to further reduce the purification cost by recycling the ELP capturing scaffold, a truncated dockerin domain with the calcium-coordinating function partially impaired was employed. We demonstrated that the truncated dockerin domain was sufficient to function as an effective affinity tag, and the target protein was purified directly from cell extracts in a single binding step followed by intein cleavage. The efficient EDTA-mediated dissociation of the bound dockerin-intein tag from the ELP-cohesin capturing scaffold was realized, and the regenerated ELP capturing scaffold was reused in another purification cycle without any decrease in the purification efficiency. This recyclable non-chromatographic based affinity method provides an attractive approach for efficient and cost-effective protein purification. © 2013 American Institute of Chemical Engineers.

  10. Proteolytic maturation of protein C upon engineering the mouse mammary gland to express furin.

    Science.gov (United States)

    Drews, R; Paleyanda, R K; Lee, T K; Chang, R R; Rehemtulla, A; Kaufman, R J; Drohan, W N; Luboń, H

    1995-11-07

    Endoproteolytic processing of the human protein C (HPC) precursor to its mature form involves cleavage of the propeptide after amino acids Lys-2-Arg-1 and removal of a Lys156-Arg157 dipeptide connecting the light and heavy chains. This processing was inefficient in the mammary gland of transgenic mice and pigs. We hypothesized that the protein processing capacity of specific animal organs may be improved by the coexpression of selected processing enzymes. We tested this by targeting expression of the human proprotein processing enzyme, named paired basic amino acid cleaving enzyme (PACE)/furin, or an enzymatically inactive mutant, PACEM, to the mouse mammary gland. In contrast to mice expressing HPC alone, or to HPC/PACEM bigenic mice, coexpression of PACE with HPC resulted in efficient conversion of the precursor to mature protein, with cleavage at the appropriate sites. These results suggest the involvement of PACE in the processing of HPC in vivo and represent an example of the engineering of animal organs into bioreactors with enhanced protein processing capacity.

  11. Engineering mammalian cell factories with SINEUP noncoding RNAs to improve translation of secreted proteins.

    Science.gov (United States)

    Patrucco, Laura; Chiesa, Andrea; Soluri, Maria Felicia; Fasolo, Francesca; Takahashi, Hazuki; Carninci, Piero; Zucchelli, Silvia; Santoro, Claudio; Gustincich, Stefano; Sblattero, Daniele; Cotella, Diego

    2015-09-15

    Whenever the function of a recombinant protein depends on post-translational processing, mammalian cells become an indispensable tool for their production. This is particularly true for biologics and therapeutic monoclonal antibodies (MAbs). Despite some drawbacks, Chinese Hamster Ovary (CHO) cells are the workhorse for MAbs production in academia and industry. Several methodologies have been adopted to improve expression and stability, including methods based on selective pressure or cell engineering. We have previously identified SINEUPs as a new functional class of natural and synthetic long non-coding RNAs that through the activity of an inverted SINEB2 element are able to promote translation of partially overlapping sense coding mRNAs. Here we show that by taking advantage of their modular structure, synthetic SINEUPs can be designed to increase production of secreted proteins. Furthermore, by experimentally validating antisense to elastin (AS-eln) RNA as a natural SINEUP, we show that SINEUP-mediated control may target extracellular proteins. These results lead us to propose synthetic SINEUPs as new versatile tools to optimize production of secreted proteins in manufacturing pipelines and natural SINEUPs as new regulatory RNAs in the secretory pathways. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Biotechnology and genetic engineering in the new drug development. Part I. DNA technology and recombinant proteins.

    Science.gov (United States)

    Stryjewska, Agnieszka; Kiepura, Katarzyna; Librowski, Tadeusz; Lochyński, Stanisław

    2013-01-01

    Pharmaceutical biotechnology has a long tradition and is rooted in the last century, first exemplified by penicillin and streptomycin as low molecular weight biosynthetic compounds. Today, pharmaceutical biotechnology still has its fundamentals in fermentation and bioprocessing, but the paradigmatic change affected by biotechnology and pharmaceutical sciences has led to an updated definition. The biotechnology revolution redrew the research, development, production and even marketing processes of drugs. Powerful new instruments and biotechnology related scientific disciplines (genomics, proteomics) make it possible to examine and exploit the behavior of proteins and molecules. Recombinant DNA (rDNA) technologies (genetic, protein, and metabolic engineering) allow the production of a wide range of peptides, proteins, and biochemicals from naturally nonproducing cells. This technology, now approximately 25 years old, is becoming one of the most important technologies developed in the 20(th) century. Pharmaceutical products and industrial enzymes were the first biotech products on the world market made by means of rDNA. Despite important advances regarding rDNA applications in mammalian cells, yeasts still represent attractive hosts for the production of heterologous proteins. In this review we describe these processes.

  13. Enhanced Missing Proteins Detection in NCI60 Cell Lines Using an Integrative Search Engine Approach.

    Science.gov (United States)

    Guruceaga, Elizabeth; Garin-Muga, Alba; Prieto, Gorka; Bejarano, Bartolomé; Marcilla, Miguel; Marín-Vicente, Consuelo; Perez-Riverol, Yasset; Casal, J Ignacio; Vizcaíno, Juan Antonio; Corrales, Fernando J; Segura, Victor

    2017-12-01

    The Human Proteome Project (HPP) aims deciphering the complete map of the human proteome. In the past few years, significant efforts of the HPP teams have been dedicated to the experimental detection of the missing proteins, which lack reliable mass spectrometry evidence of their existence. In this endeavor, an in depth analysis of shotgun experiments might represent a valuable resource to select a biological matrix in design validation experiments. In this work, we used all the proteomic experiments from the NCI60 cell lines and applied an integrative approach based on the results obtained from Comet, Mascot, OMSSA, and X!Tandem. This workflow benefits from the complementarity of these search engines to increase the proteome coverage. Five missing proteins C-HPP guidelines compliant were identified, although further validation is needed. Moreover, 165 missing proteins were detected with only one unique peptide, and their functional analysis supported their participation in cellular pathways as was also proposed in other studies. Finally, we performed a combined analysis of the gene expression levels and the proteomic identifications from the common cell lines between the NCI60 and the CCLE project to suggest alternatives for further validation of missing protein observations.

  14. Multiplex iterative plasmid engineering for combinatorial optimization of metabolic pathways and diversification of protein coding sequences.

    Science.gov (United States)

    Li, Yifan; Gu, Qun; Lin, Zhenquan; Wang, Zhiwen; Chen, Tao; Zhao, Xueming

    2013-11-15

    Engineering complex biological systems typically requires combinatorial optimization to achieve the desired functionality. Here, we present Multiplex Iterative Plasmid Engineering (MIPE), which is a highly efficient and customized method for combinatorial diversification of plasmid sequences. MIPE exploits ssDNA mediated λ Red recombineering for the introduction of mutations, allowing it to target several sites simultaneously and generate libraries of up to 10(7) sequences in one reaction. We also describe "restriction digestion mediated co-selection (RD CoS)", which enables MIPE to produce enhanced recombineering efficiencies with greatly simplified coselection procedures. To demonstrate this approach, we applied MIPE to fine-tune gene expression level in the 5-gene riboflavin biosynthetic pathway and successfully isolated a clone with 2.67-fold improved production in less than a week. We further demonstrated the ability of MIPE for highly multiplexed diversification of protein coding sequence by simultaneously targeting 23 codons scattered along the 750 bp sequence. We anticipate this method to benefit the optimization of diverse biological systems in synthetic biology and metabolic engineering.

  15. Protein engineering tests of a homology model of Plasmodium falciparum lactate dehydrogenase.

    Science.gov (United States)

    Hewitt, C O; Sessions, R B; Dafforn, T R; Holbrook, J J

    1997-01-01

    This paper describes the testing of a homology model of Plasmodium falciparum lactate dehydrogenase (pfLDH) by protein engineering. The model had been validated in structural terms. It suggests explanations of the unusual properties of pfLDH (compared with all other LDHs). These unusual features are a lack of substrate inhibition, high activity with the synthetic coenzyme 3-acetylpyridine adenine dinucleotide (APAD+) and changes in residues at previously conserved positions. pfLDH shows several amino acid insertions and deletions in an alignment with protein sequences from all other known LDHs. The most notable is a five amino acid insertion into the active-site loop. In addition, a conserved serine at position 163 is replaced by leucine. The results showed that when the unique pfLDH structural features were engineered into Bacillus stearothermophilus lactate dehydrogenase, the thermophilic enzyme acquired the properties previously uniquely associated with the malarial enzyme. We conclude that the homology model of the malarial enzyme is adequate for the prediction of successful redesigns and, in the regions tested, is accurate.

  16. Combinatorial genome and protein engineering yields monoclonal antibodies with hypergalactosylation from CHO cells.

    Science.gov (United States)

    Chung, Cheng-Yu; Wang, Qiong; Yang, Shuang; Ponce, Sean A; Kirsch, Brian J; Zhang, Hui; Betenbaugh, Michael J

    2017-12-01

    One of the key quality attributes of monoclonal antibodies is the glycan pattern and distribution. Two terminal galactose residues typically represent a small fraction of the total glycans from antibodies. However, antibodies with defined glycosylation properties including enhanced galactosylation have been shown to exhibit altered properties for these important biomedical modalities. In this study, the disruption of two α-2,3 sialyltransferases (ST3GAL4 and ST3GAL6) from Chinese Hamster Ovary (CHO) cells was combined with protein engineering of the Fc region to generate an IgG containing 80% bigalactosylated and fucosylated (G2F) glycoforms. Expression of the same single amino acid mutant (F241A) IgG in CHO cells with a triple gene knockout of fucosyltransferase (FUT8) plus ST3GAL4 and ST3GAL6 lowered the galactosylation glycoprofile to 65% bigalactosylated G2 glycans. However, overexpression of IgGs with four amino acid substitutions recovered the G2 glycoform composition approximately 80%. Combining genome and protein engineering in CHO cells will provide a new antibody production platform that enables biotechnologists to generate glycoforms standards for specific biomedical and biotechnology applications. © 2017 Wiley Periodicals, Inc.

  17. In vivo and in vitro protein ligation by naturally occurring and engineered split DnaE inteins.

    Directory of Open Access Journals (Sweden)

    A Sesilja Aranko

    Full Text Available BACKGROUND: Protein trans-splicing by naturally occurring split DnaE inteins is used for protein ligation of foreign peptide fragments. In order to widen biotechnological applications of protein trans-splicing, it is highly desirable to have split inteins with shorter C-terminal fragments, which can be chemically synthesized. PRINCIPAL FINDINGS: We report the identification of new functional split sites in DnaE inteins from Synechocystis sp. PCC6803 and from Nostoc punctiforme. One of the newly engineered split intein bearing C-terminal 15 residues showed more robust protein trans-splicing activity than naturally occurring split DnaE inteins in a foreign context. During the course of our experiments, we found that protein ligation by protein trans-splicing depended not only on the splicing junction sequences, but also on the foreign extein sequences. Furthermore, we could classify the protein trans-splicing reactions in foreign contexts with a simple kinetic model into three groups according to their kinetic parameters in the presence of various reducing agents. CONCLUSION: The shorter C-intein of the newly engineered split intein could be a useful tool for biotechnological applications including protein modification, incorporation of chemical probes, and segmental isotopic labelling. Based on kinetic analysis of the protein splicing reactions, we propose a general strategy to improve ligation yields by protein trans-splicing, which could significantly enhance the applications of protein ligation by protein trans-splicing.

  18. Genetically engineered, biarsenically labeled influenza virus allows visualization of viral NS1 protein in living cells.

    Science.gov (United States)

    Li, Yang; Lu, Xinya; Li, Junwei; Bérubé, Nathalie; Giest, Kerri-Lane; Liu, Qiang; Anderson, Deborah H; Zhou, Yan

    2010-07-01

    Real-time fluorescence imaging of viral proteins in living cells provides a valuable means to study virus-host interactions. The challenge of generating replication-competent fluorescent influenza A virus is that the segmented genome does not allow fusion of a fluorescent protein gene to any viral gene. Here, we introduced the tetracysteine (TC) biarsenical labeling system into influenza virus in order to fluorescently label viral protein in the virus life cycle. We generated infectious influenza A viruses bearing a small TC tag (CCPGCC) in the loop/linker regions of the NS1 proteins. In the background of A/Puerto Rico/8/34 (H1N1) (PR8) virus, the TC tag can be inserted into NS1 after amino acid 52 (AA52) (PR8-410), AA79 (PR8-412), or AA102 (PR8-413) or the TC tag can be inserted and replace amino acids 79 to 84 (AA79-84) (PR8-411). Although PR8-410, PR8-411, and PR8-412 viruses are attenuated than the wild-type (WT) virus to some extent in multiple-cycle infection, their growth potential is similar to that of the WT virus during a single cycle of infection, and their NS1 subcellular localization and viral protein synthesis rate are quite similar to those of the WT virus. Furthermore, labeling with membrane-permeable biarsenical dye resulted in fluorescent NS1 protein in the context of virus infection. We could exploit this strategy on NS1 protein of A/Texas/36/91 (H1N1) (Tx91) by successfully rescuing a TC-tagged virus, Tx91-445, which carries the TC tag replacement of AA79-84. The infectivity of Tx91-445 virus was similar to that of WT Tx91 during multiple cycles of replication and a single cycle of replication. The NS1 protein derived from Tx91-445 can be fluorescently labeled in living cells. Finally, with biarsenical labeling, the engineered replication-competent virus allowed us to visualize NS1 protein nuclear import in virus-infected cells in real time.

  19. Improvement of paracellular transport in the Caco-2 drug screening model using protein-engineered substrates.

    Science.gov (United States)

    DiMarco, Rebecca L; Hunt, Daniel R; Dewi, Ruby E; Heilshorn, Sarah C

    2017-06-01

    The Caco-2 assay has achieved wide popularity among pharmaceutical companies in the past two decades as an in vitro method for estimation of in vivo oral bioavailability of pharmaceutical compounds during preclinical characterization. Despite its popularity, this assay suffers from a severe underprediction of the transport of drugs which are absorbed paracellularly, that is, which pass through the cell-cell tight junctions of the absorptive cells of the small intestine. Here, we propose that simply replacing the collagen I matrix employed in the standard Caco-2 assay with an engineered matrix, we can control cell morphology and hence regulate the cell-cell junctions that dictate paracellular transport. Specifically, we use a biomimetic engineered extracellular matrix (eECM) that contains modular protein domains derived from two ECM proteins found in the small intestine, fibronectin and elastin. This eECM allows us to independently tune the density of cell-adhesive RGD ligands presented to Caco-2 cells as well as the mechanical stiffness of the eECM. We observe that lower amounts of RGD ligand presentation as well as decreased matrix stiffness results in Caco-2 morphologies that more closely resemble primary small intestinal epithelial cells than Caco-2 cells cultured on collagen. Additionally, these matrices result in Caco-2 monolayers with decreased recruitment of actin to the apical junctional complex and increased expression of claudin-2, a tight junction protein associated with higher paracellular permeability that is highly expressed throughout the small intestine. Consistent with these morphological differences, drugs known to be paracellularly transported in vivo exhibited significantly improved transport rates in this modified Caco-2 model. As expected, permeability of transcellularly transported drugs remained unaffected. Thus, we have demonstrated a method of improving the physiological accuracy of the Caco-2 assay that could be readily adopted by

  20. Non-chromatographic bioprocess engineering of a recombinant mineralizing protein for the synthesis of silica nanocapsules.

    Science.gov (United States)

    Wibowo, David; Yang, Guang-Ze; Middelberg, Anton P J; Zhao, Chun-Xia

    2017-02-01

    yield of D4S2 protein is increased fourfold in this study. The bio-produced protein D4S2 was proved to retain it emulsification and biosilicification functionalities enabling the formation of oil-core silica-shell nanocapsules at near-neutral pH and room temperature without the use of any toxic organic solvents, confirming no adverse effects due to bioprocess simplification. This work demonstrates that, through proper bioprocess engineering including the removal of critical contaminants such as DNA, a more efficient, simple, and scalable purification process can be used for the high-yield bio-production of a recombinant templating protein useful in the synthesis of bio-inspired nanomaterials. This simplified process is expected to be easily adapted to recover other mineralizing helix bundle-based functional proteins from microbial cell factories. Biotechnol. Bioeng. 2017;114: 335-343. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  1. Virus-Like Particles Derived from HIV-1 for Delivery of Nuclear Proteins: Improvement of Production and Activity by Protein Engineering.

    Science.gov (United States)

    Robert, Marc-André; Lytvyn, Viktoria; Deforet, Francis; Gilbert, Rénald; Gaillet, Bruno

    2017-01-01

    Virus-like particles (VLPs) derived from retroviruses and lentiviruses can be used to deliver recombinant proteins without the fear of causing insertional mutagenesis to the host cell genome. In this study we evaluate the potential of an inducible lentiviral vector packaging cell line for VLP production. The Gag gene from HIV-1 was fused to a gene encoding a selected protein and it was transfected into the packaging cells. Three proteins served as model: the green fluorescent protein and two transcription factors-the cumate transactivator (cTA) of the inducible CR5 promoter and the human Krüppel-like factor 4 (KLF4). The sizes of the VLPs were 120-150 nm in diameter and they were resistant to freeze/thaw cycles. Protein delivery by the VLPs reached up to 100% efficacy in human cells and was well tolerated. Gag-cTA triggered up to 1100-fold gene activation of the reporter gene in comparison to the negative control. Protein engineering was required to detect Gag-KLF4 activity. Thus, insertion of the VP16 transactivation domain increased the activity of the VLPs by eightfold. An additional 2.4-fold enhancement was obtained by inserting nuclear export signal. In conclusion, our platform produced VLPs capable of efficient protein transfer, and it was shown that protein engineering can be used to improve the activity of the delivered proteins as well as VLP production.

  2. Engineering an effective Mn-binding MRI reporter protein by subcellular targeting.

    Science.gov (United States)

    Bartelle, Benjamin B; Mana, Miyeko D; Suero-Abreu, Giselle A; Rodriguez, Joe J; Turnbull, Daniel H

    2015-12-01

    Manganese (Mn) is an effective contrast agent and biologically active metal, which has been widely used for Mn-enhanced MRI (MEMRI). The purpose of this study was to develop and test a Mn binding protein for use as a genetic reporter for MEMRI. The bacterial Mn-binding protein, MntR was identified as a candidate reporter protein. MntR was engineered for expression in mammalian cells, and targeted to different subcellular organelles, including the Golgi Apparatus where cellular Mn is enriched. Transfected HEK293 cells and B16 melanoma cells were tested in vitro and in vivo, using immunocytochemistry, MR imaging and relaxometry. Subcellular targeting of MntR to the cytosol, endoplasmic reticulum and Golgi apparatus was verified with immunocytochemistry. After targeting to the Golgi, MntR expression produced robust R1 changes and T1 contrast in cells, in vitro and in vivo. Co-expression with the divalent metal transporter DMT1, a previously described Mn-based reporter, further enhanced contrast in B16 cells in culture, but in the in vivo B16 tumor model tested was not significantly better than MntR alone. This second-generation reporter system both expands the capabilities of genetically encoded reporters for imaging with MEMRI and provides important insights into the mechanisms of Mn biology which create endogenous MEMRI contrast. © 2014 Wiley Periodicals, Inc.

  3. Engineering multifunctional protein nanoparticles by in vitro disassembling and reassembling of heterologous building blocks

    Science.gov (United States)

    Unzueta, Ugutz; Serna, Naroa; Sánchez-García, Laura; Roldán, Mónica; Sánchez-Chardi, Alejandro; Mangues, Ramón; Villaverde, Antonio; Vázquez, Esther

    2017-12-01

    The engineering of protein self-assembling at the nanoscale allows the generation of functional and biocompatible materials, which can be produced by easy biological fabrication. The combination of cationic and histidine-rich stretches in fusion proteins promotes oligomerization as stable protein-only regular nanoparticles that are composed by a moderate number of building blocks. Among other applications, these materials are highly appealing as tools in targeted drug delivery once empowered with peptidic ligands of cell surface receptors. In this context, we have dissected here this simple technological platform regarding the controlled disassembling and reassembling of the composing building blocks. By applying high salt and imidazole in combination, nanoparticles are disassembled in a process that is fully reversible upon removal of the disrupting agents. By taking this approach, we accomplish here the in vitro generation of hybrid nanoparticles formed by heterologous building blocks. This fact demonstrates the capability to generate multifunctional and/or multiparatopic or multispecific materials usable in nanomedical applications.

  4. ANTIBODY-CYTOKINE FUSION PROTEINS FOR TREATMENT OF CANCER: ENGINEERING CYTOKINES FOR IMPROVED EFFICACY AND SAFETY

    Science.gov (United States)

    Young, Patricia A.; Morrison, Sherie L.; Timmerman, John M.

    2014-01-01

    The true potential of cytokine therapies in cancer treatment is limited by the inability to deliver optimal concentrations into tumor sites due to dose-limiting systemic toxicities. To maximize the efficacy of cytokine therapy, recombinant antibody-cytokine fusion proteins have been constructed by a number of groups to harness the tumor-targeting ability of monoclonal antibodies. The aim is to guide cytokines specifically to tumor sites where they might stimulate more optimal anti-tumor immune responses while avoiding the systemic toxicities of free cytokine therapy. Antibody-cytokine fusion proteins containing IL-2, IL-12, IL-21, TNFα, and interferons α, β and γ have been constructed and have shown anti-tumor activity in pre-clinical and early phase clinical studies. Future priorities for development of this technology include optimization of tumor targeting, bioactivity of the fused cytokine, and choice of appropriate agents for combination therapies. This review is intended to serve as a framework for engineering an ideal antibody-cytokine fusion protein, focusing on previously developed constructs and their clinical trial results. PMID:25440607

  5. Overcoming low yields of plant-made antibodies by a protein engineering approach.

    Science.gov (United States)

    Zischewski, Julia; Sack, Markus; Fischer, Rainer

    2016-01-01

    The commercial development of plant-based antibody production platforms is often limited by low and variable yields, but little is known about the factors that affect antibody accumulation during and after translation. Here, we present a strategy to identify yield-limiting regions in the transcript and protein. We exchanged variable heavy chain (VH) domain sequences between two human antibodies at structurally conserved positions, thus creating ten chimeric VH domains containing sequences from M12 (∼1000 μg/g leaf fresh weight [FW]) and 4E10 (∼100 μg/g FW). After transient expression in Nicotiana benthamiana leaves, we measured mRNA and protein levels by quantitative real-time PCR and surface plasmon resonance spectroscopy, respectively. Transcript levels were similar for all constructs, but antibody levels ranged from ∼250 μg/g to over 2000 μg/g FW. Analysis of the expression levels showed that: i) 4E10 yields were only marginally increased by suppression of post-transcriptional gene silencing; ii) the CDR3 of 4E10 contains a protease site; and iii) a bipartite, yield-limiting region exists in the CDR2/CDR3. Our findings highlight the strong impact of cotranslational and posttranslational events on antibody yields and show that protein engineering is a powerful tool that can be used to overcome the remaining limitations affecting antibody production in plants. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. P185-M Protein Identification and Validation of Results in Workflows that Integrate over Various Instruments, Datasets, Search Engines

    Science.gov (United States)

    Hufnagel, P.; Glandorf, J.; Körting, G.; Jabs, W.; Schweiger-Hufnagel, U.; Hahner, S.; Lubeck, M.; Suckau, D.

    2007-01-01

    Analysis of complex proteomes often results in long protein lists, but falls short in measuring the validity of identification and quantification results on a greater number of proteins. Biological and technical replicates are mandatory, as is the combination of the MS data from various workflows (gels, 1D-LC, 2D-LC), instruments (TOF/TOF, trap, qTOF or FTMS), and search engines. We describe a database-driven study that combines two workflows, two mass spectrometers, and four search engines with protein identification following a decoy database strategy. The sample was a tryptically digested lysate (10,000 cells) of a human colorectal cancer cell line. Data from two LC-MALDI-TOF/TOF runs and a 2D-LC-ESI-trap run using capillary and nano-LC columns were submitted to the proteomics software platform ProteinScape. The combined MALDI data and the ESI data were searched using Mascot (Matrix Science), Phenyx (GeneBio), ProteinSolver (Bruker and Protagen), and Sequest (Thermo) against a decoy database generated from IPI-human in order to obtain one protein list across all workflows and search engines at a defined maximum false-positive rate of 5%. ProteinScape combined the data to one LC-MALDI and one LC-ESI dataset. The initial separate searches from the two combined datasets generated eight independent peptide lists. These were compiled into an integrated protein list using the ProteinExtractor algorithm. An initial evaluation of the generated data led to the identification of approximately 1200 proteins. Result integration on a peptide level allowed discrimination of protein isoforms that would not have been possible with a mere combination of protein lists.

  7. Branched-chain Amino Acid Biosensing Using Fluorescent Modified Engineered Leucine/Isoleucine/Valine Binding Protein

    Science.gov (United States)

    Chino, Sakura; Sakaguchi, Akane; Yamoto, Rie; Ferri, Stefano; Sode, Koji

    2007-01-01

    A novel fluorescence sensing system for branched-chain amino acids (BCAAs) was developed based on engineered leucine/isoleucine/valine-binding proteins (LIVBPs) conjugated with environmentally sensitive fluorescence probes. LIVBP was cloned from Escherichia coli and Gln149Cys, Gly227Cys, and Gln254Cys mutants were generated by genetic engineering. The mutant LIVBPs were then modified with environmentally sensitive fluorophores. Based on the fluorescence intensity change observed upon the binding of the ligands, the MIANS-conjugated Gln149Cys mutant (Gln149Cys-M) showed the highest and most sensitive response. The BCAAs Leu, Ile, and Val can each be monitored at the sub-micromolar level using Gln149Cys-M. Measurements were also carried out on a mixture of BCAFAs and revealed that Gln149Cys-M-based measurement is not significantly affected by the change in the molar ratio of Leu, Ile and Val in the sample. Its high sensitivity and group-specific molecular recognition ability make the new sensing system ideally suited for the measurement of BCAAs and the determination of the Fischer ratio, an indicator of hepatic disease involving metabolic dysfunction.

  8. Branched-chain Amino Acid Biosensing Using Fluorescent Modified Engineered Leucine/Isoleucine/Valine Binding Protein

    Directory of Open Access Journals (Sweden)

    Koji Sode

    2007-06-01

    Full Text Available A novel fluorescence sensing system for branched-chain amino acids (BCAAswas developed based on engineered leucine/isoleucine/valine-binding proteins (LIVBPsconjugated with environmentally sensitive fluorescence probes. LIVBP was cloned fromEscherichia coli and Gln149Cys, Gly227Cys, and Gln254Cys mutants were generated bygenetic engineering. The mutant LIVBPs were then modified with environmentallysensitive fluorophores. Based on the fluorescence intensity change observed upon thebinding of the ligands, the MIANS-conjugated Gln149Cys mutant (Gln149Cys-M showedthe highest and most sensitive response. The BCAAs Leu, Ile, and Val can each bemonitored at the sub-micromolar level using Gln149Cys-M. Measurements were alsocarried out on a mixture of BCAFAs and revealed that Gln149Cys-M-based measurementis not significantly affected by the change in the molar ratio of Leu, Ile and Val in thesample. Its high sensitivity and group-specific molecular recognition ability make the newsensing system ideally suited for the measurement of BCAAs and the determination of theFischer ratio, an indicator of hepatic disease involving metabolic dysfunction.

  9. Protein engineering applications of industrially exploitable enzymes: Geobacillus stearothermophilus LDH and Candida methylica FDH.

    Science.gov (United States)

    Karagüler, N G; Sessions, R B; Binay, B; Ordu, E B; Clarke, A R

    2007-12-01

    Enzymes have become important tools in several industries due to their ability to produce chirally pure and complex molecules with interesting biological properties. The NAD(+)-dependent LDH (lactate dehydrogenase) [bsLDH [Geobacillus stearothermophilus (formerly Bacillus stearothermophilus) LDH] from G. stearothermophilus and the NAD(+)-dependent FDH (formate dehydrogenase) [cmFDH (Candida methylica FDH)] enzyme from C. methylica are particularly crucial enzymes in the pharmaceutical industry and are related to each other in terms of NADH use and regeneration. LDH catalyses the interconversion of pyruvate (oxo acid) and lactate (alpha-hydroxy acid) using the NADH/NAD(+) pair as a redox cofactor. Employing LDH to reduce other oxo acids can generate chirally pure alpha-hydroxy acids of use in the production of pharmaceuticals. One important use of FDH is to regenerate the relatively expensive NADH cofactor that is used by NAD(+)-dependent oxidoreductases such as LDH. Both LDH and FDH from organisms of interest were previously cloned and overproduced. Therefore they are available at a low cost. However, both of these enzymes show disadvantages in the large-scale production of chirally pure compounds. We have applied two routes of protein engineering studies to improve the properties of these two enzymes, namely DNA shuffling and site-directed mutagenesis. Altering the substrate specificity of bsLDH by DNA shuffling and changing the coenzyme specificity of cmFDH by site-directed mutagenesis are the most successful examples of our studies. The present paper will also include the details of these examples together with some other applications of protein engineering regarding these enzymes.

  10. Discovery of Klotho peptide antagonists against Wnt3 and Wnt3a target proteins using combination of protein engineering, protein-protein docking, peptide docking and molecular dynamics simulations.

    Science.gov (United States)

    Mirza, Shaher Bano; Ekhteiari Salmas, Ramin; Fatmi, M Qaiser; Durdagi, Serdar

    2017-12-01

    The Klotho is known as lifespan enhancing protein involved in antagonizing the effect of Wnt proteins. Wnt proteins are stem cell regulators, and uninterrupted exposure of Wnt proteins to the cell can cause stem and progenitor cell senescence, which may lead to aging. Keeping in mind the importance of Klotho in Wnt signaling, in silico approaches have been applied to study the important interactions between Klotho and Wnt3 and Wnt3a (wingless-type mouse mammary tumor virus (MMTV) integration site family members 3 and 3a). The main aim of the study is to identify important residues of the Klotho that help in designing peptides which can act as Wnt antagonists. For this aim, a protein engineering study is performed for Klotho, Wnt3 and Wnt3a. During the theoretical analysis of homology models, unexpected role of number of disulfide bonds and secondary structure elements has been witnessed in case of Wnt3 and Wnt3a proteins. Different in silico experiments were carried out to observe the effect of correct number of disulfide bonds on 3D protein models. For this aim, total of 10 molecular dynamics (MD) simulations were carried out for each system. Based on the protein-protein docking simulations of selected protein models of Klotho with Wnt3 and Wnt3a, different peptides derived from Klotho have been designed. Wnt3 and Wnt3a proteins have three important domains: Index finger, N-terminal domain and a patch of ∼10 residues on the solvent exposed surface of palm domain. Protein-peptide docking of designed peptides of Klotho against three important domains of palmitoylated Wnt3 and Wnt3a yields encouraging results and leads better understanding of the Wnt protein inhibition by proposed Klotho peptides. Further in vitro studies can be carried out to verify effects of novel designed peptides as Wnt antagonists.

  11. Tissue-engineered matrices as functional delivery systems: adsorption and release of bioactive proteins from degradable composite scaffolds.

    Science.gov (United States)

    Cushnie, Emily K; Khan, Yusuf M; Laurencin, Cato T

    2010-08-01

    A tissue-engineered bone graft should imitate the ideal autograft in both form and function. However, biomaterials that have appropriate chemical and mechanical properties for grafting applications often lack biological components that may enhance regeneration. The concept of adding proteins such as growth factors to scaffolds has therefore emerged as a possible solution to improve overall graft design. In this study, we investigated this concept by loading porous hydroxyapatite-poly(lactide-co-glycolide) (HA-PLAGA) scaffolds with a model protein, cytochrome c, and then studying its release in a phosphate-buffered saline solution. The HA-PLAGA scaffold has previously been shown to be bioactive, osteoconductive, and to have appropriate physical properties for tissue engineering applications. The loading experiments demonstrated that the HA-PLAGA scaffold could also function effectively as a substrate for protein adsorption and release. Scaffold protein adsorptive loading (as opposed to physical entrapment within the matrix) was directly related to levels of scaffold HA-content. The HA phase of the scaffold facilitated protein retention in the matrix following incubation in aqueous buffer for periods up to 8 weeks. Greater levels of protein retention time may improve the protein's effective activity by increasing the probability for protein-cell interactions. The ability to control protein loading and delivery simply via composition of the HA-PLAGA scaffold offers the potential of forming robust functionalized bone grafts. (c) 2010 Wiley Periodicals, Inc.

  12. Technologies for Protein Analysis and Tissue Engineering, with Applications in Cancer

    Science.gov (United States)

    Vermesh, Udi Benjamin

    The first part of this thesis describes electrolyte transport through an array of 20 nm wide, 20 mum long SiO2 nanofluidic transistors. At sufficiently low ionic strength, the Debye screening length exceeds the channel width, and ion transport is limited by the negatively charged channel surfaces. At source-drain biases > 5 V, the current exhibits a sharp, nonlinear increase, with a 20 - 50-fold conductance enhancement. This behavior is attributed to a breakdown of the zero-slip condition. Implications for peptide sequencing as well as energy conversion devices are discussed. The next part describes a technology for the detection of the highly aggressive brain cancer glioblastoma multiforme (GBM). In this study, we used an antibody-based microarray to compare plasma samples from glioblastoma patients and healthy controls with respect to the plasma levels of 35 different proteins known to be generally associated with tumor growth, survival, invasion, migration, and immune regulation. Average-linkage hierarchical clustering of the patient data stratified the two groups effectively, permitting accurate assignment of test samples into either GBM or healthy control groups with a sensitivity and specificity as high as 90 % and 94 %, respectively. Using the same 35-protein panel, we then analyzed plasma samples from GBM patients who were treated with the chemotherapeutic drug Avastin (Bevacizumab) and were able to effectively stratify patients based on treatment-responsiveness. Finally, single-cell resolution patterning of tissue engineered structures is demonstrated. The proper functioning of engineered constructs for tissue and organ transplantation requires positioning different cell types in anatomically precise arrangements that mimic their configurations in native tissues. Toward this end, we have developed a technique that involves two microfluidic-patterning steps run perpendicularly to each other using "anchor" and "bridge" DNA oligomers to create dense arrays of

  13. Direct protein quantification in complex sample solutions by surface-engineered nanorod probes

    KAUST Repository

    Schrittwieser, Stefan

    2017-06-30

    Detecting biomarkers from complex sample solutions is the key objective of molecular diagnostics. Being able to do so in a simple approach that does not require laborious sample preparation, sophisticated equipment and trained staff is vital for point-of-care applications. Here, we report on the specific detection of the breast cancer biomarker sHER2 directly from serum and saliva samples by a nanorod-based homogeneous biosensing approach, which is easy to operate as it only requires mixing of the samples with the nanorod probes. By careful nanorod surface engineering and homogeneous assay design, we demonstrate that the formation of a protein corona around the nanoparticles does not limit the applicability of our detection method, but on the contrary enables us to conduct in-situ reference measurements, thus further strengthening the point-of-care applicability of our method. Making use of sandwich assays on top of the nanorods, we obtain a limit of detection of 110 pM and 470 pM in 10-fold diluted spiked saliva and serum samples, respectively. In conclusion, our results open up numerous applications in direct protein biomarker quantification, specifically in point-of-care settings where resources are limited and ease-of-use is of essence.

  14. Protein-engineered block-copolymers as stem cell delivery vehicles

    Science.gov (United States)

    Heilshorn, Sarah

    2015-03-01

    Stem cell transplantation is a promising therapy for a myriad of debilitating diseases and injuries; however, current delivery protocols are inadequate. Transplantation by direct injection, which is clinically preferred for its minimal invasiveness, commonly results in less than 5% cell viability, greatly inhibiting clinical outcomes. We demonstrate that mechanical membrane disruption results in significant acute loss of viability at clinically relevant injection rates. As a strategy to protect cells from these damaging forces, we show that cell encapsulation within hydrogels of specific mechanical properties will significantly improve viability. Building on these fundamental studies, we have designed a reproducible, bio-resorbable, customizable hydrogel using protein-engineering technology. In our Mixing-Induced Two-Component Hydrogel (MITCH), network assembly is driven by specific and stoichiometric peptide-peptide binding interactions. By integrating protein science methodologies with simple polymer physics models, we manipulate the polypeptide chain interactions and demonstrate the direct ability to tune the network crosslinking density, sol-gel phase behavior, and gel mechanics. This is in contrast to many other physical hydrogels, where predictable tuning of bulk mechanics from the molecular level remains elusive due to the reliance on non-specific and non-stoichiometric chain interactions for network formation. Furthermore, the hydrogel network can be easily modified to deliver a variety of bioactive payloads including growth factors, peptide drugs, and hydroxyapatite nanoparticles. Through a series of in vitro and in vivo studies, we demonstrate that these materials may significantly improve transplanted stem cell retention and function.

  15. Lipases from the genus Rhizopus: Characteristics, expression, protein engineering and application.

    Science.gov (United States)

    Yu, Xiao-Wei; Xu, Yan; Xiao, Rong

    2016-10-01

    Lipases are versatile catalysts that hydrolyze ester bonds of water-insoluble glycerides or carry out reversible reactions at the water/lipid interface. The remarkable characteristics of lipases from the genus Rhizopus are their high sn-1,3-positional specificity, enantioselectivity and activity in nonaqueous media, which make them one of the most desirable enzymes for many applications, including lipid modification and biodiesel and chiral organic compound synthesis. sn-1,3-Position-specific Rhizopus lipases are particularly useful for the production of structured triacylglycerols. Significant progress has been made regarding lipases from the genus Rhizopus, including gene sequencing, elucidation of the protein structure and catalytic function, heterologous expression and redesigning Rhizopus lipases for valuable properties, which is receiving increasing academic and industrial attention. In this review, we present a comprehensive overview of Rhizopus lipases, focusing on (a) the characteristics of Rhizopus lipases, (b) Rhizopus lipase genes and structural features, (c) strategies for heterologous expression of Rhizopus lipase genes in yeast system, (d) progress in protein engineering for the improvement of the properties of Rhizopus lipases, and (e) development of biotechnological applications. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Automating gene library synthesis by structure-based combinatorial protein engineering: examples from plant sesquiterpene synthases.

    Science.gov (United States)

    Dokarry, Melissa; Laurendon, Caroline; O'Maille, Paul E

    2012-01-01

    Structure-based combinatorial protein engineering (SCOPE) is a homology-independent recombination method to create multiple crossover gene libraries by assembling defined combinations of structural elements ranging from single mutations to domains of protein structure. SCOPE was originally inspired by DNA shuffling, which mimics recombination during meiosis, where mutations from parental genes are "shuffled" to create novel combinations in the resulting progeny. DNA shuffling utilizes sequence identity between parental genes to mediate template-switching events (the annealing and extension of one parental gene fragment on another) in PCR reassembly reactions to generate crossovers and hence recombination between parental genes. In light of the conservation of protein structure and degeneracy of sequence, SCOPE was developed to enable the "shuffling" of distantly related genes with no requirement for sequence identity. The central principle involves the use of oligonucleotides to encode for crossover regions to choreograph template-switching events during PCR assembly of gene fragments to create chimeric genes. This approach was initially developed to create libraries of hybrid DNA polymerases from distantly related parents, and later developed to create a combinatorial mutant library of sesquiterpene synthases to explore the catalytic landscapes underlying the functional divergence of related enzymes. This chapter presents a simplified protocol of SCOPE that can be integrated with different mutagenesis techniques and is suitable for automation by liquid-handling robots. Two examples are presented to illustrate the application of SCOPE to create gene libraries using plant sesquiterpene synthases as the model system. In the first example, we outline how to create an active-site library as a series of complex mixtures of diverse mutants. In the second example, we outline how to create a focused library as an array of individual clones to distil minimal combinations of

  17. Engineering of an E. coli outer membrane protein FhuA with increased channel diameter

    Directory of Open Access Journals (Sweden)

    Dworeck Tamara

    2011-08-01

    Full Text Available Abstract Background Channel proteins like FhuA can be an alternative to artificial chemically synthesized nanopores. To reach such goals, channel proteins must be flexible enough to be modified in their geometry, i.e. length and diameter. As continuation of a previous study in which we addressed the lengthening of the channel, here we report the increasing of the channel diameter by genetic engineering. Results The FhuA Δ1-159 diameter increase has been obtained by doubling the amino acid sequence of the first two N-terminal β-strands, resulting in variant FhuA Δ1-159 Exp. The total number of β-strands increased from 22 to 24 and the channel surface area is expected to increase by ~16%. The secondary structure analysis by circular dichroism (CD spectroscopy shows a high β-sheet content, suggesting the correct folding of FhuA Δ1-159 Exp. To further prove the FhuA Δ1-159 Exp channel functionality, kinetic measurement using the HRP-TMB assay (HRP = Horse Radish Peroxidase, TMB = 3,3',5,5'-tetramethylbenzidine were conducted. The results indicated a 17% faster diffusion kinetic for FhuA Δ1-159 Exp as compared to FhuA Δ1-159, well correlated to the expected channel surface area increase of ~16%. Conclusion In this study using a simple "semi rational" approach the FhuA Δ1-159 diameter was enlarged. By combining the actual results with the previous ones on the FhuA Δ1-159 lengthening a new set of synthetic nanochannels with desired lengths and diameters can be produced, broadening the FhuA Δ1-159 applications. As large scale protein production is possible our approach can give a contribution to nanochannel industrial applications.

  18. Structure and Function of a Bacterial Microcompartment Shell Protein Engineered to Bind a [4Fe-4S] Cluster.

    Science.gov (United States)

    Aussignargues, Clément; Pandelia, Maria-Eirini; Sutter, Markus; Plegaria, Jefferson S; Zarzycki, Jan; Turmo, Aiko; Huang, Jingcheng; Ducat, Daniel C; Hegg, Eric L; Gibney, Brian R; Kerfeld, Cheryl A

    2016-04-27

    Bacterial microcompartments (BMCs) are self-assembling organelles composed of a selectively permeable protein shell and encapsulated enzymes. They are considered promising templates for the engineering of designed bionanoreactors for biotechnology. In particular, encapsulation of oxidoreductive reactions requiring electron transfer between the lumen of the BMC and the cytosol relies on the ability to conduct electrons across the shell. We determined the crystal structure of a component protein of a synthetic BMC shell, which informed the rational design of a [4Fe-4S] cluster-binding site in its pore. We also solved the structure of the [4Fe-4S] cluster-bound, engineered protein to 1.8 Å resolution, providing the first structure of a BMC shell protein containing a metal center. The [4Fe-4S] cluster was characterized by optical and EPR spectroscopies; it has a reduction potential of -370 mV vs the standard hydrogen electrode (SHE) and is stable through redox cycling. This remarkable stability may be attributable to the hydrogen-bonding network provided by the main chain of the protein scaffold. The properties of the [4Fe-4S] cluster resemble those in low-potential bacterial ferredoxins, while its ligation to three cysteine residues is reminiscent of enzymes such as aconitase and radical S-adenosymethionine (SAM) enzymes. This engineered shell protein provides the foundation for conferring electron-transfer functionality to BMC shells.

  19. Two novel variants of human medium chain acyl-CoA dehydrogenase (MCAD). K364R, a folding mutation, and R256T, a catalytic-site mutation resulting in a well-folded but totally inactive protein

    DEFF Research Database (Denmark)

    O'Reilly, Linda P; Andresen, Brage S; Engel, Paul C

    2005-01-01

    the gene for K364R was overexpressed in Escherichia coli, the synthesized mutant protein only exhibited activity when the gene for chaperonin GroELS was co-overexpressed. Levels of activity correlated with the amounts of native MCAD protein visible in western blots. The R256T mutant, by contrast, displayed...... no activity either with or without chaperonin, but in this case a strong MCAD protein band was seen in the western blots throughout. The proteins were also purified, and the enzyme function and thermostability investigated. The K364R protein showed only moderate kinetic impairment, whereas the R256T protein...... was again totally inactive. Neither mutant showed marked depletion of FAD. The pure K364R protein was considerably less thermostable than wild-type MCAD. Western blots indicated that, although the R256T mutant protein is less thermostable than normal MCAD, it is much more stable than K364R. Though...

  20. Delivering Single-Walled Carbon Nanotubes to the Nucleus Using Engineered Nuclear Protein Domains.

    Science.gov (United States)

    Boyer, Patrick D; Ganesh, Sairaam; Qin, Zhao; Holt, Brian D; Buehler, Markus J; Islam, Mohammad F; Dahl, Kris Noel

    2016-02-10

    Single-walled carbon nanotubes (SWCNTs) have great potential for cell-based therapies due to their unique intrinsic optical and physical characteristics. Consequently, broad classes of dispersants have been identified that individually suspend SWCNTs in water and cell media in addition to reducing nanotube toxicity to cells. Unambiguous control and verification of the localization and distribution of SWCNTs within cells, particularly to the nucleus, is needed to advance subcellular technologies utilizing nanotubes. Here we report delivery of SWCNTs to the nucleus by noncovalently attaching the tail domain of the nuclear protein lamin B1 (LB1), which we engineer from the full-length LMNB1 cDNA. More than half of this low molecular weight globular protein is intrinsically disordered but has an immunoglobulin-fold composed of a central hydrophobic core, which is highly suitable for associating with SWCNTs, stably suspending SWCNTs in water and cell media. In addition, LB1 has an exposed nuclear localization sequence to promote active nuclear import of SWCNTs. These SWCNTs-LB1 dispersions in water and cell media display near-infrared (NIR) absorption spectra with sharp van Hove peaks and an NIR fluorescence spectra, suggesting that LB1 individually disperses nanotubes. The dispersing capability of SWCNTs by LB1 is similar to that by albumin proteins. The SWCNTs-LB1 dispersions with concentrations ≥150 μg/mL (≥30 μg/mL) in water (cell media) remain stable for ≥75 days (≥3 days) at 4 °C (37 °C). Further, molecular dynamics modeling of association of LB1 with SWCNTs reveal that the exposure of the nuclear localization sequence is independent of LB1 binding conformation. Measurements from confocal Raman spectroscopy and microscopy, NIR fluorescence imaging of SWCNTs, and fluorescence lifetime imaging microscopy show that millions of these SWCNTs-LB1 complexes enter HeLa cells, localize to the nucleus of cells, and interact with DNA. We postulate that the

  1. Sequence-engineered mRNA Without Chemical Nucleoside Modifications Enables an Effective Protein Therapy in Large Animals

    Science.gov (United States)

    Thess, Andreas; Grund, Stefanie; Mui, Barbara L; Hope, Michael J; Baumhof, Patrick; Fotin-Mleczek, Mariola; Schlake, Thomas

    2015-01-01

    Being a transient carrier of genetic information, mRNA could be a versatile, flexible, and safe means for protein therapies. While recent findings highlight the enormous therapeutic potential of mRNA, evidence that mRNA-based protein therapies are feasible beyond small animals such as mice is still lacking. Previous studies imply that mRNA therapeutics require chemical nucleoside modifications to obtain sufficient protein expression and avoid activation of the innate immune system. Here we show that chemically unmodified mRNA can achieve those goals as well by applying sequence-engineered molecules. Using erythropoietin (EPO) driven production of red blood cells as the biological model, engineered Epo mRNA elicited meaningful physiological responses from mice to nonhuman primates. Even in pigs of about 20 kg in weight, a single adequate dose of engineered mRNA encapsulated in lipid nanoparticles (LNPs) induced high systemic Epo levels and strong physiological effects. Our results demonstrate that sequence-engineered mRNA has the potential to revolutionize human protein therapies. PMID:26050989

  2. Protein design and engineering of a de novo pathway for microbial production of 1,3-propanediol from glucose.

    Science.gov (United States)

    Chen, Zhen; Geng, Feng; Zeng, An-Ping

    2015-02-01

    Protein engineering to expand the substrate spectrum of native enzymes opens new possibilities for bioproduction of valuable chemicals from non-natural pathways. No natural microorganism can directly use sugars to produce 1,3-propanediol (PDO). Here, we present a de novo route for the biosynthesis of PDO from sugar, which may overcome the mentioned limitations by expanding the homoserine synthesis pathway. The accomplishment of pathway from homoserine to PDO is achieved by protein engineering of glutamate dehydrogenase (GDH) and pyruvate decarboxylase to sequentially convert homoserine to 4-hydroxy-2-ketobutyrate and 3-hydroxypropionaldehyde. The latter is finally converted to PDO by using a native alcohol dehydrogenase. In this work, we report on experimental accomplishment of this non-natural pathway, especially by protein engineering of GDH for the key step of converting homoserine to 4-hydroxy-2-ketobutyrate. These results show the feasibility and significance of protein engineering for de novo pathway design and overproduction of desired industrial products. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Applications of Engineered DNA-Binding Molecules Such as TAL Proteins and the CRISPR/Cas System in Biology Research

    Directory of Open Access Journals (Sweden)

    Toshitsugu Fujita

    2015-09-01

    Full Text Available Engineered DNA-binding molecules such as transcription activator-like effector (TAL or TALE proteins and the clustered regularly interspaced short palindromic repeats (CRISPR and CRISPR-associated proteins (Cas (CRISPR/Cas system have been used extensively for genome editing in cells of various types and species. The sequence-specific DNA-binding activities of these engineered DNA-binding molecules can also be utilized for other purposes, such as transcriptional activation, transcriptional repression, chromatin modification, visualization of genomic regions, and isolation of chromatin in a locus-specific manner. In this review, we describe applications of these engineered DNA-binding molecules for biological purposes other than genome editing.

  4. Designing Improved Enzymes of industrial application from marine microorganisms using Protein Engineering

    Science.gov (United States)

    Prajapati, A. S.; Panchal, K.; Subramanian, R. B.; Patel, D. H.; Sudhir, A. P.; Dave, B. R.

    2015-12-01

    Global demand for energy has grown with the development of new industries, requiring constant improvement and search for new sources of energy. One of the challenges today is releasing the energy of glucose that nature has cleverly locked into lignocellulosic biomass. Potent and efficient enzyme preparations need to be developed for the enzymatic saccharification process to be more economical. Approaches like enzyme engineering, reconstitution of enzyme mixtures and bioprospecting for superior enzymes are gaining importance. The ocean is considered to be a great reservoir of biodiversity. Because enzymes have unequalled advantages, many industries are keenly interested in adapting enzymatic methods for their processes. Microbial communities in marine environments are ecologically relevant as intermediaries of energy and play an important role in nutrient regeneration cycles as decomposers of dead and decaying organic matter. The exploitation of marine bacteria in the search for improved enzymes or strategies provides a means to upgrade feasibility for lignocellulosic biomass conversion, ultimately providing means to a 'greener' technology. Several industrial enzymes are derived from terrestrial sources, whereas, marine environment which encompasses about 71 percent of the earth's surface and a vast resources for useful enzymes, remain unexplored. Marine microorganisms take active part in the mineralization of complex organic matter through degradative pathways of their metabolism. Bacteria from marine environments secrete different enzymes based on their habitat and their ecological functions. Therefore marine microbial enzymes have become the focal point of interest. Even though many of these enzymes are being isolated, the efficiency of hydrolysis is very poor. This could be overcome by altering the substrate specificity of lignocellulases. Protein engineering could prove to be useful to improve the catalytic function these enzymes.

  5. A General Method for Intracellular Protein Delivery through 'E-tag' Protein Engineering and Arginine Functionalized Gold Nanoparticles.

    Science.gov (United States)

    Mout, Rubul; Rotello, Vincent M

    2017-12-20

    In this protocol, we describe a method for direct cytosolic protein delivery that avoids endosomal entrapment of the delivered proteins. We achieved this by tagging the desired protein with an oligo glutamic acid tag (E-tag), and subsequently using carrier gold nanoparticles to deliver these E-tagged proteins. When E-tagged proteins and nanoparticles were mixed, they formed nanoassemblies, which got fused to cell membrane upon incubation and directly released the E-tagged protein into cell cytosol. We used this method to deliver a wide variety of proteins with different sizes, charges, and functions in various cell lines (Mout et al., 2017). To use this protocol, the first step is to generate the required materials (gold nanoparticles, recombinant E-tagged proteins). Laboratory-synthesis of gold nanoparticles has been previously described (Yang et al., 2011). Desired E-tagged proteins can be cloned from the corresponding genes, and expressed and purified using standard laboratory procedures. We will use E-tagged green fluorescent protein (GFP) as a reference protein here. Users can simply insert an E-tag into their protein of interest, at either terminus. To achieve maximum delivery efficiency, we suggest users testing different length of E-tags. For example, we inserted E = 0 to 20 (E0 means no E-tag insertion, and E20 means 20 glutamic acids insertion in a row) to most of the proteins we tested, and screened for optimal E-tagged length for highest delivery efficiency. E10-tagged proteins gave us the highest delivery efficiency for most of the proteins (except for Cas9, where E20 tag showed highest delivery efficiency). Once these materials are ready, it takes about ~10 min to make the E-tagged protein and nanoparticle nanoassemblies, which are immediately used for delivery. Complete delivery (~100% for GFP-E10) is achieved in less than 3 h.

  6. Engineering fluorescent proteins towards ultimate performances: lessons from the newly developed cyan variants

    Science.gov (United States)

    Mérola, Fabienne; Erard, Marie; Fredj, Asma; Pasquier, Hélène

    2016-03-01

    New fluorescent proteins (FPs) are constantly discovered from natural sources, and submitted to intensive engineering based on random mutagenesis and directed evolution. However, most of these newly developed FPs fail to achieve all the performances required for their bioimaging applications. The design of highly optimised FP-based reporters, simultaneously displaying appropriate colour, multimeric state, chromophore maturation, brightness, photostability and environmental sensitivity will require a better understanding of the structural and dynamic determinants of FP photophysics. The recent development of cyan fluorescent proteins (CFPs) like mCerulean3, mTurquoise2 and Aquamarine brings a different view on these questions, as in this particular case, a step by step evaluation of critical mutations has been performed within a family of spectrally identical and evolutionary close variants. These efforts have led to CFPs with quantum yields close to unity, near single exponential emission decays, high photostability and complete insensitivity to pH, making them ideal choices as energy transfer donors in FRET and FLIM imaging applications. During this process, it was found that a proper amino-acid choice at only two positions (148 and 65) is sufficient to transform the performances of CFPs: with the help of structural and theoretical investigations, we rationalise here how these two positions critically control the CFP photophysics, in the context of FPs derived from the Aequorea victoria species. Today, these results provide a useful toolbox for upgrading the different CFP donors carried by FRET biosensors. They also trace the route towards the de novo design of FP-based optogenetic devices that will be perfectly tailored to dedicated imaging and sensing applications.

  7. Engineer Medium and Feed for Modulating N-Glycosylation of Recombinant Protein Production in CHO Cell Culture.

    Science.gov (United States)

    Fan, Yuzhou; Kildegaard, Helene Faustrup; Andersen, Mikael Rørdam

    2017-01-01

    Chinese hamster ovary (CHO) cells have become the primary expression system for the production of complex recombinant proteins due to their long-term success in industrial scale production and generating appropriate protein N-glycans similar to that of humans. Control and optimization of protein N-glycosylation is crucial, as the structure of N-glycans can largely influence both biological and physicochemical properties of recombinant proteins. Protein N-glycosylation in CHO cell culture can be controlled and tuned by engineering medium, feed, culture process, as well as genetic elements of the cell. In this chapter, we will focus on how to carry out experiments for N-glycosylation modulation through medium and feed optimization. The workflow and typical methods involved in the experiment process will be presented.

  8. Design, Engineering and Application of an Amyloidogenic Protein, SBAFP-m1, for use in Nanotechnological Applications

    Science.gov (United States)

    Peralta, Maria del Refugio

    Nanotechnology relies on collaborations across scientific disciplines such as physics, engineering, chemistry and biology. In nanotechnology, researchers manipulate molecules on the nanometer scale for various applications, ranging from tissue engineering, nanowire synthesis, and alternative energy devices. By utilizing various biological scaffolds, namely amyloid fibrils, the work of nanometer molecular control can be achieved through the use of self-assembly systems. Here, a systematic design scheme was developed to engineer protein based amyloid fibrils and was successfully applied to the design of two, unique self-assembled monomers, SBAFP-m1 and RGAFP-m1, from naturally occurring ice binding proteins found in insects and plants. A highly idealized, in-register dimer interface was designed and experimentally synthesized and demonstrated to form micron long amyloid fibrils (Chapter 2). The strength and resistance of the designer amyloid fibrils formed by SBAFP-m1 were probed in Chapter 3. Most notably, the ultimate tensile strength of SBAFP-m1 fibrils was experimentally determined to be 2.1 +/- 1.7 GPa, on par with that of naturally occurring amyloid fibrils in literature and steel. The fibrils were found to maintain their beta-sheet structure over a wide range of temperatures, from - 80 °C to 90 °C. Fibrils were resistant to common protein denaturants like 8M urea, 2.5 M guanidine hydrochloride, 2.5 M NaCl, organic solvents (methanol, ethanol, isopropanol and acetone), and across the pH range two to 11. SBAFP-m1 was mutated to add a 5x cysteine tag to the N-terminus, allowing for gold nanoparticle conjugation along the fibril axis (Chapter 4). The gold-conjugated fibrils were then enhanced with silver to produce nanowires. Various attempts to selectively synthesize heterogeneous fibrils from SBAFP-m1 mutants were attempted in Chapter 5. An attempt to de-stabilize the homogeneous fibril assembly through unfavorable homogeneous protein interactions was not

  9. Design and Construction of Artificial Extracellular Matrix (aECM) Proteins from Escherichia coli for Skin Tissue Engineering.

    Science.gov (United States)

    Low, Pearlie S J; Tjin, Monica S; Fong, Eileen

    2015-06-11

    Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins that have incorporated functional domains derived from nature (or created de novo) have been reported. In particular, artificial extracellular matrix (aECM) proteins have been developed; these aECM proteins consist of biological domains taken from fibronectin, laminins and collagens and are combined with structural domains including elastin-like repeats, silk and collagen repeats. To date, aECM proteins have been widely investigated for applications in tissue engineering and wound repair. Recently, Tjin and coworkers developed integrin-specific aECM proteins designed for promoting human skin keratinocyte attachment and propagation. In their work, the aECM proteins incorporate cell binding domains taken from fibronectin, laminin-5 and collagen IV, as well as flanking elastin-like repeats. They demonstrated that the aECM proteins developed in their work were promising candidates for use as substrates in artificial skin. Here, we outline the design and construction of such aECM proteins as well as their purification process using the thermo-responsive characteristics of elastin.

  10. Chaperonin containing T-complex polypeptide subunit eta (CCT-eta is a specific regulator of fibroblast motility and contractility.

    Directory of Open Access Journals (Sweden)

    Latha Satish

    2010-04-01

    Full Text Available Integumentary wounds in mammalian fetuses heal without scar; this scarless wound healing is intrinsic to fetal tissues and is notable for absence of the contraction seen in postnatal (adult wounds. The precise molecular signals determining the scarless phenotype remain unclear. We have previously reported that the eta subunit of the chaperonin containing T-complex polypeptide (CCT-eta is specifically reduced in healing fetal wounds in a rabbit model. In this study, we examine the role of CCT-eta in fibroblast motility and contractility, properties essential to wound healing and scar formation. We demonstrate that CCT-eta (but not CCT-beta is underexpressed in fetal fibroblasts compared to adult fibroblasts. An in vitro wound healing assay demonstrated that adult fibroblasts showed increased cell migration in response to epidermal growth factor (EGF and platelet derived growth factor (PDGF stimulation, whereas fetal fibroblasts were unresponsive. Downregulation of CCT-eta in adult fibroblasts with short inhibitory RNA (siRNA reduced cellular motility, both basal and growth factor-induced; in contrast, siRNA against CCT-beta had no such effect. Adult fibroblasts were more inherently contractile than fetal fibroblasts by cellular traction force microscopy; this contractility was increased by treatment with EGF and PDGF. CCT-eta siRNA inhibited the PDGF-induction of adult fibroblast contractility, whereas CCT-beta siRNA had no such effect. In each of these instances, the effect of downregulating CCT-eta was to modulate the behavior of adult fibroblasts so as to more closely approximate the characteristics of fetal fibroblasts. We next examined the effect of CCT-eta modulation on alpha-smooth muscle actin (alpha-SMA expression, a gene product well known to play a critical role in adult wound healing. Fetal fibroblasts were found to constitutively express less alpha-SMA than adult cells. Reduction of CCT-eta with siRNA had minimal effect on cellular

  11. Diverse Cytopathologies in Mitochondrial Disease Are Caused by AMP-activated Protein Kinase Signaling

    OpenAIRE

    Paul B. Bokko; Francione, Lisa; Bandala-Sanchez, Esther; Ahmed, Afsar U.; Sarah J. Annesley; Huang, Xiuli; Khurana, Taruna; Kimmel, Alan R.; Fisher, Paul R.

    2007-01-01

    The complex cytopathology of mitochondrial diseases is usually attributed to insufficient ATP. AMP-activated protein kinase (AMPK) is a highly sensitive cellular energy sensor that is stimulated by ATP-depleting stresses. By antisense-inhibiting chaperonin 60 expression, we produced mitochondrially diseased strains with gene dose-dependent defects in phototaxis, growth, and multicellular morphogenesis. Mitochondrial disease was phenocopied in a gene dose-dependent manner by overexpressing a c...

  12. Extremophilic 50S Ribosomal RNA-Binding Protein L35Ae as a Basis for Engineering of an Alternative Protein Scaffold.

    Science.gov (United States)

    Lomonosova, Anna V; Ovchinnikova, Elena V; Kazakov, Alexei S; Denesyuk, Alexander I; Sofin, Alexander D; Mikhailov, Roman V; Ulitin, Andrei B; Mirzabekov, Tajib A; Permyakov, Eugene A; Permyakov, Sergei E

    2015-01-01

    Due to their remarkably high structural stability, proteins from extremophiles are particularly useful in numerous biological applications. Their utility as alternative protein scaffolds could be especially valuable in small antibody mimetic engineering. These artificial binding proteins occupy a specific niche between antibodies and low molecular weight substances, paving the way for development of innovative approaches in therapeutics, diagnostics, and reagent use. Here, the 50S ribosomal RNA-binding protein L35Ae from the extremophilic archaea Pyrococcus horikoshii has been probed for its potential to serve as a backbone in alternative scaffold engineering. The recombinant wild type L35Ae has a native-like secondary structure, extreme thermal stability (mid-transition temperature of 90°C) and a moderate resistance to the denaturation by guanidine hydrochloride (half-transition at 2.6 M). Chemical crosslinking and dynamic light scattering data revealed that the wild type L35Ae protein has a propensity for multimerization and aggregation correlating with its non-specific binding to a model cell surface of HEK293 cells, as evidenced by flow cytometry. To suppress these negative features, a 10-amino acid mutant (called L35Ae 10X) was designed, which lacks the interaction with HEK293 cells, is less susceptible to aggregation, and maintains native-like secondary structure and thermal stability. However, L35Ae 10X also shows lowered resistance to guanidine hydrochloride (half-transition at 2.0M) and is more prone to oligomerization. This investigation of an extremophile protein's scaffolding potential demonstrates that lowered resistance to charged chemical denaturants and increased propensity to multimerization may limit the utility of extremophile proteins as alternative scaffolds.

  13. Extremophilic 50S Ribosomal RNA-Binding Protein L35Ae as a Basis for Engineering of an Alternative Protein Scaffold.

    Directory of Open Access Journals (Sweden)

    Anna V Lomonosova

    Full Text Available Due to their remarkably high structural stability, proteins from extremophiles are particularly useful in numerous biological applications. Their utility as alternative protein scaffolds could be especially valuable in small antibody mimetic engineering. These artificial binding proteins occupy a specific niche between antibodies and low molecular weight substances, paving the way for development of innovative approaches in therapeutics, diagnostics, and reagent use. Here, the 50S ribosomal RNA-binding protein L35Ae from the extremophilic archaea Pyrococcus horikoshii has been probed for its potential to serve as a backbone in alternative scaffold engineering. The recombinant wild type L35Ae has a native-like secondary structure, extreme thermal stability (mid-transition temperature of 90°C and a moderate resistance to the denaturation by guanidine hydrochloride (half-transition at 2.6 M. Chemical crosslinking and dynamic light scattering data revealed that the wild type L35Ae protein has a propensity for multimerization and aggregation correlating with its non-specific binding to a model cell surface of HEK293 cells, as evidenced by flow cytometry. To suppress these negative features, a 10-amino acid mutant (called L35Ae 10X was designed, which lacks the interaction with HEK293 cells, is less susceptible to aggregation, and maintains native-like secondary structure and thermal stability. However, L35Ae 10X also shows lowered resistance to guanidine hydrochloride (half-transition at 2.0M and is more prone to oligomerization. This investigation of an extremophile protein's scaffolding potential demonstrates that lowered resistance to charged chemical denaturants and increased propensity to multimerization may limit the utility of extremophile proteins as alternative scaffolds.

  14. The Search Engine for Multi-Proteoform Complexes: An Online Tool for the Identification and Stoichiometry Determination of Protein Complexes.

    Science.gov (United States)

    Skinner, Owen S; Schachner, Luis F; Kelleher, Neil L

    2016-12-08

    Recent advances in top-down mass spectrometry using native electrospray now enable the analysis of intact protein complexes with relatively small sample amounts in an untargeted mode. Here, we describe how to characterize both homo- and heteropolymeric complexes with high molecular specificity using input data produced by tandem mass spectrometry of whole protein assemblies. The tool described is a "search engine for multi-proteoform complexes," (SEMPC) and is available for free online. The output is a list of candidate multi-proteoform complexes and scoring metrics, which are used to define a distinct set of one or more unique protein subunits, their overall stoichiometry in the intact complex, and their pre- and post-translational modifications. Thus, we present an approach for the identification and characterization of intact protein complexes from native mass spectrometry data. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

  15. Structural and functional diversity among amyloid proteins: Agents of disease, building blocks of biology, and implications for molecular engineering.

    Science.gov (United States)

    Bleem, Alissa; Daggett, Valerie

    2017-01-01

    Amyloids have long been associated with protein dysfunction and neurodegenerative diseases, but recent research has demonstrated that some organisms utilize the unique properties of the amyloid fold to create functional structures with important roles in biological processes. Additionally, new engineering approaches have taken advantage of amyloid structures for implementation in a wide variety of materials and devices. In this review, the role of amyloid in human disease is discussed and compared to the functional amyloids, which serve a largely structural purpose. We then consider the use of amyloid constructs in engineering applications, including their utility as building blocks for synthetic biology and molecular engineering. Biotechnol. Bioeng. 2017;114: 7-20. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  16. Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway

    Science.gov (United States)

    2012-01-01

    Βackground The methylotrophic yeast Pichia pastoris has become an important host organism for recombinant protein production and is able to use methanol as a sole carbon source. The methanol utilization pathway describes all the catalytic reactions, which happen during methanol metabolism. Despite the importance of certain key enzymes in this pathway, so far very little is known about possible effects of overexpressing either of these key enzymes on the overall energetic behavior, the productivity and the substrate uptake rate in P. pastoris strains. Results A fast and easy-to-do approach based on batch cultivations with methanol pulses was used to characterize different P. pastoris strains. A strain with MutS phenotype was found to be superior over a strain with Mut+ phenotype in both the volumetric productivity and the efficiency in expressing recombinant horseradish peroxidase C1A. Consequently, either of the enzymes dihydroxyacetone synthase, transketolase or formaldehyde dehydrogenase, which play key roles in the methanol utilization pathway, was co-overexpressed in MutS strains harboring either of the reporter enzymes horseradish peroxidase or Candida antarctica lipase B. Although the co-overexpression of these enzymes did not change the stoichiometric yields of the recombinant MutS strains, significant changes in the specific growth rate, the specific substrate uptake rate and the specific productivity were observed. Co-overexpression of dihydroxyacetone synthase yielded a 2- to 3-fold more efficient conversion of the substrate methanol into product, but also resulted in a reduced volumetric productivity. Co-overexpression of formaldehyde dehydrogenase resulted in a 2-fold more efficient conversion of the substrate into product and at least similar volumetric productivities compared to strains without an engineered methanol utilization pathway, and thus turned out to be a valuable strategy to improve recombinant protein production. Conclusions Co

  17. Road to the future of systems biotechnology: CRISPR-Cas-mediated metabolic engineering for recombinant protein production.

    Science.gov (United States)

    Roointan, Amir; Morowvat, Mohammad Hossein

    The rising potential for CRISPR-Cas-mediated genome editing has revolutionized our strategies in basic and practical bioengineering research. It provides a predictable and precise method for genome modification in a robust and reproducible fashion. Emergence of systems biotechnology and synthetic biology approaches coupled with CRISPR-Cas technology could change the future of cell factories to possess some new features which have not been found naturally. We have discussed the possibility and versatile potentials of CRISPR-Cas technology for metabolic engineering of a recombinant host for heterologous protein production. We describe the mechanisms involved in this metabolic engineering approach and present the diverse features of its application in biotechnology and protein production.

  18. Calcium Phosphate Scaffolds Combined with Bone Morphogenetic Proteins or Mesenchymal Stem Cells in Bone Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Han Sun

    2015-01-01

    Full Text Available Objective: The purpose of this study was to review the current status of calcium phosphate (CaP scaffolds combined with bone morphogenetic proteins (BMPs or mesenchymal stem cells (MSCs in the field of bone tissue engineering (BTE. Date Sources: Data cited in this review were obtained primarily from PubMed and Medline in publications from 1979 to 2014, with highly regarded older publications also included. The terms BTE, CaP, BMPs, and MSC were used for the literature search. Study Selection: Reviews focused on relevant aspects and original articles reporting in vitro and/or in vivo results concerning the efficiency of CaP/BMPs or CaP/MSCs composites were retrieved, reviewed, analyzed, and summarized. Results: An ideal BTE product contains three elements: Scaffold, growth factors, and stem cells. CaP-based scaffolds are popular because of their outstanding biocompatibility, bioactivity, and osteoconductivity. However, they lack stiffness and osteoinductivity. To solve this problem, composite scaffolds of CaP with BMPs have been developed. New bone formation by CaP/BMP composites can reach levels similar to those of autografts. CaP scaffolds are compatible with MSCs and CaP/MSC composites exhibit excellent osteogenesis and stiffness. In addition, a CaP/MSC/BMP scaffold can repair bone defects more effectively than an autograft. Conclusions: Novel BTE products possess remarkable osteoconduction and osteoinduction capacities, and exhibit balanced degradation with osteogenesis. Further work should yield safe, viable, and efficient materials for the repair of bone lesions.

  19. Creating an Efficient Methanol-Stable Biocatalyst by Protein and Immobilization Engineering Steps towards Efficient Biosynthesis of Biodiesel.

    Science.gov (United States)

    Gihaz, Shalev; Weiser, Diána; Dror, Adi; Sátorhelyi, Péter; Jerabek-Willemsen, Moran; Poppe, László; Fishman, Ayelet

    2016-11-23

    Two ternary sol-gel matrices, an octyltriethoxysilane-based aliphatic matrix and a phenyltriethoxysilane (PTEOS)-based aromatic matrix, were used to immobilize a methanol-stable variant of lipase from Geobacillus stearothermophilus T6 for the synthesis of biodiesel from waste oil. Superior thermal stability of the mutant versus the wildtype in methanol was confirmed by intrinsic protein fluorescence measurements. The influence of skim milk and soluble E. coli lysate proteins as bulking and stabilizing agents in conjunction with sol-gel entrapment were investigated. E. coli lysate proteins were better stabilizing agents of the purified lipase mutant than skim milk, as evidenced by reverse engineering of the aromatic-based system. This was also shown for commercial Candida antarctica lipase B (CaLB) and Thermomyces lanuginosus lipase (TLL). Uniform, dense, and nonaggregated particles imaged by scanning electron microscopy and a small particle size of 13 μm pertaining to the system comprising PTEOS and E. coli lysate proteins correlated well with high esterification activity. Combining protein and immobilization engineering resulted in a durable biocatalyst with efficient recycling ability and high biodiesel conversion rates. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Can enzyme engineering benefit from the modulation of protein motions? Lessons learned from NMR relaxation dispersion experiments.

    Science.gov (United States)

    Doucet, Nicolas

    2011-04-01

    Despite impressive progress in protein engineering and design, our ability to create new and efficient enzyme activities remains a laborious and time-consuming endeavor. In the past few years, intricate combinations of rational mutagenesis, directed evolution and computational methods have paved the way to exciting engineering examples and are now offering a new perspective on the structural requirements of enzyme activity. However, these structure-function analyses are usually guided by the time-averaged static models offered by enzyme crystal structures, which often fail to describe the functionally relevant 'invisible states' adopted by proteins in space and time. To alleviate such limitations, NMR relaxation dispersion experiments coupled to mutagenesis studies have recently been applied to the study of enzyme catalysis, effectively complementing 'structure-function' analyses with 'flexibility-function' investigations. In addition to offering quantitative, site-specific information to help characterize residue motion, these NMR methods are now being applied to enzyme engineering purposes, providing a powerful tool to help characterize the effects of controlling long-range networks of flexible residues affecting enzyme function. Recent advancements in this emerging field are presented here, with particular attention to mutagenesis reports highlighting the relevance of NMR relaxation dispersion tools in enzyme engineering.

  1. Efficient myogenic differentiation of human adipose-derived stem cells by the transduction of engineered MyoD protein

    Energy Technology Data Exchange (ETDEWEB)

    Sung, Min Sun [Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806 (Korea, Republic of); Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 305-350 (Korea, Republic of); Mun, Ji-Young [Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806 (Korea, Republic of); Kwon, Ohsuk [Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806 (Korea, Republic of); Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 305-350 (Korea, Republic of); Kwon, Ki-Sun [Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806 (Korea, Republic of); Oh, Doo-Byoung, E-mail: dboh@kribb.re.kr [Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806 (Korea, Republic of); Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 305-350 (Korea, Republic of)

    2013-07-19

    Highlights: •MyoD was engineered to contain protein transduction domain and endosome-disruptive INF7 peptide. •The engineered MyoD-IT showed efficient nuclear targeting through an endosomal escape by INF7 peptide. •By applying MyoD-IT, human adipose-derived stem cells (hASCs) were differentiated into myogenic cells. •hASCs differentiated by applying MyoD-IT fused to myotubes through co-culturing with mouse myoblasts. •Myogenic differentiation using MyoD-IT is a safe method without the concern of altering the genome. -- Abstract: Human adipose-derived stem cells (hASCs) have great potential as cell sources for the treatment of muscle disorders. To provide a safe method for the myogenic differentiation of hASCs, we engineered the MyoD protein, a key transcription factor for myogenesis. The engineered MyoD (MyoD-IT) was designed to contain the TAT protein transduction domain for cell penetration and the membrane-disrupting INF7 peptide, which is an improved version of the HA2 peptide derived from influenza. MyoD-IT showed greatly improved nuclear targeting ability through an efficient endosomal escape induced by the pH-sensitive membrane disruption of the INF7 peptide. By applying MyoD-IT to a culture, hASCs were efficiently differentiated into long spindle-shaped myogenic cells expressing myosin heavy chains. Moreover, these cells differentiated by an application of MyoD-IT fused to myotubes with high efficiency through co-culturing with mouse C2C12 myoblasts. Because internalized proteins can be degraded in cells without altering the genome, the myogenic differentiation of hASCs using MyoD-IT would be a safe and clinically applicable method.

  2. Yeast Interacting Proteins Database: YOR020C, YOR020C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available YOR020C HSP10 Mitochondrial matrix co-chaperonin that inhibits the ATPase activity of Hsp60p, a mitochondrial...this bait as prey (3) YOR020C HSP10 Mitochondrial matrix co-chaperonin that inhibits the ATPase activity...gene name HSP10 Bait description Mitochondrial matrix co-chaperonin that inhibits the ATPase activity...gene name HSP10 Prey description Mitochondrial matrix co-chaperonin that inhibits the ATPase activity

  3. Integrated Proteomic Pipeline Using Multiple Search Engines for a Proteogenomic Study with a Controlled Protein False Discovery Rate.

    Science.gov (United States)

    Park, Gun Wook; Hwang, Heeyoun; Kim, Kwang Hoe; Lee, Ju Yeon; Lee, Hyun Kyoung; Park, Ji Yeong; Ji, Eun Sun; Park, Sung-Kyu Robin; Yates, John R; Kwon, Kyung-Hoon; Park, Young Mok; Lee, Hyoung-Joo; Paik, Young-Ki; Kim, Jin Young; Yoo, Jong Shin

    2016-11-04

    In the Chromosome-Centric Human Proteome Project (C-HPP), false-positive identification by peptide spectrum matches (PSMs) after database searches is a major issue for proteogenomic studies using liquid-chromatography and mass-spectrometry-based large proteomic profiling. Here we developed a simple strategy for protein identification, with a controlled false discovery rate (FDR) at the protein level, using an integrated proteomic pipeline (IPP) that consists of four engrailed steps as follows. First, using three different search engines, SEQUEST, MASCOT, and MS-GF+, individual proteomic searches were performed against the neXtProt database. Second, the search results from the PSMs were combined using statistical evaluation tools including DTASelect and Percolator. Third, the peptide search scores were converted into E-scores normalized using an in-house program. Last, ProteinInferencer was used to filter the proteins containing two or more peptides with a controlled FDR of 1.0% at the protein level. Finally, we compared the performance of the IPP to a conventional proteomic pipeline (CPP) for protein identification using a controlled FDR of <1% at the protein level. Using the IPP, a total of 5756 proteins (vs 4453 using the CPP) including 477 alternative splicing variants (vs 182 using the CPP) were identified from human hippocampal tissue. In addition, a total of 10 missing proteins (vs 7 using the CPP) were identified with two or more unique peptides, and their tryptic peptides were validated using MS/MS spectral pattern from a repository database or their corresponding synthetic peptides. This study shows that the IPP effectively improved the identification of proteins, including alternative splicing variants and missing proteins, in human hippocampal tissues for the C-HPP. All RAW files used in this study were deposited in ProteomeXchange (PXD000395).

  4. Protein cages and synthetic polymers: a fruitful symbiosis for drug delivery applications, bionanotechnology and materials science.

    Science.gov (United States)

    Rother, Martin; Nussbaumer, Martin G; Renggli, Kasper; Bruns, Nico

    2016-11-07

    Protein cages are hollow protein nanoparticles, such as viral capsids, virus-like particles, ferritin, heat-shock proteins and chaperonins. They have well-defined capsule-like structures with a monodisperse size. Their protein subunits can be modified by genetic engineering at predetermined positions, allowing for example site-selective introduction of attachment points for functional groups, catalysts or targeting ligands on their outer surface, in their interior and between subunits. Therefore, protein cages have been extensively explored as functional entities in bionanotechnology, as drug-delivery or gene-delivery vehicles, as nanoreactors or as templates for the synthesis of organic and inorganic nanomaterials. The scope of functionalities and applications of protein cages can be significantly broadened if they are combined with synthetic polymers on their surface or within their interior. For example, PEGylation reduces the immunogenicity of protein cage-based delivery systems and active targeting ligands can be attached via polymer chains to favour their accumulation in diseased tissue. Polymers within protein cages offer the possibility of increasing the loading density of drug molecules, nucleic acids, magnetic resonance imaging contrast agents or catalysts. Moreover, the interaction of protein cages and polymers can be used to modulate the size and shape of some viral capsids to generate structures that do not occur with native viruses. Another possibility is to use the interior of polymer cages as a confined reaction space for polymerization reactions such as atom transfer radical polymerization or rhodium-catalysed polymerization of phenylacetylene. The protein nanoreactors facilitate a higher degree of control over polymer synthesis. This review will summarize the hybrid structures that have been synthesized by polymerizing from protein cage-bound initiators, by conjugating polymers to protein cages, by embedding protein cages into bulk polymeric

  5. Biological properties of coral GFP-type proteins provide clues for engineering novel optical probes and biosensors

    Science.gov (United States)

    Salih, Anya; Larkum, Anthony W.; Cronin, Thomas W.; Wiedenmann, Joerg; Szymczak, Ron; Cox, Guy C.

    2004-06-01

    In recent years, a variety of Green Fluorescent Protein (GFP)-like pigments have been discovered from corals and other marine organisms. They are widely used to expand the range of available GFP-type proteins in imaging applications, such as in vivo markers for gene expression and protein localization studies, FRET-based (Förster resonance energy transfer) multicolor imaging and biosensors. They have known diverse optical and biochemical properties but their in vivo spectral properties and biological function in marine organisms is only beginning to be understood. We have investigated their spectral diversity, optical properties and cellular microstructure in corals of the Great Barrier Reef with the aim of elucidating their photo-biological function/s as well as to identify novel proteins suitable for GFP-based technologies. We found numerous spectral variants, with emissions covering almost the full range of the visible spectrum. Many of these GFP-like proteins, especially in corals from the more extreme habitats, such as sun-exposed shallows or in deep water, showed a range of light-related spectral characteristics: high photostability, spectral tuning for energy transfer and dynamic photo-induced transformation properties. Intra-cellularly they were organized into spectral donor-acceptor pairs or even arrays, tuned for FRET. Coral color proteins thus offer an exciting potential to expand the use of the available GFPs in bio-imaging applications and as a basis for improved protein engineering.

  6. Engineering FKBP-Based Destabilizing Domains to Build Sophisticated Protein Regulation Systems.

    Directory of Open Access Journals (Sweden)

    Wenlin An

    Full Text Available Targeting protein stability with small molecules has emerged as an effective tool to control protein abundance in a fast, scalable and reversible manner. The technique involves tagging a protein of interest (POI with a destabilizing domain (DD specifically controlled by a small molecule. The successful construction of such fusion proteins may, however, be limited by functional interference of the DD epitope with electrostatic interactions required for full biological function of proteins. Another drawback of this approach is the remaining endogenous protein. Here, we combined the Cre-LoxP system with an advanced DD and generated a protein regulation system in which the loss of an endogenous protein, in our case the tumor suppressor PTEN, can be coupled directly with a conditionally fine-tunable DD-PTEN. This new system will consolidate and extend the use of DD-technology to control protein function precisely in living cells and animal models.

  7. Quantitative Characterization of Configurational Space Sampled by HIV-1 Nucleocapsid Using Solution NMR, X-ray Scattering and Protein Engineering.

    Science.gov (United States)

    Deshmukh, Lalit; Schwieters, Charles D; Grishaev, Alexander; Clore, G Marius

    2016-06-03

    Nucleic-acid-related events in the HIV-1 replication cycle are mediated by nucleocapsid, a small protein comprising two zinc knuckles connected by a short flexible linker and flanked by disordered termini. Combining experimental NMR residual dipolar couplings, solution X-ray scattering and protein engineering with ensemble simulated annealing, we obtain a quantitative description of the configurational space sampled by the two zinc knuckles, the linker and disordered termini in the absence of nucleic acids. We first compute the conformational ensemble (with an optimal size of three members) of an engineered nucleocapsid construct lacking the N- and C-termini that satisfies the experimental restraints, and then validate this ensemble, as well as characterize the disordered termini, using the experimental data from the full-length nucleocapsid construct. The experimental and computational strategy is generally applicable to multidomain proteins. Differential flexibility within the linker results in asymmetric motion of the zinc knuckles which may explain their functionally distinct roles despite high sequence identity. One of the configurations (populated at a level of ≈40 %) closely resembles that observed in various ligand-bound forms, providing evidence for conformational selection and a mechanistic link between protein dynamics and function. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Use of protein-engineered fabrics to identify design rules for integrin ligand clustering in biomaterials.

    Science.gov (United States)

    Benitez, Patrick L; Mascharak, Shamik; Proctor, Amy C; Heilshorn, Sarah C

    2016-01-01

    While ligand clustering is known to enhance integrin activation, this insight has been difficult to apply to the design of implantable biomaterials because the local and global ligand densities that enable clustering-enhanced integrin signaling were unpredictable. Here, two general design principles for biomaterial ligand clustering are elucidated. First, clustering ligands enhances integrin-dependent signals when the global ligand density, i.e., the ligand density across the cellular length scale, is near the ligand's effective dissociation constant (KD,eff). Second, clustering ligands enhances integrin activation when the local ligand density, i.e., the ligand density across the length scale of individual focal adhesions, is less than an overcrowding threshold. To identify these principles, we fabricated a series of elastin-like, electrospun fabrics with independent control over the local (0 to 122 000 ligands μm(-2)) and global (0 to 71 000 ligand μm(-2)) densities of an arginine-glycine-aspartate (RGD) ligand. Antibody blocking studies confirmed that human umbilical vein endothelial cell adhesion to these protein-engineered biomaterials was primarily due to αVβ3 integrin binding. Clustering ligands enhanced cell proliferation, focal adhesion number, and focal adhesion kinase expression near the ligand's KD,eff of 12 000 RGD μm(-2). Near this global ligand density, cells on ligand-clustered fabrics behaved similarly to cells grown on fabrics with significantly larger global ligand densities but without clustering. However, this enhanced ligand-clustering effect was not observed above a threshold cut-off concentration. At a local ligand density of 122 000 RGD μm(-2), cell division, focal adhesion number, and focal adhesion kinase expression were significantly reduced relative to fabrics with identical global ligand density and lesser local ligand densities. Thus, when clustering results in overcrowding of ligands, integrin receptors are no longer

  9. Free-Standing Metal Oxide Nanoparticle Superlattices Constructed with Engineered Protein Containers Show in Crystallo Catalytic Activity.

    Science.gov (United States)

    Lach, Marcel; Künzle, Matthias; Beck, Tobias

    2017-12-11

    The construction of defined nanostructured catalysts is challenging. In previous work, we established a strategy to assemble binary nanoparticle superlattices with oppositely charged protein containers as building blocks. Here, we show that these free-standing nanoparticle superlattices are catalytically active. The metal oxide nanoparticles inside the protein scaffold are accessible for a range of substrates and show oxidase-like and peroxidase-like activity. The stable superlattices can be reused for several reaction cycles. In contrast to bulk nanoparticle-based catalysts, which are prone to aggregation and difficult to characterize, nanoparticle superlattices based on engineered protein containers provide an innovative synthetic route to structurally defined heterogeneous catalysts with control over nanoparticle size and composition. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Improving protein secretion by engineering components of the bacterial translocation machinery

    NARCIS (Netherlands)

    Braun, P.G; Gerritse, G; van Dijl, J.M; Quax, Wim

    The increased insight into the mechanism of bacterial protein translocation has resulted in new concepts for the production of heterologous proteins. The periplasm of Gram-negative bacteria is revealed to have a role as a 'protein construction compartment', which can be used to fold complex

  11. Protein engineering of recombinant human bone morphogenetic protein 2 with higher interaction with Ca phosphate based scaffold used for osteogenesis.

    Science.gov (United States)

    Bayat, Mohammad; Shojaei, Sahar; Bahrami, Naghmeh; Mohamadnia, Abdolreza; Shojaei, Pegah; Bahrami, Naeim

    2017-10-01

    The aim of the present study was to assess the recombinant bonemorphogenetic protein 2 (RHBMP-2) with higher substantively and solubility for use in calcium phosphate scaffolds for better release in differentiation of mesenchymal stem cells to osteoblast cells. Using bioinformatics tools, two mutations (p. L10D and p. S12E) were chosen and applied in BMP2 CDS sequence to increase interaction with calcium derived composite. The new recombinant mutated sequence (BMP2mut ) was synthesized and then subcloned to expression vector pBV220. Experimental data regarded functional protein expression in E. coli. Since no modification was made in the active sites of proteins namely β-sheets and α-helixes, not only was there any change in the specific activity occurred in the specific activity of the enzyme in comparison to its commercial counterpart, but also mesenchymal osteogenesis occurred more efficient on biphasic CaP scaffold model. As we hypothesized, use of negatively charged amino acids such as aspartate and glutamate in protein loops increased the interactions of BMP2-Ca2+ and resulted in its slower and more sustained released from CaP scaffolds compare to commercial RHBMP2. Our data suggested that new BMP2mut have greater osteoinductive capacity than RHBMP2 in the same time and dose than RHBMP2. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2799-2805, 2017. © 2017 Wiley Periodicals, Inc.

  12. PERMutation Using Transposase Engineering (PERMUTE): A Simple Approach for Constructing Circularly Permuted Protein Libraries.

    Science.gov (United States)

    Jones, Alicia M; Atkinson, Joshua T; Silberg, Jonathan J

    2017-01-01

    Rearrangements that alter the order of a protein's sequence are used in the lab to study protein folding, improve activity, and build molecular switches. One of the simplest ways to rearrange a protein sequence is through random circular permutation, where native protein termini are linked together and new termini are created elsewhere through random backbone fission. Transposase mutagenesis has emerged as a simple way to generate libraries encoding different circularly permuted variants of proteins. With this approach, a synthetic transposon (called a permuteposon) is randomly inserted throughout a circularized gene to generate vectors that express different permuted variants of a protein. In this chapter, we outline the protocol for constructing combinatorial libraries of circularly permuted proteins using transposase mutagenesis, and we describe the different permuteposons that have been developed to facilitate library construction.

  13. A Structural Biology and Protein Engineering Approach to the Development of Antidotes Against the Inhibition of Human Acetylcholinesterase by OP-based Nerve Agents

    Science.gov (United States)

    2015-05-01

    AD_________________ Award Number: W81XWH-13-1-0064 TITLE: A Structural Biology and Protein Engineering Approach to the Development of Antidotes...against the Inhibition of Human Acetylcholinesterase by OP-based Nerve Agents PRINCIPAL INVESTIGATOR: Joel L. Sussman CONTRACTING ORGANIZATION...REPORT DATE May 2015 2. REPORT TYPE Final 3. DATES COVERED 11 Feb 2013 - 10 Feb 2015 4. TITLE AND SUBTITLE A Structural Biology and Protein Engineering

  14. Engineering and Characterization of Peptides and Proteins at Surfaces and Interfaces: A Case Study in Surface-Sensitive Vibrational Spectroscopy.

    Science.gov (United States)

    Ding, Bei; Jasensky, Joshua; Li, Yaoxin; Chen, Zhan

    2016-06-21

    Understanding molecular structures of interfacial peptides and proteins impacts many research fields by guiding the advancement of biocompatible materials, new and improved marine antifouling coatings, ultrasensitive and highly specific biosensors and biochips, therapies for diseases related to protein amyloid formation, and knowledge on mechanisms for various membrane proteins and their interactions with ligands. Developing methods for measuring such unique systems, as well as elucidating the structure and function relationship of such biomolecules, has been the goal of our lab at the University of Michigan. We have made substantial progress to develop sum frequency generation (SFG) vibrational spectroscopy into a powerful technique to study interfacial peptides and proteins, which lays a foundation to obtain unique and valuable insights when using SFG to probe various biologically relevant systems at the solid/liquid interface in situ in real time. One highlighting feature of this Account is the demonstration of the power of combining SFG with other techniques and methods such as ATR-FTIR, surface engineering, MD simulation, liquid crystal sensing, and isotope labeling in order to study peptides and proteins at interfaces. It is necessary to emphasize that SFG plays a major role in these studies, while other techniques and methods are supplemental. The central role of SFG is to provide critical information on interfacial peptide and protein structure (e.g., conformation and orientation) in order to elucidate how surface engineering (e.g., to vary the structure) can ultimately affect surface function (e.g., to optimize the activity). This Account focuses on the most significant recent progress in research on interfacial peptides and proteins carried out by our group including (1) the development of SFG analysis methods to determine orientations of regular as well as disrupted secondary structures, and the successful demonstration and application of an isotope

  15. Elucidating Bottlenecks to the Efficient Preparation of AB5-Hexamer Mucosal Adjuvant Protein LTm by Genetic Engineering.

    Science.gov (United States)

    Liu, Di; Hu, Fabiao; Wang, Wenpeng; Wu, Dong; He, Xiujuan; Zheng, Wenyun; Liu, Haipeng; Ma, Xingyuan

    2017-08-28

    Escherichia coli heat-labile enterotoxin (LT) and its non-toxic mutant (LTm) are well-known powerful mucosal adjuvants and immunogens. However, the yields of these adjuvants from genetically engineered strains remain at extremely low levels, thereby hindering their extensive application in fundamental and clinical research. Therefore, efficient production of these adjuvant proteins from genetically engineered microbes is a huge challenge in the field of molecular biology. In order to explore the expression bottlenecks of LTm in E. coli, we constructed a series of recombinant plasmids based on various considerations and gene expression strategies. After comparing the protein expression among strains containing different recombinant plasmids, the signal sequence was found to be critical for the expression of LTm and its subunits. When the signal sequence was present, the strong hydrophobicity and instability of this amino acid sequence greatly restricted the generation of subunits. However, when the signal sequence was removed, abundantly expressed subunits formed inactive inclusion bodies that could not be assembled into the hexameric native form, although the inclusion body subunits could be refolded and the biological activity recovered in vitro. Therefore, the dilemma choice of signal sequence formed bottlenecks in the expression of LTm. These results reveal the expression bottlenecks of LTm, provide guidance for the preparation of LTm and its subunits, and certainly help to promote efficient preparation of this mucosal adjuvant protein.

  16. Genome-wide mapping of mutations at single-nucleotide resolution for protein, metabolic and genome engineering.

    Science.gov (United States)

    Garst, Andrew D; Bassalo, Marcelo C; Pines, Gur; Lynch, Sean A; Halweg-Edwards, Andrea L; Liu, Rongming; Liang, Liya; Wang, Zhiwen; Zeitoun, Ramsey; Alexander, William G; Gill, Ryan T

    2017-01-01

    Improvements in DNA synthesis and sequencing have underpinned comprehensive assessment of gene function in bacteria and eukaryotes. Genome-wide analyses require high-throughput methods to generate mutations and analyze their phenotypes, but approaches to date have been unable to efficiently link the effects of mutations in coding regions or promoter elements in a highly parallel fashion. We report that CRISPR-Cas9 gene editing in combination with massively parallel oligomer synthesis can enable trackable editing on a genome-wide scale. Our method, CRISPR-enabled trackable genome engineering (CREATE), links each guide RNA to homologous repair cassettes that both edit loci and function as barcodes to track genotype-phenotype relationships. We apply CREATE to site saturation mutagenesis for protein engineering, reconstruction of adaptive laboratory evolution experiments, and identification of stress tolerance and antibiotic resistance genes in bacteria. We provide preliminary evidence that CREATE will work in yeast. We also provide a webtool to design multiplex CREATE libraries.

  17. A divide-and-conquer approach to determine the Pareto frontier for optimization of protein engineering experiments.

    Science.gov (United States)

    He, Lu; Friedman, Alan M; Bailey-Kellogg, Chris

    2012-03-01

    In developing improved protein variants by site-directed mutagenesis or recombination, there are often competing objectives that must be considered in designing an experiment (selecting mutations or breakpoints): stability versus novelty, affinity versus specificity, activity versus immunogenicity, and so forth. Pareto optimal experimental designs make the best trade-offs between competing objectives. Such designs are not "dominated"; that is, no other design is better than a Pareto optimal design for one objective without being worse for another objective. Our goal is to produce all the Pareto optimal designs (the Pareto frontier), to characterize the trade-offs and suggest designs most worth considering, but to avoid explicitly considering the large number of dominated designs. To do so, we develop a divide-and-conquer algorithm, Protein Engineering Pareto FRontier (PEPFR), that hierarchically subdivides the objective space, using appropriate dynamic programming or integer programming methods to optimize designs in different regions. This divide-and-conquer approach is efficient in that the number of divisions (and thus calls to the optimizer) is directly proportional to the number of Pareto optimal designs. We demonstrate PEPFR with three protein engineering case studies: site-directed recombination for stability and diversity via dynamic programming, site-directed mutagenesis of interacting proteins for affinity and specificity via integer programming, and site-directed mutagenesis of a therapeutic protein for activity and immunogenicity via integer programming. We show that PEPFR is able to effectively produce all the Pareto optimal designs, discovering many more designs than previous methods. The characterization of the Pareto frontier provides additional insights into the local stability of design choices as well as global trends leading to trade-offs between competing criteria. Copyright © 2011 Wiley Periodicals, Inc.

  18. Remarkable alkaline stability of an engineered protein A as immunoglobulin affinity ligand: C domain having only one amino acid substitution

    Science.gov (United States)

    Minakuchi, Kazunobu; Murata, Dai; Okubo, Yuji; Nakano, Yoshiyuki; Yoshida, Shinichi

    2013-01-01

    Protein A affinity chromatography is the standard purification process for the capture of therapeutic antibodies. The individual IgG-binding domains of protein A (E, D, A, B, C) have highly homologous amino acid sequences. From a previous report, it has been assumed that the C domain has superior resistance to alkaline conditions compared to the other domains. We investigated several properties of the C domain as an IgG-Fc capture ligand. Based on cleavage site analysis of a recombinant protein A using a protein sequencer, the C domain was found to be the only domain to have neither of the potential alkaline cleavage sites. Circular dichroism (CD) analysis also indicated that the C domain has good physicochemical stability. Additionally, we evaluated the amino acid substitutions at the Gly-29 position of the C domain, as the Z domain (an artificial B domain) acquired alkaline resistance through a G29A mutation. The G29A mutation proved to increase the alkaline resistance of the C domain, based on BIACORE analysis, although the improvement was significantly smaller than that observed for the B domain. Interestingly, a number of other amino acid mutations at the same position increased alkaline resistance more than did the G29A mutation. This result supports the notion that even a single mutation on the originally alkali-stable C domain would improve its alkaline stability. An engineered protein A based on this C domain is expected to show remarkable performance as an affinity ligand for immunoglobulin. PMID:23868198

  19. Structural and dynamic changes associated with beneficial engineered single-amino-acid deletion mutations in enhanced green fluorescent protein.

    Science.gov (United States)

    Arpino, James A J; Rizkallah, Pierre J; Jones, D Dafydd

    2014-08-01

    Single-amino-acid deletions are a common part of the natural evolutionary landscape but are rarely sampled during protein engineering owing to limited and prejudiced molecular understanding of mutations that shorten the protein backbone. Single-amino-acid deletion variants of enhanced green fluorescent protein (EGFP) have been identified by directed evolution with the beneficial effect of imparting increased cellular fluorescence. Biophysical characterization revealed that increased functional protein production and not changes to the fluorescence parameters was the mechanism that was likely to be responsible. The structure EGFP(D190Δ) containing a deletion within a loop revealed propagated changes only after the deleted residue. The structure of EGFP(A227Δ) revealed that a `flipping' mechanism was used to adjust for residue deletion at the end of a β-strand, with amino acids C-terminal to the deletion site repositioning to take the place of the deleted amino acid. In both variants new networks of short-range and long-range interactions are generated while maintaining the integrity of the hydrophobic core. Both deletion variants also displayed significant local and long-range changes in dynamics, as evident by changes in B factors compared with EGFP. Rather than being detrimental, deletion mutations can introduce beneficial structural effects through altering core protein properties, folding and dynamics, as well as function.

  20. Photo-oxidation of tyrosine in a bio-engineered bacterioferritin 'reaction centre'-a protein model for artificial photosynthesis.

    Science.gov (United States)

    Hingorani, Kastoori; Pace, Ron; Whitney, Spencer; Murray, James W; Smith, Paul; Cheah, Mun Hon; Wydrzynski, Tom; Hillier, Warwick

    2014-10-01

    The photosynthetic reaction centre (RC) is central to the conversion of solar energy into chemical energy and is a model for bio-mimetic engineering approaches to this end. We describe bio-engineering of a Photosystem II (PSII) RC inspired peptide model, building on our earlier studies. A non-photosynthetic haem containing bacterioferritin (BFR) from Escherichia coli that expresses as a homodimer was used as a protein scaffold, incorporating redox-active cofactors mimicking those of PSII. Desirable properties include: a di-nuclear metal binding site which provides ligands for bivalent metals, a hydrophobic pocket at the dimer interface which can bind a photosensitive porphyrin and presence of tyrosine residues proximal to the bound cofactors, which can be utilised as efficient electron-tunnelling intermediates. Light-induced electron transfer from proximal tyrosine residues to the photo-oxidised ZnCe6(•+), in the modified BFR reconstituted with both ZnCe6 and Mn(II), is presented. Three site-specific tyrosine variants (Y25F, Y58F and Y45F) were made to localise the redox-active tyrosine in the engineered system. The results indicate that: presence of bound Mn(II) is necessary to observe tyrosine oxidation in all BFR variants; Y45 the most important tyrosine as an immediate electron donor to the oxidised ZnCe6(•+) and that Y25 and Y58 are both redox-active in this system, but appear to function interchangebaly. High-resolution (2.1Å) crystal structures of the tyrosine variants show that there are no mutation-induced effects on the overall 3-D structure of the protein. Small effects are observed in the Y45F variant. Here, the BFR-RC represents a protein model for artificial photosynthesis. Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.

  1. Engineering Designed Proteins for Light Capture, Energy Transfer, and Emissive Sensing In Vivo

    Science.gov (United States)

    Mancini, Joshua A.

    Proteins that are used for photosynthetic light harvesting and biological signaling are critical to life. These types of proteins act as scaffolds that hold small, sometimes metal-containing organic molecules in precise locations for light absorption and successive use. For signaling proteins, this energy can be used to induce a photoisomerization of the small molecule that can turn on or off a signaling cascade that controls the physiology of an organism. Alternatively, photosynthetic light-harvesting proteins funnel this energy in a directional manner towards a charge separating catalytic component that can change this light energy into chemical energy. The protein environment also serves to tune the photophysical properties of the small molecules. This is seen extensively with the linear tetrapyrroles that are used in both photosynthetic and signaling proteins. Many efforts have been made to harness these natural proteins for societal use, including improving photophysical properties and interfacing capabilities with manmade catalytic components. Several methods of achieving improvement have entailed structurally guided mutation and directed evolution. However, these methods all have their limitations due to the inherent complexity and fragility of the natural proteins. This work presents an alternative more robust method to natural proteins. My thesis states: that man-made proteins, known as maquettes, employing basic rules of protein folding, can be designed to become light harvesting and signaling proteins that can be assembled fully in vivo providing an alternative, robust, and versatile platform for meeting the diverse array of societal "green chemistry" and biomedical needs. This in vivo assembly is carried out by interacting with cyanobacterial protein and pigment machinery, both as stand-alone units and as protein fusions with natural antenna complexes. Additionally, this work offers insight for fast and tight binding of circular and linear tetrapyrroles

  2. The use of unnatural amino acids to study and engineer protein function.

    Science.gov (United States)

    Neumann-Staubitz, Petra; Neumann, Heinz

    2016-06-01

    The expansion of the genetic code for the incorporation of unnatural amino acids (UAAs) in proteins of bacteria, yeasts, mammalian cells or whole animals provides molecular and structural biologists with an amazing kit of novel tools. UAAs can be used to investigate the structure and dynamics of proteins, to study their interactions or to control their activity in living cells. Incorporation of UAAs with bioorthogonal reactivity facilitates the site-specific installation of labels for spectroscopy and microscopy. Light-activatable crosslinker UAAs can be used to trap interacting molecules in living cells with a precision almost at the structural level. Post-translational modifications such as lysine acetylation and serine phosphorylation can be directly encoded to analyse their impact on protein function, and caging groups can be installed on critical residues to create light-activatable proteins. In this review we highlight recent applications of this technology to investigate protein function. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Engineering light-inducible nuclear localization signals for precise spatiotemporal control of protein dynamics in living cells.

    Science.gov (United States)

    Niopek, Dominik; Benzinger, Dirk; Roensch, Julia; Draebing, Thomas; Wehler, Pierre; Eils, Roland; Di Ventura, Barbara

    2014-07-14

    The function of many eukaryotic proteins is regulated by highly dynamic changes in their nucleocytoplasmic distribution. The ability to precisely and reversibly control nuclear translocation would, therefore, allow dissecting and engineering cellular networks. Here we develop a genetically encoded, light-inducible nuclear localization signal (LINuS) based on the LOV2 domain of Avena sativa phototropin 1. LINuS is a small, versatile tag, customizable for different proteins and cell types. LINuS-mediated nuclear import is fast and reversible, and can be tuned at different levels, for instance, by introducing mutations that alter AsLOV2 domain photo-caging properties or by selecting nuclear localization signals (NLSs) of various strengths. We demonstrate the utility of LINuS in mammalian cells by controlling gene expression and entry into mitosis with blue light.

  4. Engineering formation of multiple recombinant Eut protein nanocompartments in E. coli.

    Science.gov (United States)

    Held, Mark; Kolb, Alexander; Perdue, Sarah; Hsu, Szu-Yi; Bloch, Sarah E; Quin, Maureen B; Schmidt-Dannert, Claudia

    2016-04-11

    Compartmentalization of designed metabolic pathways within protein based nanocompartments has the potential to increase reaction efficiency in multi-step biosynthetic reactions. We previously demonstrated proof-of-concept of this aim by targeting a functional enzyme to single cellular protein nanocompartments, which were formed upon recombinant expression of the Salmonella enterica LT2 ethanolamine utilization bacterial microcompartment shell proteins EutS or EutSMNLK in Escherichia coli. To optimize this system, increasing overall encapsulated enzyme reaction efficiency, factor(s) required for the production of more than one nanocompartment per cell must be identified. In this work we report that the cupin domain protein EutQ is required for assembly of more than one nanocompartment per cell. Overexpression of EutQ results in multiple nanocompartment assembly in our recombinant system. EutQ specifically interacts with the shell protein EutM in vitro via electrostatic interactions with the putative cytosolic face of EutM. These findings lead to the theory that EutQ could facilitate multiple nanocompartment biogenesis by serving as an assembly hub for shell proteins. This work offers insights into the biogenesis of Eut bacterial microcompartments, and also provides an improved platform for the production of protein based nanocompartments for targeted encapsulation of enzyme pathways.

  5. Express your LOV: an engineered flavoprotein as a reporter for protein expression and purification.

    Directory of Open Access Journals (Sweden)

    Jayde A Gawthorne

    Full Text Available In this work, we describe the utility of Light, Oxygen, or Voltage-sensing (LOV flavoprotein domains from plant phototropins as a reporter for protein expression and function. Specifically, we used iLOV, an enhanced and more photostable variant of LOV. A pET-based plasmid for protein expression was constructed, encoding a C terminal iLOV-octahistidine (His8-tag and a HRV 3C protease cleavage recognition site. Ten different proteins, with various sub-cellular locations, were cloned into the plasmid, creating iLOV-His8 tag fusions. To test protein expression and how iLOV could be used as a reporter, the proteins were expressed in three different cell lines, in four different culture media, at two different temperatures. To establish whether the presence of the iLOV tag could have an impact on the functionality, one of the proteins, EspG, was over-expressed and purified. EspG is an "effector" protein normally produced by enterohemorrhagic E. coli strains and "injected" into host cells via the T3SS. We tested functionality of EspG-iLOV fusion by performing functional studies of EspG in mammalian host cells. When EspG-iLOV was microinjected into the host cell, the Golgi apparatus was completely disrupted as had previously been observed for EspG.

  6. An engineered genetic selection for ternary protein complexes inspired by a natural three-component hitchhiker mechanism.

    Science.gov (United States)

    Lee, Hyeon-Cheol; Portnoff, Alyse D; Rocco, Mark A; DeLisa, Matthew P

    2014-12-22

    The bacterial twin-arginine translocation (Tat) pathway is well known to translocate correctly folded monomeric and dimeric proteins across the tightly sealed cytoplasmic membrane. We identified a naturally occurring heterotrimer, the Escherichia coli aldehyde oxidoreductase PaoABC, that is co-translocated by the Tat translocase according to a ternary "hitchhiker" mechanism. Specifically, the PaoB and PaoC subunits, each devoid of export signals, are escorted to the periplasm in a piggyback fashion by the Tat signal peptide-containing subunit PaoA. Moreover, export of PaoA was blocked when either PaoB or PaoC was absent, revealing a surprising interdependence for export that is not seen for classical secretory proteins. Inspired by this observation, we created a bacterial three-hybrid selection system that links the formation of ternary protein complexes with antibiotic resistance. As proof-of-concept, a bispecific antibody was employed as an adaptor that physically crosslinked one antigen fused to a Tat export signal with a second antigen fused to TEM-1 β-lactamase (Bla). The resulting non-covalent heterotrimer was exported in a Tat-dependent manner, delivering Bla to the periplasm where it hydrolyzed β-lactam antibiotics. Collectively, these results highlight the remarkable flexibility of the Tat system and its potential for studying and engineering ternary protein interactions in living bacteria.

  7. Genetic engineering of the glucocorticoid receptor by fusion with the herpes viral protein VP22 causes selective loss of transactivation.

    Science.gov (United States)

    Soden, J; Stevens, A; Ray, D W

    2002-03-01

    The development of methods for engineering proteins with novel properties opens the way to manipulating intracellular processes in a therapeutically useful way. Glucocorticoids, acting via glucocorticoid receptors (GR), are potent anti-inflammatory agents, acting to oppose nuclear factor kappa B (NF kappa B) function. The herpes viral protein, VP22, has been reported to confer intercellular trafficking activity on 'cargo' proteins, potentially facilitating gene therapy with intracellular proteins. VP22GR, resulting from the addition of VP22 to the N terminal of GR, was equipotent with the wild-type GR in opposing NF kappa B p65-driven expression of an NF kappa B reporter gene. Surprisingly, VP22GR was incapable of inducing transactivation of positive glucocorticoid reporter genes (MMTV-luc and TAT3-luc). Furthermore, the VP22GR had powerful dominant negative activity on both endogenous and exogenous GR transactivation. VP22GR was cytoplasmic in quiescent cells, and after hormone addition underwent nuclear translocation to share the same distribution as the GR. The ability of the VP22GR to selectively confer and enhance glucocorticoid-dependent transrepression of NF kappa B may be of use therapeutically in e.g. transplant rejection, inflammatory arthritis or asthma.

  8. Effect of Genomic Integration Location on Heterologous Protein Expression and Metabolic Engineering in E. coli.

    Science.gov (United States)

    Englaender, Jacob A; Jones, J Andrew; Cress, Brady F; Kuhlman, Thomas E; Linhardt, Robert J; Koffas, Mattheos A G

    2017-04-21

    Chromosomal integration offers a selection-free alternative to DNA plasmids for expression of foreign proteins and metabolic pathways. Episomal plasmid DNA is convenient but has drawbacks including increased metabolic burden and the requirement for selection in the form of antibiotics. E. coli has long been used for the expression of foreign proteins and for the production of valuable metabolites by expression of complete metabolic pathways. The gene encoding the fluorescent reporter protein mCherry was integrated into four genomic loci on the E. coli chromosome to measure protein expression at each site. Expression levels ranged from 25% to 500% compared to the gene expressed on a high-copy plasmid. Modular expression of DNA is one of the most commonly used methods for optimizing metabolite production by metabolic engineering. By combining a recently developed method for integration of large synthetic DNA constructs into the genome, we were able to integrate two foreign pathways into the same four genomic loci. We have demonstrated that only one of the genomic loci resulted in the production of violacein, and that all four loci produced trans-cinnamic acid from the TAL pathway.

  9. Fabrication of Calixarene Based Protein Scaffold by Electrospin Coating for Tissue Engineering.

    Science.gov (United States)

    Cagil, Esra Maltas; Ozcan, Fatih; Ertul, Seref

    2018-08-01

    In this study, calixarene was synthesized by using different functional groups as p-tert-butyl-Calix[4]arene ester and amides. Calixarene nanofibers were produced by electrospin coating. Protein immobilization onto the calixarene nanofibers was carried out with human serum albumin (HSA). The maximum amount of binding on produced three different calixarene nanofibers (DE, 2-AMP and 3-AMP) was compared by using a fluorescence technique for protein analysis. Result showed that maximum binding amount was found to be as 177.85 mg cm-2 for 3-AMP surface. The protein binding was also characterized by using SEM, TEM, AFM and FT-IR. From obtained results, calixarene-albumin nanofiber was also fabricated by spin coating using 3-AMP which has ability max binding of protein.

  10. Soluble eggshell membrane: A natural protein to improve the properties of biomaterials used for tissue engineering applications.

    Science.gov (United States)

    Sah, Mahesh Kumar; Rath, Subha Narayan

    2016-10-01

    Extracellular matrix (ECM) acts as an instructing template for the cells contained in tissues. It plays a vital role in regulating cellular behavior by holding and interacting with various growth factors and signaling molecules. The ECM materials are either directly derived from a natural origin, or synthesized mimicking the natural ECM. In this review, we have addressed the ECM derived from eggshell membrane (ESM). The development of porous structures from natural biopolymers, such as ESM holds a number of advantages for tissue engineering applications. By using ESM in tissue engineering application, the cells attach and function to make a required tissue. Thereafter, the scaffold provides mechanical support as well as a platform for cellular interaction, hence, forming a fully functional tissue. The present review summarizes the structure-function relationship of ESM and advancement in its processing methods; the contribution of its soluble form (soluble eggshell membrane protein, SEP) in the development of promising hybrid biomaterials; and the recent advancement of their applications. In addition, this comprehensive review highlights the use of ESM for guided tissue regeneration; promising future applications of SEP in tissue engineering and regenerative medicine. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Site-Specific Antibody–Drug Conjugates: The Nexus of Bioorthogonal Chemistry, Protein Engineering, and Drug Development

    Science.gov (United States)

    2015-01-01

    Antibody–drug conjugates (ADCs) combine the specificity of antibodies with the potency of small molecules to create targeted drugs. Despite the simplicity of this concept, generation of clinically successful ADCs has been very difficult. Over the past several decades, scientists have learned a great deal about the constraints on antibodies, linkers, and drugs as they relate to successful construction of ADCs. Once these components are in hand, most ADCs are prepared by nonspecific modification of antibody lysine or cysteine residues with drug-linker reagents, which results in heterogeneous product mixtures that cannot be further purified. With advances in the fields of bioorthogonal chemistry and protein engineering, there is growing interest in producing ADCs by site-specific conjugation to the antibody, yielding more homogeneous products that have demonstrated benefits over their heterogeneous counterparts in vivo. Here, we chronicle the development of a multitude of site-specific conjugation strategies for assembly of ADCs and provide a comprehensive account of key advances and their roots in the fields of bioorthogonal chemistry and protein engineering. PMID:25494884

  12. Carbon nanotube interaction with extracellular matrix proteins producing scaffolds for tissue engineering

    Science.gov (United States)

    Tonelli, Fernanda MP; Santos, Anderson K; Gomes, Katia N; Lorençon, Eudes; Guatimosim, Silvia; Ladeira, Luiz O; Resende, Rodrigo R

    2012-01-01

    In recent years, significant progress has been made in organ transplantation, surgical reconstruction, and the use of artificial prostheses to treat the loss or failure of an organ or bone tissue. In recent years, considerable attention has been given to carbon nanotubes and collagen composite materials and their applications in the field of tissue engineering due to their minimal foreign-body reactions, an intrinsic antibacterial nature, biocompatibility, biodegradability, and the ability to be molded into various geometries and forms such as porous structures, suitable for cell ingrowth, proliferation, and differentiation. Recently, grafted collagen and some other natural and synthetic polymers with carbon nanotubes have been incorporated to increase the mechanical strength of these composites. Carbon nanotube composites are thus emerging as potential materials for artificial bone and bone regeneration in tissue engineering. PMID:22923989

  13. Crescendo: A Protein Sequence Database Search Engine for Tandem Mass Spectra

    Science.gov (United States)

    Wang, Jianqi; Zhang, Yajie; Yu, Yonghao

    2015-07-01

    A search engine that discovers more peptides reliably is essential to the progress of the computational proteomics. We propose two new scoring functions (L- and P-scores), which aim to capture similar characteristics of a peptide-spectrum match (PSM) as Sequest and Comet do. Crescendo, introduced here, is a software program that implements these two scores for peptide identification. We applied Crescendo to test datasets and compared its performance with widely used search engines, including Mascot, Sequest, and Comet. The results indicate that Crescendo identifies a similar or larger number of peptides at various predefined false discovery rates (FDR). Importantly, it also provides a better separation between the true and decoy PSMs, warranting the future development of a companion post-processing filtering algorithm.

  14. Crescendo: A Protein Sequence Database Search Engine for Tandem Mass Spectra.

    Science.gov (United States)

    Wang, Jianqi; Zhang, Yajie; Yu, Yonghao

    2015-07-01

    A search engine that discovers more peptides reliably is essential to the progress of the computational proteomics. We propose two new scoring functions (L- and P-scores), which aim to capture similar characteristics of a peptide-spectrum match (PSM) as Sequest and Comet do. Crescendo, introduced here, is a software program that implements these two scores for peptide identification. We applied Crescendo to test datasets and compared its performance with widely used search engines, including Mascot, Sequest, and Comet. The results indicate that Crescendo identifies a similar or larger number of peptides at various predefined false discovery rates (FDR). Importantly, it also provides a better separation between the true and decoy PSMs, warranting the future development of a companion post-processing filtering algorithm.

  15. Improved 1, 2, 4-butanetriol production from an engineered Escherichia coli by co-expression of different chaperone proteins.

    Science.gov (United States)

    Lu, Xinyao; He, Shuying; Zong, Hong; Song, Jian; Chen, Wen; Zhuge, Bin

    2016-09-01

    1, 2, 4-Butanetriol (BT) is a high-value non-natural chemical and has important applications in polymers, medical production and military industry. In the constructed BT biosynthesis pathway from xylose in Escherichia coli, the xylose dehydrogenase (Xdh) and the benzoylformate decarboxylase (MdlC) are heterologous enzymes and the activity of MdlC is the key limiting factor for BT production. In this study, six chaperone protein systems were introduced into the engineered E. coli harboring the recombinant BT pathway. The chaperone GroES-GroEL was beneficial to Xdh activity but had a negative effect on MdlC activity and BT titer. The plasmid pTf16 containing the tig gene (trigger factor) was beneficial to Xdh and MdlC activities and improved the BT titer from 0.42 to 0.56 g/l from 20 g/l xylose. However, co-expression of trigger factor and GroES-GroEL simultaneously reduced the activity of MdlC and had no effect on the BT production. The plasmid pKJE7 harboring dnaK-dnaJ-grpE showed significant negative effects on these enzyme activities and cell growth, leading to completely restrained the BT production. Similarly, co-expression of DnaKJ-GrpPE and GroES-GroEL simultaneously reduced Xdh and MdlC activities and decreased the BT titer by 45.2 %. The BT production of the engineered E. coli harboring pTf16 was further improved to the highest level at 1.01 g/l under pH control (pH 7). This work showed the potential application of chaperone proteins in microorganism engineering to get high production of target compounds as an effective and valuable tool.

  16. Engineering adhesive proteins enhanced by self-assembling peptide and enzyme cross-linking

    OpenAIRE

    JAMIE JOSEPH CASTILLO

    2017-01-01

    Conventional adhesives efficiently work on dry surfaces but fail in the presence of water. Currently, there is a great need for adhesives in surgical procedures and underwater construction. Nature gives a hint on how to develop wet adhesives. Mussels survive harsh underwater conditions by secreting an adhesive which attaches to different surfaces. However, natural extraction was found uneconomical. Recombinant engineering approach is a promising alternative method for large-scale adhesive pro...

  17. Highly sensitive detection of protein biomarkers via nuclear magnetic resonance biosensor with magnetically engineered nanoferrite particles.

    Science.gov (United States)

    Jeun, Minhong; Park, Sungwook; Lee, Hakho; Lee, Kwan Hyi

    Magnetic-based biosensors are attractive for on-site detection of biomarkers due to the low magnetic susceptibility of biological samples. Here, we report a highly sensitive magnetic-based biosensing system that is composed of a miniaturized nuclear magnetic resonance (NMR) device and magnetically engineered nanoferrite particles (NFPs). The sensing performance, also identified as the transverse relaxation (R2) rate, of the NMR device is directly related to the magnetic properties of the NFPs. Therefore, we developed magnetically engineered NFPs (MnMg-NFP) and used them as NMR agents to exhibit a significantly improved R2 rate. The magnetization of the MnMg-NFPs was increased by controlling the Mn and Mg cation concentration and distribution during the synthesis process. This modification of the Mn and Mg cation directly contributed to improving the R2 rate. The miniaturized NMR system, combined with the magnetically engineered MnMg-NFPs, successfully detected a small amount of infectious influenza A H1N1 nucleoprotein with high sensitivity and stability.

  18. Chinese hamster ovary (CHO) host cell engineering to increase sialylation of recombinant therapeutic proteins by modulating sialyltransferase expression.

    Science.gov (United States)

    Lin, Nan; Mascarenhas, Joaquina; Sealover, Natalie R; George, Henry J; Brooks, Jeanne; Kayser, Kevin J; Gau, Brian; Yasa, Isil; Azadi, Parastoo; Archer-Hartmann, Stephanie

    2015-01-01

    N-Glycans of human proteins possess both α2,6- and α2,3-linked terminal sialic acid (SA). Recombinant glycoproteins produced in Chinese hamster overy (CHO) only have α2,3-linkage due to the absence of α2,6-sialyltransferase (St6gal1) expression. The Chinese hamster ST6GAL1 was successfully overexpressed using a plasmid expression vector in three recombinant immunoglobulin G (IgG)-producing CHO cell lines. The stably transfected cell lines were enriched for ST6GAL1 overexpression using FITC-Sambucus nigra (SNA) lectin that preferentially binds α2,6-linked SA. The presence of α2,6-linked SA was confirmed using a novel LTQ Linear Ion Trap Mass Spectrometry (LTQ MS) method including MSn fragmentation in the enriched ST6GAL1 Clone 27. Furthermore, the total SA (mol/mol) in IgG produced by the enriched ST6GAL1 Clone 27 increased by 2-fold compared to the control. For host cell engineering, the CHOZN(®) GS host cell line was transfected and enriched for ST6GAL1 overexpression. Single-cell clones were derived from the enriched population and selected based on FITC-SNA staining and St6gal1 expression. Two clones ("ST6GAL1 OE Clone 31 and 32") were confirmed for the presence of α2,6-linked SA in total host cell protein extracts. ST6GAL1 OE Clone 32 was subsequently used to express SAFC human IgG1. The recombinant IgG expressed in this host cell line was confirmed to have α2,6-linked SA and increased total SA content. In conclusion, overexpression of St6gal1 is sufficient to produce recombinant proteins with increased sialylation and more human-like glycoprofiles without combinatorial engineering of other sialylation pathway genes. This work represents our ongoing effort of glycoengineering in CHO host cell lines for the development of "bio-better" protein therapeutics and cell culture vaccine production. © 2015 American Institute of Chemical Engineers.

  19. Enhancing E. coli tolerance towards oxidative stress via engineering its global regulator cAMP receptor protein (CRP.

    Directory of Open Access Journals (Sweden)

    Souvik Basak

    Full Text Available Oxidative damage to microbial hosts often occurs under stressful conditions during bioprocessing. Classical strain engineering approaches are usually both time-consuming and labor intensive. Here, we aim to improve E. coli performance under oxidative stress via engineering its global regulator cAMP receptor protein (CRP, which can directly or indirectly regulate redox-sensing regulators SoxR and OxyR, and other ~400 genes in E. coli. Error-prone PCR technique was employed to introduce modifications to CRP, and three mutants (OM1~OM3 were identified with improved tolerance via H(2O(2 enrichment selection. The best mutant OM3 could grow in 12 mM H(2O(2 with the growth rate of 0.6 h(-1, whereas the growth of wild type was completely inhibited at this H(2O(2 concentration. OM3 also elicited enhanced thermotolerance at 48°C as well as resistance against cumene hydroperoxide. The investigation about intracellular reactive oxygen species (ROS, which determines cell viability, indicated that the accumulation of ROS in OM3 was always lower than in WT with or without H(2O(2 treatment. Genome-wide DNA microarray analysis has shown not only CRP-regulated genes have demonstrated great transcriptional level changes (up to 8.9-fold, but also RpoS- and OxyR-regulated genes (up to 7.7-fold. qRT-PCR data and enzyme activity assay suggested that catalase (katE could be a major antioxidant enzyme in OM3 instead of alkyl hydroperoxide reductase or superoxide dismutase. To our knowledge, this is the first work on improving E. coli oxidative stress resistance by reframing its transcription machinery through its native global regulator. The positive outcome of this approach may suggest that engineering CRP can be successfully implemented as an efficient strain engineering alternative for E. coli.

  20. Expression of cardiac proteins in neonatal cardiomyocytes on PGS/fibrinogen core/shell substrate for Cardiac tissue engineering.

    Science.gov (United States)

    Ravichandran, Rajeswari; Venugopal, Jayarama Reddy; Sundarrajan, Subramanian; Mukherjee, Shayanti; Sridhar, Radhakrishnan; Ramakrishna, Seeram

    2013-08-20

    Heart failure due to myocardial infarction remains the leading cause of death worldwide owing to the inability of myocardial tissue regeneration. The aim of this study is to develop a core/shell fibrous cardiac patch having desirable mechanical properties and biocompatibility to engineer the infarcted myocardium. We fabricated poly(glycerol sebacate)/fibrinogen (PGS/fibrinogen) core/shell fibers with core as elastomeric PGS provides suitable mechanical properties comparable to that of native tissue and shell as fibrinogen to promote cell-biomaterial interactions. The PGS/fibrinogen core/shell fibers and fibrinogen nanofibers were characterized by SEM, contact angle and tensile testing to analyze the fiber morphology, wettability, and mechanical properties of the scaffold. The cell-scaffold interactions were analyzed using isolated neonatal cardiomyocytes for cell proliferation, confocal analysis for the expression of marker proteins α-actinin, Troponin-T, β-myosin heavy chain and connexin 43 and SEM analysis for cell morphology. We observed PGS/fibrinogen core/shell fibers had a Young's modulus of about 3.28 ± 1.7 MPa, which was comparable to that of native myocardium. Neonatal cardiomyocytes cultured on these scaffolds showed normal expression of cardiac specific marker proteins α-actinin, Troponin, β-myosin heavy chain and connexin 43 to prove PGS/fibrinogen core/shell fibers have potential for cardiac tissue engineering. Results indicated that neonatal cardiomyocytes formed predominant gap junctions and expressed cardiac specific marker proteins on PGS/fibrinogen core/shell fibers compared to fibrinogen nanofibers, indicating PGS/fibrinogen core/shell fibers may serve as a suitable cardiac patch for the regeneration of infarcted myocardium. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  1. Engineering Encodable Lanthanide-Binding Tags (LBTs) into Loop Regions of Proteins

    Science.gov (United States)

    Barthelmes, Katja; Reynolds, Anne M.; Peisach, Ezra; Jonker, Hendrik R. A.; DeNunzio, Nicholas J.; Allen, Karen N.; Imperiali, Barbara; Schwalbe, Harald

    2011-01-01

    Lanthanide-binding-tags (LBTs) are valuable tools for investigation of protein structure, function, and dynamics by NMR spectroscopy, X-ray crystallography and luminescence studies. We have inserted LBTs into three different loop positions (denoted L, R, and S) of the model protein interleukin-1β and varied the length of the spacer between the LBT and the protein (denoted 1-3). Luminescence studies demonstrate that all nine constructs bind Tb3+ tightly in the low nanomolar range. No significant change in the fusion protein occurs from insertion of the LBT, as shown by two X-ray crystallographic structures of the IL1β-S1 and IL1β-L3 constructs and for the remaining constructs by comparing 1H-15N-HSQC NMR spectra with wild-type IL1β. Additionally, binding of LBT-loop IL1β proteins to their native binding partner in vitro remains unaltered. X-ray crystallographic phasing was successful using only the signal from the bound lanthanide. Large residual dipolar couplings (RDCs) could be determined by NMR spectroscopy for all LBT-loop-constructs and revealed that the LBT-2 series were rigidly incorporated into the interleukin-1β structure. The paramagnetic NMR spectra of loop-LBT mutant IL1β-R2 were assigned and the Δχ tensor components were calculated based on RDCs and pseudocontact shifts (PCSs). A structural model of the IL1β-R2 construct was calculated using the paramagnetic restraints. The current data provide support that encodable LBTs serve as versatile biophysical tags when inserted into loop regions of proteins of known structure or predicted via homology modelling. PMID:21182275

  2. Functional heterologous protein expression by genetically engineered probiotic yeast Saccharomyces boulardii.

    Directory of Open Access Journals (Sweden)

    Lauren E Hudson

    Full Text Available Recent studies have suggested the potential of probiotic organisms to be adapted for the synthesis and delivery of oral therapeutics. The probiotic yeast Saccharomyces boulardii would be especially well suited for this purpose due to its ability, in contrast to probiotic prokaryotes, to perform eukaryotic post translational modifications. This probiotic yeast thus has the potential to express a broad array of therapeutic proteins. Currently, however, use of wild type (WT S. boulardii relies on antibiotic resistance for the selection of transformed yeast. Here we report the creation of auxotrophic mutant strains of S. boulardii that can be selected without antibiotics and demonstrate that these yeast can express functional recombinant protein even when recovered from gastrointestinal immune tissues in mice. A UV mutagenesis approach was employed to generate three uracil auxotrophic S. boulardii mutants that show a low rate of reversion to wild type growth. These mutants can express recombinant protein and are resistant in vitro to low pH, bile acid salts, and anaerobic conditions. Critically, oral gavage experiments using C57BL/6 mice demonstrate that mutant S. boulardii survive and are taken up into gastrointestinal immune tissues on a similar level as WT S. boulardii. Mutant yeast recovered from gastrointestinal immune tissues furthermore retain expression of functional recombinant protein. These data show that auxotrophic mutant S. boulardii can safely express recombinant protein without antibiotic selection and can deliver recombinant protein to gastrointestinal immune tissues. These auxotrophic mutants of S. boulardii pave the way for future experiments to test the ability of S. boulardii to deliver therapeutics and mediate protection against gastrointestinal disorders.

  3. Nuclear Engineering of Microalgae for High Yield Secretion of Recombinant Proteins

    DEFF Research Database (Denmark)

    Ramos Martinez, Erick Miguel

    Photosynthetic microorganism like microalgae and cyanobacteria are considered as emerging biotechnology platforms for production of recombinant proteins and other high-value biomolecules with a wide range of applications. Moreover, microalgae offer significant advantages compared with other...... the potential of microalgae as a cell factory for secretion of recombinant proteins. The second research project presented in this thesis aimed to establish a new robust method to allow in vivo measurements of metabolic enzyme activities in cyanobacteria, with a hope that the method would facilitate further...

  4. Chimeric Cyanovirin-MPER Recombinantly Engineered Proteins Cause Cell-Free Virolysis of HIV-1

    OpenAIRE

    Contarino, Mark; Bastian, Arangassery R.; Kalyana Sundaram, Ramalingam Venkat; McFadden, Karyn; Duffy, Caitlin; Gangupomu, Vamshi; Baker, Michelle; Abrams, Cameron; Chaiken, Irwin

    2013-01-01

    Human immunodeficiency virus (HIV) is the primary etiologic agent responsible for the AIDS pandemic. In this work, we used a chimeric recombinant protein strategy to test the possibility of irreversibly destroying the HIV-1 virion using an agent that simultaneously binds the Env protein and viral membrane. We constructed a fusion of the lectin cyanovirin-N (CVN) and the gp41 membrane-proximal external region (MPER) peptide with a variable-length (Gly4Ser)x linker (where x is 4 or 8) between t...

  5. Introduction of a covalent histidine-heme linkage in a hemoglobin: a promising tool for heme protein engineering.

    Science.gov (United States)

    Rice, Selena L; Preimesberger, Matthew R; Johnson, Eric A; Lecomte, Juliette T J

    2014-12-01

    The hemoglobins of the cyanobacteria Synechococcus and Synechocystis (GlbNs) are capable of spontaneous and irreversible attachment of the b heme to the protein matrix. The reaction, which saturates the heme 2-vinyl by addition of a histidine residue, is reproduced in vitro by preparing the recombinant apoprotein, adding ferric heme, and reducing the iron to the ferrous state. Spontaneous covalent attachment of the heme is potentially useful for protein engineering purposes. Thus, to explore whether the histidine-heme linkage can serve in such applications, we attempted to introduce it in a test protein. We selected as our target the heme domain of Chlamydomonas eugametos LI637 (CtrHb), a eukaryotic globin that exhibits less than 50% sequence identity with the cyanobacterial GlbNs. We chose two positions, 75 in the FG corner and 111 in the H helix, to situate a histidine near a vinyl group. We characterized the proteins with gel electrophoresis, absorbance spectroscopy, and NMR analysis. Both T111H and L75H CtrHbs reacted upon reduction of the ferric starting material containing cyanide as the distal ligand to the iron. With L75H CtrHb, nearly complete (>90%) crosslinking was observed to the 4-vinyl as expected from the X-ray structure of wild-type CtrHb. Reaction of T111H CtrHb also occurred at the 4-vinyl, in a 60% yield indicating a preference for the flipped heme orientation in the starting material. The work suggests that the His-heme modification will be applicable to the design of proteins with a non-dissociable heme group. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Surface Immobilization of Human Arginase-1 with an Engineered Ice Nucleation Protein Display System in E. coli.

    Directory of Open Access Journals (Sweden)

    Zhen Zhang

    Full Text Available Ice nucleation protein (INP is frequently used as a surface anchor for protein display in gram-negative bacteria. Here, MalE and TorA signal peptides, and three charged polypeptides, 6×Lys, 6×Glu and 6×Asp, were anchored to the N-terminus of truncated INP (InaK-N to improve its surface display efficiency for human Arginase1 (ARG1. Our results indicated that the TorA signal peptide increased the surface translocation of non-protein fused InaK-N and human ARG1 fused InaK-N (InaK-N/ARG1 by 80.7% and 122.4%, respectively. Comparably, the MalE signal peptide decreased the display efficiencies of both the non-protein fused InaK-N and InaK-N/ARG1. Our results also suggested that the 6×Lys polypeptide significantly increased the surface display efficiency of K6-InaK-N/ARG1 by almost 2-fold, while also practically abolishing the surface translocation of non-protein fused InaK-N, indicating the interesting roles of charged polypeptides in bacteria surface display systems. Cell surface-immobilized K6-InaK-N/ARG1 presented an arginase activity of 10.7 U/OD600 under the optimized conditions of 40°C, pH 10.0 and 1 mM Mn2+, which could convert more than 95% of L-Arginine (L-Arg to L-Ornithine (L-Orn in 16 hours. The engineered InaK-Ns expanded the INP surface display system, which aided in the surface immobilization of human ARG1 in E. coli cells.

  7. Engineering Permissive Insertion Sites in the Bacteriophage Phi29 DNA-Linked Terminal Protein.

    Directory of Open Access Journals (Sweden)

    Pablo Gella

    Full Text Available Many different DNA delivery vehicles have been developed and tested, all with their advantages and disadvantages. The bacteriophage phi29 terminal protein (TP is covalently linked to the 5' ends of the phage genome during the DNA replication process. Our approach is to utilize this TP as a platform to incorporate different protein or peptide modules that can target the DNA to the interior of the cell, to the nucleus, or even to subcellular compartments. In order to be able to insert different peptide modules on the TP sequence to endow it with desired functions and/or eliminate unwanted regions of the protein, we have carried out a transposition screening to detect insertion-permissive points on the sequence of the TP. We report the functional characterization of 12 insertion mutants of the TP, and the identification of one site at position 38 that allows the insertion of peptides up to 17 amino acids in length while maintaining the ability of the TP to support DNA amplification in vitro. A protein with one insertion at that position containing a cysteine residue, a linker, and a thrombin recognition site was purified and its amplification activity was optimized.

  8. Fueling the engine: induction of AMP-activated protein kinase in trout skeletal muscle by swimming

    NARCIS (Netherlands)

    Magnoni, L.J.; Palstra, A.P.; Planas, J.V.

    2014-01-01

    AMP-activated protein kinase (AMPK) is well known to be induced by exercise and to mediate important metabolic changes in the skeletal muscle of mammals. Despite the physiological importance of exercise as a modulator of energy use by locomotory muscle, the regulation of this enzyme by swimming has

  9. Nuclear Engineering of Microalgae for High Yield Secretion of Recombinant Proteins

    DEFF Research Database (Denmark)

    Ramos Martinez, Erick Miguel

    to the glycomodules, accumulation of a fusion protein was dramatically increased by up to 12 folds, with the maximum yield of 15 mg L-1. Characterization of the secreted Venus showed the presence of glycosylations and increased resistance to proteolytic degradation. The results from this thesis demonstrate...

  10. Resolution of Disulfide Heterogeneity in Nogo Receptor 1 Fusion Proteins by Molecular Engineering

    Energy Technology Data Exchange (ETDEWEB)

    P Weinreb; D Wen; F Qian; C Wildes; E Garber; L Walus; M Jung; J Wang; J Relton; et al.

    2011-12-31

    NgRI (Nogo-66 receptor) is part of a signalling complex that inhibits axon regeneration in the central nervous system. Truncated soluble versions of NgRI have been used successfully to promote axon regeneration in animal models of spinal-cord injury, raising interest in this protein as a potential therapeutic target. The LRR (leucine-rich repeat) regions in NgRI are flanked by N- and C-terminal disulfide-containing 'cap' domains (LRRNT and LRRCT respectively). In the present work we show that, although functionally active, the NgRI(310)-Fc fusion protein contains mislinked and heterogeneous disulfide patterns in the LRRCT domain, and we report the generation of a series of variant molecules specifically designed to prevent this heterogeneity. Using these variants we explored the effects of modifying the NgRI truncation site or the spacing between the NgRI and Fc domains, or replacing cysteines within the NgRI or IgG hinge regions. One variant, which incorporates replacements of Cys{sup 266} and Cys{sup 309} with alanine residues, completely eliminated disulfide scrambling while maintaining functional in vitro and in vivo efficacy. This modified NgRI-Fc molecule represents a significantly improved candidate for further pharmaceutical development, and may serve as a useful model for the optimization of other IgG fusion proteins made from LRR proteins.

  11. Structure engineering of filled protein microbeads to tailor release of oil droplets in gastric digestion

    NARCIS (Netherlands)

    Leusden, van P.; Hartog, den G.J.M.; Bast, A.; Postema, M.; Linden, van der E.; Sagis, L.M.C.

    2016-01-01

    Oil-soluble components can be encapsulated in an O/W1/W2 microsystem, in which they are dissolved in oil droplets dispersed in a gelled microbead (W1), which forms a barrier between the oil droplets and the aqueous continuous phase (W2). We investigated the rate and mechanism of breakdown of protein

  12. Developing Progranulin-Loaded Protein-Engineered Hydrogel for Post-Traumatic Osteoarthritis

    Science.gov (United States)

    2017-09-01

    cells were harvested at 4 °C and 4000 rpm for 25 minutes. The cells were re-suspended in lysis buffer (50 mM sodium phosphate dibasic pH 8, 6 M...the previous literature. We are discovering that these E5C are micellar systems, which may prove to be unique in assemblies from other protein

  13. Exploring the folding pathway of green fluorescent protein through disulfide engineering.

    Science.gov (United States)

    Pitman, Derek J; Banerjee, Shounak; Macari, Stephen J; Castaldi, Christopher A; Crone, Donna E; Bystroff, Christopher

    2015-03-01

    We have introduced two disulfide crosslinks into the loop regions on opposite ends of the beta barrel in superfolder green fluorescent protein (GFP) in order to better understand the nature of its folding pathway. When the disulfide on the side opposite the N/C-termini is formed, folding is 2× faster, unfolding is 2000× slower, and the protein is stabilized by 16 kJ/mol. But when the disulfide bond on the side of the termini is formed we see little change in the kinetics and stability. The stabilization upon combining the two crosslinks is approximately additive. When the kinetic effects are broken down into multiple phases, we observe Hammond behavior in the upward shift of the kinetic m-value of unfolding. We use these results in conjunction with structural analysis to assign folding intermediates to two parallel folding pathways. The data are consistent with a view that the two fastest transition states of folding are "barrel closing" steps. The slower of the two phases passes through an intermediate with the barrel opening occurring between strands 7 and 8, while the faster phase opens between 9 and 4. We conclude that disulfide crosslink-induced perturbations in kinetics are useful for mapping the protein folding pathway. © 2014 The Protein Society.

  14. Tier-1 assays for assessing the toxicity of insecticidal proteins produced by genetically engineered plants to non-target arthropods.

    Science.gov (United States)

    Li, Yun-He; Romeis, Jörg; Wu, Kong-Ming; Peng, Yu-Fa

    2014-04-01

    In assessing an insect-resistant genetically engineered (IRGE) crop before its commercialization, researchers normally use so-called "Tier-1 assays" as the initial step to determine the effects of the crop on non-target organisms. In these tests, the insecticidal proteins (IPs) produced by the IRGEs are added to the diets of test organisms in the laboratory. Test organisms in such assays can be directly exposed to much higher concentrations of the test IPs than they would encounter in the field. The results of Tier-1 assays are thus more conservative than those generated in studies in which the organisms are exposed to the IPs by feeding on IRGE plant tissue or in the case of predators or parasites, by feeding on invertebrate prey or hosts that have fed on IRGE plant tissue. In this report, we consider three important factors that must be considered in Tier-1 assays: (i) methods for delivery of the IP to the test organisms; (ii) the need for and selection of compounds used as positive controls; and (iii) methods for monitoring the concentration, stability and bioactivity of the IP during the assay. We also analyze the existing data from Tier-1 assays regarding the toxicity of Bt Cry proteins to non-target arthropod species. The data indicate that the widely used Bt proteins have no direct toxicity to non-target organisms. © 2013 Institute of Zoology, Chinese Academy of Sciences.

  15. Integrated cell and process engineering for improved transient production of a "difficult-to-express" fusion protein by CHO cells.

    Science.gov (United States)

    Johari, Yusuf B; Estes, Scott D; Alves, Christina S; Sinacore, Marty S; James, David C

    2015-12-01

    Based on an optimized electroporation protocol, we designed a rapid, milliliter-scale diagnostic transient production assay to identify limitations in the ability of Chinese hamster ovary (CHO) cells to produce a model "difficult-to-express" homodimeric Fc-fusion protein, Sp35Fc, that exhibited very low volumetric titer and intracellular formation of disulfide-bonded oligomeric aggregates post-transfection. As expression of Sp35Fc induced an unfolded protein response in transfected host cells, we utilized the transient assay to compare, in parallel, multiple functionally diverse strategies to engineer intracellular processing of Sp35Fc in order to increase production and reduce aggregation as two discrete design objectives. Specifically, we compared the effect of (i) co-expression of ER-resident molecular chaperones (BiP, PDI, CypB) or active forms of UPR transactivators (ATF6c, XBP1s) at varying recombinant gene load, (ii) addition of small molecules known to act as chemical chaperones (PBA, DMSO, glycerol, betaine, TMAO) or modulate UPR signaling (PERK inhibitor GSK2606414) at varying concentration, (iii) a reduction in culture temperature to 32°C. Using this information, we designed a biphasic, Sp35Fc-specific transient manufacturing process mediated by lipofection that utilized CypB co-expression at an optimal Sp35Fc:CypB gene ratio of 5:1 to initially maximize transfected cell proliferation, followed by addition of a combination of PBA (0.5 mM) and glycerol (1% v/v) at the onset of stationary phase to maximize cell specific production and eliminate Sp35Fc aggregation. Using this optimal, engineered process transient Sp35Fc production was significantly increased sixfold over a 12 day production process with no evidence of disulfide-bonded aggregates. Finally, transient production in clonally derived sub-populations (derived from parental CHO host) screened for a heritably improved capability to produce Sp35Fc was also significantly improved by the optimized

  16. Evaluation of Biological Toxicity of CdTe Quantum Dots with Different Coating Reagents according to Protein Expression of Engineering Escherichia coli

    Directory of Open Access Journals (Sweden)

    Wei Xu

    2015-01-01

    Full Text Available The results obtained from toxicity assessment of quantum dots (QDs can be used to establish guidelines for the application of QDs in bioimaging. This paper focused on the design of a novel method to evaluate the toxicity of CdTe QDs using engineering Escherichia coli as a model. The toxicity of mercaptoacetic acid (MPA, glutathione (GSH, and L-cysteine (Cys capped CdTe QDs was analyzed according to the heterologous protein expression in BL21/DE3, engineering Escherichia coli extensively used for protein expression. The results showed that the MPA-CdTe QDs had more serious toxicity than the other two kinds of CdTe QDs. The microscopic images and SEM micrographs further proved that both the proliferation and the protein expression of engineering Escherichia coli were inhibited after treatment with MPA-CdTe QDs. The proposed method is important to evaluate biological toxicity of both QDs and other nanoparticles.

  17. Shedding light on disulfide bond formation: engineering a redox switch in green fluorescent protein

    DEFF Research Database (Denmark)

    Østergaard, H.; Henriksen, A.; Hansen, Flemming G.

    2001-01-01

    To visualize the formation of disulfide bonds in living cells, a pair of redox-active cysteines was introduced into the yellow fluorescent variant of green fluorescent protein. Formation of a disulfide bond between the two cysteines was fully reversible and resulted in a >2-fold decrease in the i......To visualize the formation of disulfide bonds in living cells, a pair of redox-active cysteines was introduced into the yellow fluorescent variant of green fluorescent protein. Formation of a disulfide bond between the two cysteines was fully reversible and resulted in a >2-fold decrease...... as a structural reorganization of residues in the immediate chromophore environment. By combining this information with spectroscopic data, we propose a detailed mechanism accounting for the observed redox state-dependent fluorescence. The redox potential of the cysteine couple was found to be within...

  18. Lighting up G protein-coupled purinergic receptors with engineered fluorescent ligands

    Science.gov (United States)

    Ciruela, Francisco; Fernández-Dueñas, Víctor; Jacobson, Kenneth A.

    2015-01-01

    The use of G protein-coupled receptors fluorescent ligands is undergoing continuous expansion. In line with this, fluorescent agonists and antagonists of high affinity for G protein-coupled adenosine and P2Y receptors have been shown to be useful pharmacological probe compounds. Fluorescent ligands for A1R, A2AR, and A3R (adenosine receptors) and P2Y2R, P2Y4R, P2Y6R, and P2Y14R (nucleotide receptors) have been reported. Such ligands have been successfully applied to drug discovery and to GPCR characterization by flow cytometry, fluorescence correlation spectroscopy, fluorescence microscopy, fluorescence polarization, fluorescence resonance energy transfer and scanning confocal microscopy. Here we summarize recently reported and readily available representative fluorescent ligands of purinergic receptors. In addition, we pay special attention on the use of this family of fluorescent ligands revealing two main aspects of purinergic receptor biology, namely ligand binding and receptor oligomerization. PMID:25890205

  19. A Chimeric UDP-Glucose Pyrophosphorylase Produced by Protein Engineering Exhibits Sensitivity to Allosteric Regulators

    Directory of Open Access Journals (Sweden)

    Matías D. Asención Diez

    2013-05-01

    Full Text Available In bacteria, glycogen or oligosaccharide accumulation involves glucose-1-phosphate partitioning into either ADP-glucose (ADP-Glc or UDP-Glc. Their respective synthesis is catalyzed by allosterically regulated ADP-Glc pyrophosphorylase (EC 2.7.7.27, ADP-Glc PPase or unregulated UDP-Glc PPase (EC 2.7.7.9. In this work, we characterized the UDP-Glc PPase from Streptococcus mutans. In addition, we constructed a chimeric protein by cutting the C-terminal domain of the ADP-Glc PPase from Escherichia coli and pasting it to the entire S. mutans UDP-Glc PPase. Both proteins were fully active as UDP-Glc PPases and their kinetic parameters were measured. The chimeric enzyme had a slightly higher affinity for substrates than the native S. mutans UDP-Glc PPase, but the maximal activity was four times lower. Interestingly, the chimeric protein was sensitive to regulation by pyruvate, 3-phosphoglyceric acid and fructose-1,6-bis-phosphate, which are known to be effectors of ADP-Glc PPases from different sources. The three compounds activated the chimeric enzyme up to three-fold, and increased the affinity for substrates. This chimeric protein is the first reported UDP-Glc PPase with allosteric regulatory properties. In addition, this is a pioneer work dealing with a chimeric enzyme constructed as a hybrid of two pyrophosphorylases with different specificity toward nucleoside-diphospho-glucose and our results turn to be relevant for a deeper understanding of the evolution of allosterism in this family of enzymes.

  20. Introduction of Culex Toxicity into Bacillus thuringiensis Cry4Ba by Protein Engineering

    OpenAIRE

    Abdullah, Mohd Amir F.; Alzate, Oscar; Mohammad, Marwan; McNall, Rebecca J.; Adang, Michael J.; Dean, Donald H.

    2003-01-01

    Bacillus thuringiensis mosquitocidal toxin Cry4Ba has no significant natural activity against Culex quinquefasciatus or Culex pipiens (50% lethal concentrations [LC50], >80,000 and >20,000 ng/ml, respectively). We introduced amino acid substitutions in three putative loops of domain II of Cry4Ba. The mutant proteins were tested on four different species of mosquitoes, Aedes aegypti, Anopheles quadrimaculatus, C. quinquefasciatus, and C. pipiens. Putative loop 1 and 2 exchanges eliminated acti...

  1. Engineering Human TMJ Discs with Protein-Releasing 3D-Printed Scaffolds.

    Science.gov (United States)

    Legemate, K; Tarafder, S; Jun, Y; Lee, C H

    2016-07-01

    The temporomandibular joint (TMJ) disc is a heterogeneous fibrocartilaginous tissue positioned between the mandibular condyle and glenoid fossa of the temporal bone, with important roles in TMJ functions. Tissue engineering TMJ discs has emerged as an alternative approach to overcoming limitations of current treatments for TMJ disorders. However, the anisotropic collagen orientation and inhomogeneous fibrocartilaginous matrix distribution present challenges in the tissue engineering of functional TMJ discs. Here, we developed 3-dimensional (3D)-printed anatomically correct scaffolds with region-variant microstrand alignment, mimicking anisotropic collagen alignment in the TMJ disc and corresponding mechanical properties. Connective tissue growth factor (CTGF) and transforming growth factor beta 3 (TGFβ3) were then delivered in the scaffolds by spatially embedding CTGF- or TGFβ3-encapsulated microspheres (µS) to reconstruct the regionally variant fibrocartilaginous matrix in the native TMJ disc. When cultured with human mesenchymal stem/progenitor cells (MSCs) for 6 wk, 3D-printed scaffolds with CTGF/TGFβ3-µS resulted in a heterogeneous fibrocartilaginous matrix with overall distribution of collagen-rich fibrous structure in the anterior/posterior (AP) bands and fibrocartilaginous matrix in the intermediate zone, reminiscent of the native TMJ disc. High dose of CTGF/TGFβ3-µS (100 mg µS/g of scaffold) showed significantly more collagen II and aggrecan in the intermediate zone than a low dose (50 mg µS/g of scaffold). Similarly, a high dose of CTGF/TGFβ3-µS yielded significantly higher collagen I expression in the AP bands compared with the low-dose and empty µS. From stress relaxation tests, the ratio of relaxation modulus to instantaneous modulus was significantly smaller with CTGF/TGFβ3-µS than empty µS. Similarly, a significantly higher coefficient of viscosity was achieved with the high dose of CTGF/TGFβ3-µS compared with the low-dose and empty

  2. Chaperonin containing T-complex polypeptide subunit eta is a potential marker of joint contracture: an experimental study in the rat.

    Science.gov (United States)

    He, Ronghan; Wang, Zhe; Lu, Yunxiang; Huang, Junqi; Ren, Jianhua; Wang, Kun

    2015-11-01

    Joint contracture is a fibroproliferative disorder that restricts joint mobility, resulting in tissue degeneration and deformity. However, the etiology of joint contracture is still unknown. Chaperonin containing T-complex polypeptide subunit eta (CCT-eta) is reported to increase in fibrotic diseases. The purpose of this study was to investigate whether CCT-eta is implicated in joint contracture and to determine the role of CCT-eta in the progression of joint contracture by analyzing a rat model. We immobilized the left knee joint of rat by internal fixation for 8 weeks. The non-immobilized right leg served as a control. The range of motion (ROM) of the knee was investigated. Fibroblasts were obtained from the posterior joint capsule of the joints. The outcome was followed by quantitative real-time polymerase chain reaction (qRT-PCR), Western blot, fibroblast migration assay, and collagen assay. The effect of CCT-eta on the functions of fibroblasts was observed by utilizing a short inhibitory RNA (siRNA) targeting CCT-eta. The ROM of the immobilized joints was significantly limited compared to the contralateral joints (p contracture disease.

  3. A New Molecular Mechanism To Engineer Protean Agonism at a G Protein-Coupled Receptor.

    Science.gov (United States)

    De Min, Anna; Matera, Carlo; Bock, Andreas; Holze, Janine; Kloeckner, Jessica; Muth, Mathias; Traenkle, Christian; De Amici, Marco; Kenakin, Terry; Holzgrabe, Ulrike; Dallanoce, Clelia; Kostenis, Evi; Mohr, Klaus; Schrage, Ramona

    2017-04-01

    Protean agonists are of great pharmacological interest as their behavior may change in magnitude and direction depending on the constitutive activity of a receptor. Yet, this intriguing phenomenon has been poorly described and understood, due to the lack of stable experimental systems and design strategies. In this study, we overcome both limitations: First, we demonstrate that modulation of the ionic strength in a defined experimental set-up allows for analysis of G protein-coupled receptor activation in the absence and presence of a specific amount of spontaneous receptor activity using the muscarinic M2 acetylcholine receptor as a model. Second, we employ this assay system to show that a dualsteric design principle, that is, molecular probes, carrying two pharmacophores to simultaneously adopt orthosteric and allosteric topography within a G protein-coupled receptor, may represent a novel approach to achieve protean agonism. We pinpoint three molecular requirements within dualsteric compounds that elicit protean agonism at the muscarinic M2 acetylcholine receptor. Using radioligand-binding and functional assays, we posit that dynamic ligand binding may be the mechanism underlying protean agonism of dualsteric ligands. Our findings provide both new mechanistic insights into the still enigmatic phenomenon of protean agonism and a rationale for the design of such compounds for a G protein-coupled receptor. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

  4. Decontamination of prion protein (BSE301V) using a genetically engineered protease.

    Science.gov (United States)

    Dickinson, J; Murdoch, H; Dennis, M J; Hall, G A; Bott, R; Crabb, W D; Penet, C; Sutton, J M; Raven, N D H

    2009-05-01

    A previous study has demonstrated the potential of alkaline proteases to inactivate bovine spongiform encephalopathy (BSE301V). Here we explored the use of MC3, a genetically engineered variant of Bacillus lentus subtilisin. MC3 was used to digest BSE301V infectious mouse brain homogenate (iMBH). MC3 eliminated all detectable 6H4-immunoreactive material at pH 10 and 12; however, Proteinase K was only partially effective at pH 12. When bioassayed in VM mice, MC3- and Proteinase K-digested iMBH gave respectively 66.6% and 22.7% survival rates. Using a titration series for disease incubation, this equates to a >7log reduction in infectivity for MC3 and >6log reduction for Proteinase K. This study demonstrates the potential for thermostable proteases to be developed as effective inactivation processes for prion agents in healthcare management.

  5. Expression of protein engineered NADP{sup +}-dependent xylitol dehydrogenase increases ethanol production from xylose in recombinant Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Matsushika, Akinori; Inoue, Hiroyuki; Murakami, Katsuji; Takimura, Osamu; Sawayama, Shigeki [National Institute of Advanced Industrial Science and Technology, Hiroshima (Japan). Biomass Technology Research Center; Watanabe, Seiya; Kodaki, Tsutomu; Makino, Keisuke [Kyoto Univ. (Japan). Inst. of Advanced Energy

    2008-11-15

    A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis has the ability to convert xylose to ethanol together with the unfavorable excretion of xylitol, which may be due to cofactor imbalance between NADPH-preferring XR and NAD{sup +}-dependent XDH. To reduce xylitol formation, we have already generated several XDH mutants with a reversal of coenzyme specificity toward NADP{sup +}. In this study, we constructed a set of recombinant S. cerevisiae strains with xylose-fermenting ability, including protein-engineered NADP{sup +}-dependent XDH-expressing strains. The most positive effect on xylose-to-ethanol fermentation was found by using a strain named MA-N5, constructed by chromosomal integration of the gene for NADP{sup +}-dependent XDH along with XR and endogenous xylulokinase genes. The MA-N5 strain had an increase in ethanol production and decrease in xylitol excretion compared with the reference strain expressing wild-type XDH when fermenting not only xylose but also mixed sugars containing glucose and xylose. Furthermore, the MA-N5 strain produced ethanol with a high yield of 0.49 g of ethanol/g of total consumed sugars in the nonsulfuric acid hydrolysate of wood chips. The results demonstrate that glucose and xylose present in the lignocellulosic hydrolysate can be efficiently fermented by this redox-engineered strain. (orig.)

  6. Expression of protein engineered NADP+-dependent xylitol dehydrogenase increases ethanol production from xylose in recombinant Saccharomyces cerevisiae.

    Science.gov (United States)

    Matsushika, Akinori; Watanabe, Seiya; Kodaki, Tsutomu; Makino, Keisuke; Inoue, Hiroyuki; Murakami, Katsuji; Takimura, Osamu; Sawayama, Shigeki

    2008-11-01

    A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis has the ability to convert xylose to ethanol together with the unfavorable excretion of xylitol, which may be due to cofactor imbalance between NADPH-preferring XR and NAD(+)-dependent XDH. To reduce xylitol formation, we have already generated several XDH mutants with a reversal of coenzyme specificity toward NADP(+). In this study, we constructed a set of recombinant S. cerevisiae strains with xylose-fermenting ability, including protein-engineered NADP(+)-dependent XDH-expressing strains. The most positive effect on xylose-to-ethanol fermentation was found by using a strain named MA-N5, constructed by chromosomal integration of the gene for NADP(+)-dependent XDH along with XR and endogenous xylulokinase genes. The MA-N5 strain had an increase in ethanol production and decrease in xylitol excretion compared with the reference strain expressing wild-type XDH when fermenting not only xylose but also mixed sugars containing glucose and xylose. Furthermore, the MA-N5 strain produced ethanol with a high yield of 0.49 g of ethanol/g of total consumed sugars in the nonsulfuric acid hydrolysate of wood chips. The results demonstrate that glucose and xylose present in the lignocellulosic hydrolysate can be efficiently fermented by this redox-engineered strain.

  7. Engineering specific chemical modification sites into a collagen-like protein from Streptococcus pyogenes.

    Science.gov (United States)

    Stoichevska, Violet; Peng, Yong Y; Vashi, Aditya V; Werkmeister, Jerome A; Dumsday, Geoff J; Ramshaw, John A M

    2017-03-01

    Recombinant bacterial collagens provide a new opportunity for safe biomedical materials. They are readily expressed in Escherichia coli in good yield and can be readily purified by simple approaches. However, recombinant proteins are limited in that direct secondary modification during expression is generally not easily achieved. Thus, inclusion of unusual amino acids, cyclic peptides, sugars, lipids, and other complex functions generally needs to be achieved chemically after synthesis and extraction. In the present study, we have illustrated that bacterial collagens that have had their sequences modified to include cysteine residue(s), which are not normally present in bacterial collagen-like sequences, enable a range of specific chemical modification reactions to be produced. Various model reactions were shown to be effective for modifying the collagens. The ability to include alkyne (or azide) functions allows the extensive range of substitutions that are available via "click" chemistry to be accessed. When bifunctional reagents were used, some crosslinking occurred to give higher molecular weight polymeric proteins, but gels were not formed. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 806-813, 2017. © 2016 Wiley Periodicals, Inc.

  8. Interfacial protein engineering for spray-dried emulsions - part II: oxidative stability.

    Science.gov (United States)

    Damerau, Annelie; Moisio, Timo; Partanen, Riitta; Forssell, Pirkko; Lampi, Anna-Maija; Piironen, Vieno

    2014-02-01

    The aim of this work was to investigate how the oxidative stability of encapsulated oil is affected by the humidity response of a Na-caseinate-maltodextrin matrix. Furthermore, the effect of modification of the interfacial Na-caseinate layer through cross-linking was studied. For this purpose, two model spray-dried emulsions containing sunflower oil, maltodextrin, and either non-cross-linked or cross-linked Na-caseinate were stored at different relative humidities (RHs; ∼0%, 11%, 33%, 54%, and 75%). Increasing RH improved the oxidative stability of the spray-dried emulsions. This behaviour was mainly linked to the loss of individual powder particles upon caking and collapsing of the matrix at RH 75%. Oxidation of non-encapsulated surface lipids with a proportion of ca. 5% of total lipids was only twofold compared to total lipids. Excess protein on particle surfaces may have delayed oxidation, e.g., by its radical scavenging activity. Under several storage conditions, cross-linking of the protein slightly improved the oxidative stability. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. Thioredoxin reductase from barley: Structure, recognition of thioredoxin, protein engineering and catalytic mechanism

    DEFF Research Database (Denmark)

    Kirkensgaard, Kristine Groth

    ) and a protein disulfide substrate, respectively. This comparison suggested some overlapping interactions as amino acid residues from HvTrxh2 involved in the binding of BASI are also interacting with the FAD and NADPH domains of HvNTR2. The model of HvNTR2:HvTrxh2 served as a guideline for a comprehensive...... proteins. Attempts to crystallise the HvNTR2:HvTrxh2 complex were unfruitful, possibly due to sample heterogeneity as indicated by molecular weight and isoelectric point determinations. Since no crystal structure of the HvNTR2:HvTrxh2 complex was obtained, a homology model based on the crystal structures...... of HvNTR2 and HvTrxh2 was built, using the structure of Escherichia coli NTR covalently bound to Trx (EcNTR:EcTrx) as a template (Lennon et al., 2000). The model provides new insight into how eukaryotic LMW NTRs in general bind Trx and suggests major differences in the NTR:Trx binding interface of Hv...

  10. Progranulin derived engineered protein Atsttrin suppresses TNF-α-mediated inflammation in intervertebral disc degenerative disease.

    Science.gov (United States)

    Ding, Hong; Wei, Jianlu; Zhao, Yunpeng; Liu, Yi; Liu, Lian; Cheng, Lei

    2017-12-12

    Atsttrin, an engineered molecule composed of three fragments of progranulin(PGRN), exerts comparable anti-inflammation ability. Intervertebral disc degeneration (IDD) is involved in inflammation in which TNF-α plays a key role. This study aims to examine the effect and the mechanism of Atsttrin in the pathogenesis of intervertebral disc degeneration. For this purpose, we took advantage of murine and human intervertebral disc (IVD) and examined the expression of TNF-α in IVD tissues using immunohistochemistry and TNF-α level in peripheral sera by ELISA assay. Moreover, murine IVD was taken to undergo the Safranin O and HE staining. Furthermore, primary human nucleus pulposus cells were used for immunohistochemistry staining, fluorescent staining, Western Blot, ELISA assay and RT-PCR assay. Herein we found TNF-α expression was elevated in intervertebral disc and peripheral sera in patients with IDD. Interestingly, Atsttrin effectively inhibited TNF-α-mediated catabolism in murine disc by ex vivo study. TNF-α-induced inflammatory cytokines were strongly reduced in presence of Atsttrin in primary human disc. Mechanism study indicated Atsttrin protected against intervertebral disc degeneration by inhibiting TNF-α-induced inflammation. These findings show that Atsttrin is a potential molecular target for disc degenerative diseases.

  11. Interaction of Biliverdin Chromophore with Near-Infrared Fluorescent Protein BphP1-FP Engineered from Bacterial Phytochrome

    Directory of Open Access Journals (Sweden)

    Olesya V. Stepanenko

    2017-05-01

    Full Text Available Near-infrared (NIR fluorescent proteins (FPs designed from PAS (Per-ARNT-Sim repeats and GAF (cGMP phosphodiesterase/adenylate cyclase/FhlA transcriptional activator domains of bacterial phytochromes covalently bind biliverdin (BV chromophore via one or two Cys residues. We studied BV interaction with a series of NIR FP variants derived from the recently reported BphP1-FP protein. The latter was engineered from a bacterial phytochrome RpBphP1, and has two reactive Cys residues (Cys15 in the PAS domain and Cys256 in the GAF domain, whereas its mutants contain single Cys residues either in the PAS domain or in the GAF domain, or no Cys residues. We characterized BphP1-FP and its mutants biochemically and spectroscopically in the absence and in the presence of denaturant. We found that all BphP1-FP variants are monomers. We revealed that spectral properties of the BphP1-FP variants containing either Cys15 or Cys256, or both, are determined by the covalently bound BV chromophore only. Consequently, this suggests an involvement of the inter-monomeric allosteric effects in the BV interaction with monomers in dimeric NIR FPs, such as iRFPs. Likely, insertion of the Cys15 residue, in addition to the Cys256 residue, in dimeric NIR FPs influences BV binding by promoting the BV chromophore covalent cross-linking to both PAS and GAF domains.

  12. Interaction of Biliverdin Chromophore with Near-Infrared Fluorescent Protein BphP1-FP Engineered from Bacterial Phytochrome.

    Science.gov (United States)

    Stepanenko, Olesya V; Stepanenko, Olga V; Kuznetsova, Irina M; Shcherbakova, Daria M; Verkhusha, Vladislav V; Turoverov, Konstantin K

    2017-05-08

    Near-infrared (NIR) fluorescent proteins (FPs) designed from PAS (Per-ARNT-Sim repeats) and GAF (cGMP phosphodiesterase/adenylate cyclase/FhlA transcriptional activator) domains of bacterial phytochromes covalently bind biliverdin (BV) chromophore via one or two Cys residues. We studied BV interaction with a series of NIR FP variants derived from the recently reported BphP1-FP protein. The latter was engineered from a bacterial phytochrome RpBphP1, and has two reactive Cys residues (Cys15 in the PAS domain and Cys256 in the GAF domain), whereas its mutants contain single Cys residues either in the PAS domain or in the GAF domain, or no Cys residues. We characterized BphP1-FP and its mutants biochemically and spectroscopically in the absence and in the presence of denaturant. We found that all BphP1-FP variants are monomers. We revealed that spectral properties of the BphP1-FP variants containing either Cys15 or Cys256, or both, are determined by the covalently bound BV chromophore only. Consequently, this suggests an involvement of the inter-monomeric allosteric effects in the BV interaction with monomers in dimeric NIR FPs, such as iRFPs. Likely, insertion of the Cys15 residue, in addition to the Cys256 residue, in dimeric NIR FPs influences BV binding by promoting the BV chromophore covalent cross-linking to both PAS and GAF domains.

  13. Barley alpha-amylase/subtilisin inhibitor: structure, biophysics and protein engineering

    DEFF Research Database (Denmark)

    Nielsen, P.K.; Bønsager, Birgit Christine; Fukuda, Kenji

    2004-01-01

    -type trypsin inhibitor family of the beta-trefoil fold proteins. Diverse approaches including site-directed mutagenesis, hybrid constructions, and crystallography have been used to characterise the structures and contact residues in the AMY2/BASI complex. The three-dimensional structure of the AMY2/BASI......Bifunctional alpha-amylase/subtilisin inhibitors have been implicated in plant defence and regulation of endogenous alpha-amylase action. The barley alpha-amylase/subtilisin inhibitor (BASI) inhibits the barley alpha-amylase 2 (AMY2) and subtilisin-type serine proteases. BASI belongs to the Kunitz...... Ca2+-modulated kinetics of the AMY2/BASl interaction and found that the complex formation involves minimal structural changes. The modulation of the interaction by calcium ions makes it unique among the currently known binding mechanisms of proteinaceous alpha-amylase inhibitors....

  14. Mapping the structure of amyloid nucleation precursors by protein engineering kinetic analysis.

    Science.gov (United States)

    Ruzafa, David; Varela, Lorena; Azuaga, Ana I; Conejero-Lara, Francisco; Morel, Bertrand

    2014-02-21

    Understanding the early molecular mechanisms governing amyloid aggregation is crucial to learn how to prevent it. Here, we used a site-directed mutagenesis approach to explore the molecular mechanism of nucleation of amyloid structure in the N47A Spc-SH3 domain. The changes in the native state stability produced by a series of mutations on each structural element of the domain were uncorrelated with the changes in the aggregation rates, although the overall aggregation mechanism was not altered. Analysis of the thioflavin T initial rates based on a simple kinetic model allowed us to extract thermodynamic magnitudes of the precursor states of nucleation and map the regions of the protein participating in the structure of the amyloidogenic precursors. This structure differs from that of the folding transition state of the SH3 domains, strongly suggesting that the regions of the conformational landscape leading to amyloid formation are divergent from those leading to the native fold.

  15. Protein engineering through chemical synthesis: design and synthesis of betabellins 4 and 5

    Energy Technology Data Exchange (ETDEWEB)

    Daniels, S.B.; Bobe, F.W.; Reddy, P.A.; Richardson, J.S.; Richardson, D.C.; Erickson, B.W.

    1986-05-01

    Betabellin (beta-barrel bell-shaped protein) was designed to provide a framework for the construction of binding and active sites. Its two identical peptide chains are COOH-terminally cross-linked by a symmetric diamino acid. Each chain should fold into an amphiphilic 4-stranded anti-parallel beta-pleated sheet. Each 32-residue chain of betabellin 4 is shown below, where Abu = 4-aminobutyric acid and Phi = 4-iodophenylalanine. Cross-linker X is 3,5-bis(aminoethyl)benzoic acid (Bab). A disulfide bridge between the two Cys-21 residues increases structural rigidity. Betabellin 5 contains D-amino acids at six positions in each chain (Pro, Asn). Betabellins 4 and 5 were synthesized from Boc/sub 2/Bab-Abu-NH-resin by simultaneous solid-phase assembly of both chains and were purified by gel filtration and centrifugal partition chromatography. CD and NMR studies indicate substantial beta-sheet structure.

  16. A Rational Engineering Strategy for Designing Protein A-Binding Camelid Single-Domain Antibodies.

    Directory of Open Access Journals (Sweden)

    Kevin A Henry

    Full Text Available Staphylococcal protein A (SpA and streptococcal protein G (SpG affinity chromatography are the gold standards for purifying monoclonal antibodies (mAbs in therapeutic applications. However, camelid VHH single-domain Abs (sdAbs or VHHs are not bound by SpG and only sporadically bound by SpA. Currently, VHHs require affinity tag-based purification, which limits their therapeutic potential and adds considerable complexity and cost to their production. Here we describe a simple and rapid mutagenesis-based approach designed to confer SpA binding upon a priori non-SpA-binding VHHs. We show that SpA binding of VHHs is determined primarily by the same set of residues as in human mAbs, albeit with an unexpected degree of tolerance to substitutions at certain core and non-core positions and some limited dependence on at least one residue outside the SpA interface, and that SpA binding could be successfully introduced into five VHHs against three different targets with no adverse effects on expression yield or antigen binding. Next-generation sequencing of llama, alpaca and dromedary VHH repertoires suggested that species differences in SpA binding may result from frequency variation in specific deleterious polymorphisms, especially Ile57. Thus, the SpA binding phenotype of camelid VHHs can be easily modulated to take advantage of tag-less purification techniques, although the frequency with which this is required may depend on the source species.

  17. Human milk protein production in xenografts of genetically engineered bovine mammary epithelial stem cells.

    Science.gov (United States)

    Martignani, Eugenio; Eirew, Peter; Accornero, Paolo; Eaves, Connie J; Baratta, Mario

    2010-10-19

    In the bovine species milk production is well known to correlate with mammary tissue mass. However, most advances in optimizing milk production relied on improvements of breeding and husbandry practices. A better understanding of the cells that generate bovine mammary tissue could facilitate important advances in milk production and have global economic impact. With this possibility in mind, we show that a mammary stem cell population can be functionally identified and isolated from the bovine mammary gland. We also demonstrate that this stem cell population may be a promising target for manipulating the composition of cow's milk using gene transfer. We show that the in vitro colony-forming cell assay for detecting normal primitive bipotent and lineage-restricted human mammary clonogenic progenitors are applicable to bovine mammary cells. Similarly, the ability of normal human mammary stem cells to regenerate functional bilayered structures in collagen gels placed under the kidney capsule of immunodeficient mice is shared by a subset of bovine mammary cells that lack aldehyde dehydrogenase activity. We also find that this activity is a distinguishing feature of luminal-restricted bovine progenitors. The regenerated structures recapitulate the organization of bovine mammary tissue, and milk could be readily detected in these structures when they were assessed by immunohistochemical analysis. Transplantation of the bovine cells transduced with a lentivirus encoding human β-CASEIN led to expression of the transgene and secretion of the product by their progeny regenerated in vivo. These findings point to a common developmental hierarchy shared by human and bovine mammary glands, providing strong evidence of common mechanisms regulating the maintenance and differentiation of mammary stem cells from both species. These results highlight the potential of novel engineering and transplant strategies for a variety of commercial applications including the production of

  18. Bright blue-shifted fluorescent proteins with Cys in the GAF domain engineered from bacterial phytochromes: fluorescence mechanisms and excited-state dynamics

    NARCIS (Netherlands)

    Hontani, Yusaku; Shcherbakova, Daria M.; Baloban, Mikhail; Zhu, Jingyi; Verkhusha, Vladislav V.; Kennis, John T. M.

    2016-01-01

    Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes (BphPs) are of great interest for in vivo imaging. They utilize biliverdin (BV) as a chromophore, which is a heme degradation product, and therefore they are straightforward to use in mammalian tissues. Here, we

  19. Engineering the cellular protein secretory pathway for enhancement of recombinant tissue plasminogen activator expression in Chinese hamster ovary cells: effects of CERT and XBP1s genes.

    Science.gov (United States)

    Rahimpour, Azam; Vaziri, Behrouz; Moazzami, Reza; Nematollahi, Leila; Barkhordari, Farzaneh; Kokabee, Leila; Adeli, Ahmad; Mahboudi, Fereidoun

    2013-08-01

    Cell line development is the most critical and also the most time-consuming step in the production of recombinant therapeutic proteins. In this regard, a variety of vector and cell engineering strategies have been developed for generating high-producing mammalian cells; however, the cell line engineering approach seems to show various results on different recombinant protein producer cells. In order to improve the secretory capacity of a recombinant tissue plasminogen activator (t-PA)-producing Chinese hamster ovary (CHO) cell line, we developed cell line engineering approaches based on the ceramide transfer protein (CERT) and X-box binding protein 1 (XBP1) genes. For this purpose, CERT S132A, a mutant form of CERT that is resistant to phosphorylation, and XBP1s were overexpressed in a recombinant t-PA-producing CHO cell line. Overexpression of CERT S132A increased the specific productivity of t-PA-producing CHO cells up to 35%. In contrast, the heterologous expression of XBP1s did not affect the t-PA expression rate. Our results suggest that CERTS132A- based secretion engineering could be an effective strategy for enhancing recombinant t- PA production in CHO cells.

  20. Fueling the engine: induction of AMP-activated protein kinase in trout skeletal muscle by swimming.

    Science.gov (United States)

    Magnoni, Leonardo J; Palstra, Arjan P; Planas, Josep V

    2014-05-15

    AMP-activated protein kinase (AMPK) is well known to be induced by exercise and to mediate important metabolic changes in the skeletal muscle of mammals. Despite the physiological importance of exercise as a modulator of energy use by locomotory muscle, the regulation of this enzyme by swimming has not been investigated in fish. We found that sustained swimming (40 days at 0.75 body lengths s(-1)) increased AMPK activity in red and white trout skeletal muscle (3.9- and 2.2-fold, respectively) as well as the expression of AMPK target genes involved in energy use: lipoprotein lipase and citrate synthase in red and white muscle and CPT1β1b and PGC-1α in red muscle. Furthermore, electrical pulse stimulation of cultured trout myotubes increased AMPK activity and glucose uptake (1.9- and 1.2-fold, respectively) in an AMPK-dependent manner. These results suggest that AMPK may play an important mediatory role in the metabolic adaptation to swimming in fish skeletal muscle. © 2014. Published by The Company of Biologists Ltd.

  1. Protein engineering to develop a redox insensitive endothelial nitric oxide synthase

    Directory of Open Access Journals (Sweden)

    Ruslan Rafikov

    2014-01-01

    Full Text Available The zinc tetrathiolate (ZnS4 cluster is an important structural feature of endothelial nitric oxide synthase (eNOS. The cluster is located on the dimeric interface and four cysteine residues (C94 and C99 from two adjacent subunits form a cluster with a Zn ion in the center of a tetrahedral configuration. Due to its high sensitivity to oxidants this cluster is responsible for eNOS dimer destabilization during periods of redox stress. In this work we utilized site directed mutagenesis to replace the redox sensitive cysteine residues in the ZnS4 cluster with redox stable tetra-arginines. Our data indicate that this C94R/C99R eNOS mutant is active. In addition, this mutant protein is insensitive to dimer disruption and inhibition when challenged with hydrogen peroxide (H2O2. Further, the overexpression of the C94R/C99R mutant preserved the angiogenic response in endothelial cells challenged with H2O2. The over-expression of the C94R/C99R mutant preserved the ability of endothelial cells to migrate towards vascular endothelial growth factor (VEGF and preserved the endothelial monolayer in a scratch wound assay. We propose that this dimer stable eNOS mutant could be utilized in the treatment of diseases in which there is eNOS dysfunction due to high levels of oxidative stress.

  2. Chaperonin-GroEL as a Smart Hydrophobic Drug Delivery and Tumor Targeting Molecular Machine for Tumor Therapy.

    Science.gov (United States)

    Yuan, Yi; Du, Chong; Sun, Cuiji; Zhu, Jin; Wu, Shan; Zhang, Yinlong; Ji, Tianjiao; Lei, Jianlin; Yang, Yinmo; Gao, Ning; Nie, Guangjun

    2018-02-14

    The targeted delivery of hydrophobic therapeutic drugs to tumors is one of the major challenges in drug development. The use of natural proteins as drug delivery vehicles holds great promise due to various functionalities of proteins. In the current study, we exploited a natural protein, GroEL, which possesses a double layer cage structure, as a hydrophobic drug container, which is switchable by ATP binding to a hydrophilic status, to design a novel and intelligent hydrophobic drug delivery molecular machine with a controlled drug release profile. When loaded with the hydrophobic antitumor drug, Doxorubicin (Dox), GroEL was able to shield the drug from the aqueous phase of blood, releasing the drug once in the presence of a critical concentration of ATP at the tumor site. Unexpectedly, we found that GroEL has a specific affinity for the cell structural protein, plectin, which is expressed at abnormally elevated levels on the membranes of tumor cells but not in normal cells. This finding, in combination with the ATP sensitivity, makes GroEL a superior natural tumor targeting nanocarrier. Our data show that GroEL-Dox is able to effectively, and highly selectively, deliver the hydrophobic drug to fast growing tumors without overt adverse effects on the major organs. GroEL is therefore a promising drug delivery platform that can overcome the obstacles to hydrophobic drug targeting and delivery.

  3. Acceleration through passive destabilization: protein folding in a weak hydrophobic environment

    Science.gov (United States)

    Jewett, Andrew; Baumketner, Andrij; Shea, Joan-Emma

    2004-03-01

    The GroEL chaperonin is a biomolecule which assists the folding of an extremely diverse range of proteins in Eubacteria. Some proteins undergo many rounds of ATP-regulated binding and dissociation from GroEL/ES before folding. It has been proposed that transient stress from ATP-regulated binding and release from GroEL/ES frees frustrated proteins from misfolded conformations. However recent evidence suggests that chaperonin-accelerated protein folding can take place entirely within a mutated GroEL+ES cavity that is unable to open and release the protein. Using molecular dynamics, we demonstrate that static confinement within a weakly hydrophobic (attractive) cavity (similar to the interior of the cavity formed by the GroEL+ES complex) is sufficient to significantly accelerate the folding of a highly frustrated protein-like heteropolymer. Our frustrated molecule benifits kinetically from a static hydrophobic environment that destabilizes misfolded conformations. This may shed light on the mechanisms used by other chaperones which do not depend on ATP.

  4. Engineered Context-Sensitive Agonism: Tissue-Selective Drug Signaling through a G Protein-Coupled Receptor.

    Science.gov (United States)

    Seemann, Wiebke K; Wenzel, Daniela; Schrage, Ramona; Etscheid, Justine; Bödefeld, Theresa; Bartol, Anna; Warnken, Mareille; Sasse, Philipp; Klöckner, Jessica; Holzgrabe, Ulrike; DeAmici, Marco; Schlicker, Eberhard; Racké, Kurt; Kostenis, Evi; Meyer, Rainer; Fleischmann, Bernd K; Mohr, Klaus

    2017-02-01

    Drug discovery strives for selective ligands to achieve targeted modulation of tissue function. Here we introduce engineered context-sensitive agonism as a postreceptor mechanism for tissue-selective drug action through a G protein-coupled receptor. Acetylcholine M2-receptor activation is known to mediate, among other actions, potentially dangerous slowing of the heart rate. This unwanted side effect is one of the main reasons that limit clinical application of muscarinic agonists. Herein we show that dualsteric (orthosteric/allosteric) agonists induce less cardiac depression ex vivo and in vivo than conventional full agonists. Exploration of the underlying mechanism in living cells employing cellular dynamic mass redistribution identified context-sensitive agonism of these dualsteric agonists. They translate elevation of intracellular cAMP into a switch from full to partial agonism. Designed context-sensitive agonism opens an avenue toward postreceptor pharmacologic selectivity, which even works in target tissues operated by the same subtype of pharmacologic receptor. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

  5. Engineering a Biocompatible Scaffold with Either Micrometre or Nanometre Scale Surface Topography for Promoting Protein Adsorption and Cellular Response

    Directory of Open Access Journals (Sweden)

    Xuan Le

    2013-01-01

    Full Text Available Surface topographical features on biomaterials, both at the submicrometre and nanometre scales, are known to influence the physicochemical interactions between biological processes involving proteins and cells. The nanometre-structured surface features tend to resemble the extracellular matrix, the natural environment in which cells live, communicate, and work together. It is believed that by engineering a well-defined nanometre scale surface topography, it should be possible to induce appropriate surface signals that can be used to manipulate cell function in a similar manner to the extracellular matrix. Therefore, there is a need to investigate, understand, and ultimately have the ability to produce tailor-made nanometre scale surface topographies with suitable surface chemistry to promote favourable biological interactions similar to those of the extracellular matrix. Recent advances in nanoscience and nanotechnology have produced many new nanomaterials and numerous manufacturing techniques that have the potential to significantly improve several fields such as biological sensing, cell culture technology, surgical implants, and medical devices. For these fields to progress, there is a definite need to develop a detailed understanding of the interaction between biological systems and fabricated surface structures at both the micrometre and nanometre scales.

  6. Synthetic Nanochaperones Facilitate Refolding of Denatured Proteins.

    Science.gov (United States)

    Ma, Fei-He; An, Yingli; Wang, Jianzu; Song, Yiqing; Liu, Yang; Shi, Linqi

    2017-10-24

    The folding process of a protein is inherently error-prone, owing to the large number of possible conformations that a protein chain can adopt. Partially folded or misfolded proteins typically expose hydrophobic surfaces and tend to form dysfunctional protein aggregates. Therefore, materials that can stabilize unfolded proteins and then efficiently assist them refolding to its bioactive form are of significant interest. Inspired by natural chaperonins, we have synthesized a series of polymeric nanochaperones that can facilitate the refolding of denatured proteins with a high recovery efficiency (up to 97%). Such nanochaperones possess phase-separated structure with hydrophobic microdomains on the surface. This structure allows nanochaperones to stabilize denatured proteins by binding them to the hydrophobic microdomains. We have also investigated the mechanism by which nanochaperones assist the protein refolding and established the design principles of nanochaperones in order to achieve effective recovery of a certain protein from their denatured forms. With a carefully designed composition of the microdomains according to the surface properties of the client proteins, the binding affinity between the hydrophobic microdomain and the denatured protein molecules can be tuned precisely, which enables the self-sorting of the polypeptides and the refolding of the proteins into their bioactive states. This work provides a feasible and effective strategy to recover inclusion bodies to their bioactive forms, which has potential to reduce the cost of the manufacture of recombinant proteins significantly.

  7. Single-molecule detection of chaperonin dynamics through polarization rotation modulation of CdSe QD luminescence imaging

    Energy Technology Data Exchange (ETDEWEB)

    Tani, Toshiro, E-mail: ttani@cc.tuat.ac.jp [Division of Advanced Applied Physics, Tokyo University of Agriculture and Technology, Institute of Technology, Naka-cho 2-24-16, Kogane-i, Tokyo 184-8588 (Japan); Oda, Masaru [Division of Advanced Applied Physics, Tokyo University of Agriculture and Technology, Institute of Technology, Naka-cho 2-24-16, Kogane-i, Tokyo 184-8588 (Japan); Araki, Daisuke; Miyashita, Tatsuki; Nakajima, Koudai [Department of Applied Physics, Tokyo University of Agriculture and Technology, Institute of Technology, Naka-cho 2-24-16, Kogane-i, Tokyo 184-8588 (Japan); Arita, Mayuno [Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Institute of Technology, Naka-cho 2-24-16, Kogane-i, Tokyo 184-8588 (Japan); Yohda, Masafumi [Division of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Institute of Technology, Naka-cho 2-24-16, Kogane-i, Tokyo 184-8588 (Japan)

    2014-08-01

    We report our recent trials examining the single-molecule three-dimensional (3D) detection of protein conformational dynamics at room temperature. Using molecular chaperones as model proteins and cadmium selenide (CdSe) semiconductor quantum dots (QDs) as nanometer-scale probes, we monitored the temporal evolution of ATP-induced conformation changes with a total internal reflection fluorescence (TIRF) microscopy imaging technique in buffer solutions. The two-dimensional (2D) degenerate nature of the emission dipoles of the QDs, due to the uniaxial wurtzite crystal structure, made it possible to capture the 3D orientation using a polarization modulation technique in real time. The temporal resolution was half the period of analyzer rotation. Although still insufficient, the obtained signals suggest possible 3D detection of specific motions, which supports the two-step conformational changes triggered by ATP attachment. - Highlights: • We report our recent trials examining the single-molecule three-dimensional (3D) detection of protein conformational dynamics at room temperature. • Using molecular chaperones as model proteins and cadmium selenide (CdSe) semiconductor quantum dots (QDs) as nanometer-scale probes, we monitored the temporal evolution of ATP-induced conformation changes with a total internal reflection fluorescence (TIRF) microscopy imaging technique in buffer solutions. • The two-dimensional (2D) degenerate nature of the emission dipoles of the QDs, due to the uniaxial wurtzite crystal structure, made it possible to capture the 3D orientation using a polarization modulation technique in real time. • The temporal resolution was half the period of analyzer rotation. • Although still insufficient, the obtained signals suggest possible 3D detection of specific motions, which supports the two-step conformational changes triggered by ATP attachment.

  8. "Velcro" engineering of high affinity CD47 ectodomain as signal regulatory protein α (SIRPα) antagonists that enhance antibody-dependent cellular phagocytosis.

    Science.gov (United States)

    Ho, Chia Chi M; Guo, Nan; Sockolosky, Jonathan T; Ring, Aaron M; Weiskopf, Kipp; Özkan, Engin; Mori, Yasuo; Weissman, Irving L; Garcia, K Christopher

    2015-05-15

    CD47 is a cell surface protein that transmits an anti-phagocytic signal, known as the "don't-eat-me" signal, to macrophages upon engaging its receptor signal regulatory protein α (SIRPα). Molecules that antagonize the CD47-SIRPα interaction by binding to CD47, such as anti-CD47 antibodies and the engineered SIRPα variant CV1, have been shown to facilitate macrophage-mediated anti-tumor responses. However, these strategies targeting CD47 are handicapped by large antigen sinks in vivo and indiscriminate cell binding due to ubiquitous expression of CD47. These factors reduce bioavailability and increase the risk of toxicity. Here, we present an alternative strategy to antagonize the CD47-SIRPα pathway by engineering high affinity CD47 variants that target SIRPα, which has restricted tissue expression. CD47 proved to be refractive to conventional affinity maturation techniques targeting its binding interface with SIRPα. Therefore, we developed a novel engineering approach, whereby we augmented the existing contact interface via N-terminal peptide extension, coined "Velcro" engineering. The high affinity variant (Velcro-CD47) bound to the two most prominent human SIRPα alleles with greatly increased affinity relative to wild-type CD47 and potently antagonized CD47 binding to SIRPα on human macrophages. Velcro-CD47 synergizes with tumor-specific monoclonal antibodies to enhance macrophage phagocytosis of tumor cells in vitro, with similar potency as CV1. Finally, Velcro-CD47 interacts specifically with a subset of myeloid-derived cells in human blood, whereas CV1 binds all myeloid, lymphoid, and erythroid populations interrogated. This is consistent with the restricted expression of SIRPα compared with CD47. Herein, we have demonstrated that "Velcro" engineering is a powerful protein-engineering tool with potential applications to other systems and that Velcro-CD47 could be an alternative adjuvant to CD47-targeting agents for cancer immunotherapy. © 2015 by

  9. “Velcro” Engineering of High Affinity CD47 Ectodomain as Signal Regulatory Protein α (SIRPα) Antagonists That Enhance Antibody-dependent Cellular Phagocytosis*

    Science.gov (United States)

    Ho, Chia Chi M.; Guo, Nan; Sockolosky, Jonathan T.; Ring, Aaron M.; Weiskopf, Kipp; Özkan, Engin; Mori, Yasuo; Weissman, Irving L.; Garcia, K. Christopher

    2015-01-01

    CD47 is a cell surface protein that transmits an anti-phagocytic signal, known as the “don't-eat-me” signal, to macrophages upon engaging its receptor signal regulatory protein α (SIRPα). Molecules that antagonize the CD47-SIRPα interaction by binding to CD47, such as anti-CD47 antibodies and the engineered SIRPα variant CV1, have been shown to facilitate macrophage-mediated anti-tumor responses. However, these strategies targeting CD47 are handicapped by large antigen sinks in vivo and indiscriminate cell binding due to ubiquitous expression of CD47. These factors reduce bioavailability and increase the risk of toxicity. Here, we present an alternative strategy to antagonize the CD47-SIRPα pathway by engineering high affinity CD47 variants that target SIRPα, which has restricted tissue expression. CD47 proved to be refractive to conventional affinity maturation techniques targeting its binding interface with SIRPα. Therefore, we developed a novel engineering approach, whereby we augmented the existing contact interface via N-terminal peptide extension, coined “Velcro” engineering. The high affinity variant (Velcro-CD47) bound to the two most prominent human SIRPα alleles with greatly increased affinity relative to wild-type CD47 and potently antagonized CD47 binding to SIRPα on human macrophages. Velcro-CD47 synergizes with tumor-specific monoclonal antibodies to enhance macrophage phagocytosis of tumor cells in vitro, with similar potency as CV1. Finally, Velcro-CD47 interacts specifically with a subset of myeloid-derived cells in human blood, whereas CV1 binds all myeloid, lymphoid, and erythroid populations interrogated. This is consistent with the restricted expression of SIRPα compared with CD47. Herein, we have demonstrated that “Velcro” engineering is a powerful protein-engineering tool with potential applications to other systems and that Velcro-CD47 could be an alternative adjuvant to CD47-targeting agents for cancer immunotherapy

  10. Optimization of Search Engines and Postprocessing Approaches to Maximize Peptide and Protein Identification for High-Resolution Mass Data.

    Science.gov (United States)

    Tu, Chengjian; Sheng, Quanhu; Li, Jun; Ma, Danjun; Shen, Xiaomeng; Wang, Xue; Shyr, Yu; Yi, Zhengping; Qu, Jun

    2015-11-06

    The two key steps for analyzing proteomic data generated by high-resolution MS are database searching and postprocessing. While the two steps are interrelated, studies on their combinatory effects and the optimization of these procedures have not been adequately conducted. Here, we investigated the performance of three popular search engines (SEQUEST, Mascot, and MS Amanda) in conjunction with five filtering approaches, including respective score-based filtering, a group-based approach, local false discovery rate (LFDR), PeptideProphet, and Percolator. A total of eight data sets from various proteomes (e.g., E. coli, yeast, and human) produced by various instruments with high-accuracy survey scan (MS1) and high- or low-accuracy fragment ion scan (MS2) (LTQ-Orbitrap, Orbitrap-Velos, Orbitrap-Elite, Q-Exactive, Orbitrap-Fusion, and Q-TOF) were analyzed. It was found combinations involving Percolator achieved markedly more peptide and protein identifications at the same FDR level than the other 12 combinations for all data sets. Among these, combinations of SEQUEST-Percolator and MS Amanda-Percolator provided slightly better performances for data sets with low-accuracy MS2 (ion trap or IT) and high accuracy MS2 (Orbitrap or TOF), respectively, than did other methods. For approaches without Percolator, SEQUEST-group performs the best for data sets with MS2 produced by collision-induced dissociation (CID) and IT analysis; Mascot-LFDR gives more identifications for data sets generated by higher-energy collisional dissociation (HCD) and analyzed in Orbitrap (HCD-OT) and in Orbitrap Fusion (HCD-IT); MS Amanda-Group excels for the Q-TOF data set and the Orbitrap Velos HCD-OT data set. Therefore, if Percolator was not used, a specific combination should be applied for each type of data set. Moreover, a higher percentage of multiple-peptide proteins and lower variation of protein spectral counts were observed when analyzing technical replicates using Percolator

  11. A Robust and Engineerable Self-Assembling Protein Template for the Synthesis and Patterning of Ordered Nanoparticle Arrays

    Science.gov (United States)

    McMillan, R. Andrew; Howard, Jeanie; Zaluzec, Nestor J.; Kagawa, Hiromi K.; Li, Yi-Fen; Paavola, Chad D.; Trent, Jonathan D.

    2004-01-01

    Self-assembling biomolecules that form highly ordered structures have attracted interest as potential alternatives to conventional lithographic processes for patterning materials. Here we introduce a general technique for patterning materials on the nanoscale using genetically modified protein cage structures called chaperonins that self-assemble into crystalline templates. Constrained chemical synthesis of transition metal nanoparticles is specific to templates genetically functionalized with poly-Histidine sequences. These arrays of materials are ordered by the nanoscale structure of the crystallized protein. This system may be easily adapted to pattern a variety of materials given the rapidly growing list of peptide sequences selected by screening for specificity for inorganic materials.

  12. Comparison between medium-chain acyl-CoA dehydrogenase mutant proteins overexpressed in bacterial and mammalian cells

    DEFF Research Database (Denmark)

    Jensen, T G; Bross, P; Andresen, B S

    1995-01-01

    Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is a potentially lethal inherited defect in the beta-oxidation of fatty acids. By comparing the behaviour of five missense MCAD mutant proteins expressed in COS cells and in Escherichia coli, we can define some of these as "pure folding mutants......." Upon expression in E. coli, these mutant proteins produce activity levels in the range of the wild-type enzyme only if the chaperonins GroESL are co-overproduced. When overexpressed in COS cells, the pure folding mutants display enzyme activities comparable to the wild-type enzyme. The results suggest...

  13. Generation of arming yeasts with active proteins and peptides via cell surface display system: cell surface engineering, bio-arming technology.

    Science.gov (United States)

    Kuroda, Kouichi; Ueda, Mitsuyoshi

    2014-01-01

    The cell surface display system in yeast enables the innovative strategy for improving cellular functions in a wide range of applications such as biofuel production, bioremediation, synthesis of valuable chemicals, recovery of rare metal ions, development of biosensors, and high-throughput screening of proteins/peptides library. Display of enzymes for polysaccharide degradation enables the construction of metabolically engineered whole-cell biocatalyst owing to the accessibility of the displayed enzymes to high-molecular-weight polysaccharides. In addition, along with fluorescence-based activity evaluation, fluorescence-activated cell sorting (FACS), and yeast cell chip, the cell surface display system is an effective molecular tool for high-throughput screening of mutated proteins/peptides library. In this article, we describe the methods for cell surface display of proteins/peptides of interest on yeast, evaluation of display efficiency, and harvesting of the displayed proteins/peptides from cell surface.

  14. Endoglucanase enzyme protein engineering by site-directed mutagenesis to improve the enzymatic properties and its expression in yeast

    Directory of Open Access Journals (Sweden)

    Farnaz Nikzad Jamnani

    2013-11-01

    Full Text Available Introduction: Fossil fuel is an expensive and finite energy source. Therefore, the use of renewable energy and biofuels production has been taken into consideration. One of the most suitable raw materials for biofuels is cellulosic compounds. Only microorganisms that contain cellulose enzymes can decompose cellulose and fungus of Trichodermareesei is the most important producer of this enzyme. Methods: In this study the nucleotide sequence of endoglucanase II, which is the starter of attack to cellulose chains, synthesized from amino acid sequence of this enzyme in fungus T.reesei and based on codon usage in the host; yeast Pichiapastoris. To produce optimized enzyme and to decrease the production time and enzyme price, protein engineering will be used. There are some methods to improve the enzymatic properties like site-directed mutagenesis in which amino-acid replacement occur. In this study two mutations were induced in endoglucanase enzyme gene by PCR in which free syctein positions 169 and 393 were switched to valine and histidine respectively. Then this gene was inserted into the pPinka expression vector and cloned in Escherichia coli. The recombinant plasmids were transferred into P.pastoris competent cells with electroporation, recombinant yeasts were cultured in BMMY medium and induced with methanol. Results: The sequencing of gene proved the induction of the two mutations and the presence of recombinant enzyme was confirmed by dinitrosalicilic acid method and SDS-PAGE. Conclusion: Examination of biochemical properties revealed that the two mutations simultaneously decreased catalytic power, thermal stability and increased the affinity of enzyme and substrate.

  15. Polymer-based protein engineering grown ferrocene-containing redox polymers improve current generation in an enzymatic biofuel cell.

    Science.gov (United States)

    Campbell, Alan S; Murata, Hironobu; Carmali, Sheiliza; Matyjaszewski, Krzysztof; Islam, Mohammad F; Russell, Alan J

    2016-12-15

    Enzymatic biofuel cells (EBFCs) are capable of generating electricity from physiologically present fuels making them promising power sources for the future of implantable devices. The potential application of such systems is limited, however, by inefficient current generation. Polymer-based protein engineering (PBPE) offers a unique method to tailor enzyme function through tunable modification of the enzyme surface with functional polymers. In this study, we report on the modification of glucose oxidase (GOX) with ferrocene-containing redox polymers to increase current generation efficiency in an enzyme-modified anode. Poly(N-(3-dimethyl(ferrocenyl)methylammonium bromide)propyl acrylamide) (pFcAc) was grown from covalently attached, water-soluble initiator molecules on the surface of GOX in a "grafting-from" approach using atom transfer radical polymerization (ATRP). The covalently-coupled ferrocene-containing polymers on the enzyme surface promoted the effective "wiring" of the GOX active site to an external electrode. The resulting GOX-pFcAc conjugates generated over an order of magnitude increase in current generation efficiency and a 4-fold increase in maximum EBFC power density (≈1.7µWcm(-2)) with similar open circuit voltage (0.27V) compared to native GOX when physically adsorbed onto paddle-shaped electrodes made up of electrospun polyacrylonitrile fibers coated with gold nanoparticles and multi-wall carbon nanotubes. The formation of electroactive enzyme-redox polymer conjugates using PBPE represents a powerful new tool for the improvement of mediated enzyme-based bioelectronics without the need for free redox mediators or anode/cathode compartmentalization. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  16. Efficiently engineered cell sheet using a complex of polyethylenimine–alginate nanocomposites plus bone morphogenetic protein 2 gene to promote new bone formation

    Science.gov (United States)

    Jin, Han; Zhang, Kai; Qiao, Chunyan; Yuan, Anliang; Li, Daowei; Zhao, Liang; Shi, Ce; Xu, Xiaowei; Ni, Shilei; Zheng, Changyu; Liu, Xiaohua; Yang, Bai; Sun, Hongchen

    2014-01-01

    Regeneration of large bone defects is a common clinical problem. Recently, stem cell sheet has been an emerging strategy in bone tissue engineering. To enhance the osteogenic potential of stem cell sheet, we fabricated bone morphogenetic protein 2 (BMP-2) gene-engineered cell sheet using a complex of polyethylenimine–alginate (PEI–al) nanocomposites plus human BMP-2 complementary(c)DNA plasmid, and studied its osteogenesis in vitro and in vivo. PEI–al nanocomposites carrying BMP-2 gene could efficiently transfect bone marrow mesenchymal stem cells. The cell sheet was made by culturing the cells in medium containing vitamin C for 10 days. Assays on the cell culture showed that the genetically engineered cells released the BMP-2 for at least 14 days. The expression of osteogenesis-related gene was increased, which demonstrated that released BMP-2 could effectively induce the cell sheet osteogenic differentiation in vitro. To further test the osteogenic potential of the cell sheet in vivo, enhanced green fluorescent protein or BMP-2-producing cell sheets were treated on the cranial bone defects. The results indicated that the BMP-2-producing cell sheet group was more efficient than other groups in promoting bone formation in the defect area. Our results suggested that PEI–al nanocomposites efficiently deliver the BMP-2 gene to bone marrow mesenchymal stem cells and that BMP-2 gene-engineered cell sheet is an effective way for promoting bone regeneration. PMID:24855355

  17. Efficiently engineered cell sheet using a complex of polyethylenimine-alginate nanocomposites plus bone morphogenetic protein 2 gene to promote new bone formation.

    Science.gov (United States)

    Jin, Han; Zhang, Kai; Qiao, Chunyan; Yuan, Anliang; Li, Daowei; Zhao, Liang; Shi, Ce; Xu, Xiaowei; Ni, Shilei; Zheng, Changyu; Liu, Xiaohua; Yang, Bai; Sun, Hongchen

    2014-01-01

    Regeneration of large bone defects is a common clinical problem. Recently, stem cell sheet has been an emerging strategy in bone tissue engineering. To enhance the osteogenic potential of stem cell sheet, we fabricated bone morphogenetic protein 2 (BMP-2) gene-engineered cell sheet using a complex of polyethylenimine-alginate (PEI-al) nanocomposites plus human BMP-2 complementary(c)DNA plasmid, and studied its osteogenesis in vitro and in vivo. PEI-al nanocomposites carrying BMP-2 gene could efficiently transfect bone marrow mesenchymal stem cells. The cell sheet was made by culturing the cells in medium containing vitamin C for 10 days. Assays on the cell culture showed that the genetically engineered cells released the BMP-2 for at least 14 days. The expression of osteogenesis-related gene was increased, which demonstrated that released BMP-2 could effectively induce the cell sheet osteogenic differentiation in vitro. To further test the osteogenic potential of the cell sheet in vivo, enhanced green fluorescent protein or BMP-2-producing cell sheets were treated on the cranial bone defects. The results indicated that the BMP-2-producing cell sheet group was more efficient than other groups in promoting bone formation in the defect area. Our results suggested that PEI-al nanocomposites efficiently deliver the BMP-2 gene to bone marrow mesenchymal stem cells and that BMP-2 gene-engineered cell sheet is an effective way for promoting bone regeneration.

  18. Beta-tricalcium phosphate particles as a controlled release carrier of osteogenic proteins for bone tissue engineering.

    Science.gov (United States)

    Hu, Junli; Hou, Yaping; Park, Hyejin; Lee, Min

    2012-07-01

    Beta-tricalcium phosphate (β-TCP) has been widely used as bone substitutes and delivery carriers of osteogenic proteins. However, low protein carrying capacity and agent burst release profiles of β-TCP limit their usage. This study investigates strategies to enhance protein carrying capacity of β-TCP particles with reduced initial burst by surface etching in citric acid solution or by creating apatite coatings with the simulate body fluid immersion approach. The release kinetics of protein from the modified β-TCP particles was investigated using Nel-like molecule-1 (Nell-1), a novel osteogenic protein, as a model protein. Although chemical etching treatments reduced the initial burst release of protein from the particles, a rapid burst release was observed with high protein dose. In contrast, the burst release of protein was significantly reduced by the apatite coating and a high protein dose was successfully delivered over a prolonged period from the apatite-coated particles. Protein release was further modulated by simultaneously delivering proteins from two different substrates: acid-etched and apatite-coated particles. The bioactivity of the protein was preserved during the loading procedure onto the particles. In addition, protein-loaded particles maintained biological activity in the lyophilized state over 4 weeks. These findings suggest that the protein carrying capacity of β-TCP can be modulated by surface modification, which has a potential for use as a protein carrier with controlled release. Copyright © 2012 Wiley Periodicals, Inc.

  19. Theory and simulation of explicit solvent effects on protein folding in vitro and in vivo

    Science.gov (United States)

    England, Jeremy L.

    The aim of this work is to develop theoretical tools for understanding what happens to water that is confined in amphipathic cavities, and for testing the consequences of this understanding for protein folding in vitro and in vivo. We begin in the first chapter with a brief review of the theoretical and simulation literature on the hydrophobic effect and the aqueous solvation of charged species that also puts forward a simple theoretical framework within which various solvation phenomena reported in past studies may be unified. Subsequently, in the second chapter we also review past computational and theoretical work on the specific question of how chaperonin complexes assist the folding of their substrates. With the context set, we turn in Chapter 3 to the case of an open system with water trapped between hydrophobic plates that experiences a uniform electric field normal to and between the plates. Classic bulk theory of electrostriction in polarizable fluids tells us that the electric field should cause an increase in local water density as it rises, yet some simulations have suggested the opposite. We present a mean-field Potts model we have developed to explain this discrepancy, and show how such a simple, coarse-grained lattice description can capture the fundamental consequences of the fact that external electric fields can frustrate the hydrogen bond network in confined water. Chapter 4 continues to pursue the issue of solvent evacuation between hydrophobic plates, but focuses on the impact of chemical denaturants on hydrophobic effects using molecular dynamics simulations of hydrophobic dewetting. We find that while urea and guanidinium have similar qualitative effects at the bulk level, they seem to differ in the microscopic mechanism by which they denature proteins, although both inhibit the onset of dewetting. Lastly, Chapters 5 and 6 examine the potential importance of solvent-mediated forces to protein folding in vivo. Chapter 5 develops a Landau

  20. An inverse metabolic engineering approach for the design of an improved host platform for over-expression of recombinant proteins in Escherichia coli.

    Science.gov (United States)

    Ghosh, Chaitali; Gupta, Rashmi; Mukherjee, Krishna Jyoti

    2012-07-03

    A useful goal for metabolic engineering would be to generate non-growing but metabolically active quiescent cells which would divert the metabolic fluxes towards product formation rather than growth. However, for products like recombinant proteins, which are intricately coupled to the growth process it is difficult to identify the genes that need to be knocked-out/knocked-in to get this desired phenotype. To circumvent this we adopted an inverse metabolic engineering strategy which would screen for the desired phenotype and thus help in the identification of genetic targets which need to be modified to get overproducers of recombinant protein. Such quiescent cells would obviate the need for high cell density cultures and increase the operational life span of bioprocesses. A novel strategy for generating a library, consisting of randomly down regulated metabolic pathways in E. coli was designed by cloning small genomic DNA fragments in expression vectors. Some of these DNA fragments got inserted in the reverse orientation thereby generating anti-sense RNA upon induction. These anti-sense fragments would hybridize to the sense mRNA of specific genes leading to gene 'silencing'. This library was first screened for slow growth phenotype and subsequently for enhanced over-expression ability. Using Green Fluorescent Protein (GFP) as a reporter protein on second plasmid, we were able to identify metabolic blocks which led to significant increase in expression levels. Thus down-regulating the ribB gene (3, 4 dihydroxy-2-butanone-4-phosphate synthase) led to a 7 fold increase in specific product yields while down regulating the gene kdpD (histidine kinase) led to 3.2 fold increase in specific yields. We have designed a high throughput screening approach which is a useful tool in the repertoire of reverse metabolic engineering strategies for the generation of improved hosts for recombinant protein expression.

  1. Engineering of Chimeric Protein Based on E Protein Domain III of Tick-Borne Encephalitis Virus and OmpF Porin of Yersinia pseudotuberculosis.

    Science.gov (United States)

    Stenkova, Anna M; Chopenko, Natalia S; Davydova, Ludmila A; Mazeika, Andrey N; Bystritskaya, Evgeniya P; Portnyagina, Olga Y; Anastyuk, Stanislav D; Kulbatskii, Dmitrii S; Lyukmanova, Ekaterina N; Dolgikh, Dmitriy A; Kostetsky, Eduard Y; Sanina, Nina M

    2017-07-24

    Tick-borne encephalitis poses a serious public health threat in the endemic regions. The disease treatment is restricted to symptomatic therapy, so great expectations are in the development of the prophylactic and therapeutic vaccines. The domain III of E protein of the tick-borne encephalitis virus is the main antigenic domain which includes virus-specific epitopes recognized by neutralizing antibodies. We have expressed, isolated and characterized the chimeric protein based on the fusion of domain III of E protein of the tick-borne encephalitis virus and bacterial porin OmpF from Yersinia pseudotuberculosis. The bacterial partner protein was used for decreasing toxicity and increasing immunogenicity of antigen. The chimeric protein was successfully expressed by the E. coli cells and purified using IMAC methodology. The purified protein was recognized with immunoblots by anti-E protein of tick-borne encephalitis virus monoclonal antibodies. Furthermore, the protein was able to elicit antibody response against domain III of E protein in immunized mice. The newly obtained chimeric antigen could be valuable for the development of the preventing tick-borne encephalitis subunit vaccines. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  2. Reversible non-stick behaviour of a bacterial protein polymer provides a tuneable molecular mimic for cell and tissue engineering.

    Science.gov (United States)

    Roque, Ana I; Soliakov, Andrei; Birch, Mark A; Philips, Sion R; Shah, Deepan S H; Lakey, Jeremy H

    2014-05-01

    Yersina pestis, the bubonic plague bacterium, is coated with a polymeric protein hydrogel for protection from host defences. The protein, which is robust and non-stick, resembles structures found in many eukaryotic extracellular-matrix proteins. Cells grown on the natural polymer cannot adhere and grow poorly; however, when cell-adhesion motifs are inserted into the protein, the cells proliferate. © The Authors, 2014. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Engineer medium and feed for modulating N-glycosylation of recombinant protein production in CHO cell culture

    DEFF Research Database (Denmark)

    Fan, Yuzhou; Kildegaard, Helene Faustrup; Andersen, Mikael Rørdam

    2017-01-01

    Chinese hamster ovary (CHO) cells have become the primary expression system for the production of complex recombinant proteins due to their long-term success in industrial scale production and generating appropriate protein N-glycans similar to that of humans. Control and optimization of protein N...

  4. High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor

    DEFF Research Database (Denmark)

    Cherezov, Vadim; Rosenbaum, Daniel M; Hanson, Michael A

    2007-01-01

    Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors constitute the largest family of eukaryotic signal transduction proteins that communicate across the membrane. We report the crystal structure of a human beta2-adrenergic receptor-T4 lysozyme fusion protein bound...... to the partial inverse agonist carazolol at 2.4 angstrom resolution. The structure provides a high-resolution view of a human G protein-coupled receptor bound to a diffusible ligand. Ligand-binding site accessibility is enabled by the second extracellular loop, which is held out of the binding cavity by a pair...

  5. Structure-based protein engineering for thermostable and alkaliphilic enhancement of endo-β-1,4-xylanase for applications in pulp bleaching.

    Science.gov (United States)

    Boonyapakron, Katewadee; Jaruwat, Aritsara; Liwnaree, Benjamas; Nimchua, Thidarat; Champreda, Verawat; Chitnumsub, Penchit

    2017-10-10

    In the pulp bleaching industry, enzymes with robust activity at high pH and temperatures are desirable for facilitating the pre-bleaching process with simplified processing and minimal use of chlorinated compounds. To engineer an enzyme for this purpose, we determined the crystal structure of the Xyn12.2 xylanase, a xylan-hydrolyzing enzyme derived from the termite gut symbiont metagenome, as the basis for structure-based protein engineering to improve Xyn12.2 stability in high heat and alkaline conditions. Engineered cysteine pairs that generated exterior disulfide bonds increased the kcat of Xyn12.2 variants and melting temperature at all tested conditions. These improvements led to up to 4.2-fold increases in catalytic efficiency at pH 9.0, 50°C for 1h and up to 3-fold increases at 60°C. The most effective variants, XynTT and XynTTTE, exhibited 2-3-fold increases in bagasse hydrolysis at pH 9.0 and 60°C compared to the wild-type enzyme. Overall, engineering arginines and phenylalanines for increased pKa and hydrogen bonding improved enzyme catalytic efficiency at high stringency conditions. These modifications were the keys to enhancing thermostability and alkaliphilicity in our enzyme variants, with XynTT and XynTTTE being especially promising for their application to the pulp and paper industry. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. miRNA engineering of CHO cells facilitates production of difficult-to-express proteins and increases success in cell line development.

    Science.gov (United States)

    Fischer, Simon; Marquart, Kim F; Pieper, Lisa A; Fieder, Juergen; Gamer, Martin; Gorr, Ingo; Schulz, Patrick; Bradl, Harald

    2017-07-01

    In recent years, coherent with growing biologics portfolios also the number of complex and thus difficult-to-express (DTE) therapeutic proteins has increased considerably. DTE proteins challenge bioprocess development and can include various therapeutic protein formats such as monoclonal antibodies (mAbs), multi-specific affinity scaffolds (e.g., bispecific antibodies), cytokines, or fusion proteins. Hence, the availability of robust and versatile Chinese hamster ovary (CHO) host cell factories is fundamental for high-yielding bioprocesses. MicroRNAs (miRNAs) have emerged as potent cell engineering tools to improve process performance of CHO manufacturing cell lines. However, there has not been any report demonstrating the impact of beneficial miRNAs on industrial cell line development (CLD) yet. To address this question, we established novel CHO host cells constitutively expressing a pro-productive miRNA: miR-557. Novel host cells were tested in two independent CLD campaigns using two different mAb candidates including a normal as well as a DTE antibody. Presence of miR-557 significantly enhanced each process step during CLD in a product independent manner. Stable expression of miR-557 increased the probability to identify high-producing cell clones. Furthermore, production cell lines derived from miR-557 expressing host cells exhibited significantly increased final product yields in fed-batch cultivation processes without compromising product quality. Strikingly, cells co-expressing miR-557 and a DTE antibody achieved a twofold increase in product titer compared to clones co-expressing a negative control miRNA. Thus, host cell engineering using miRNAs represents a promising tool to overcome limitations in industrial CLD especially with regard to DTE proteins. Biotechnol. Bioeng. 2017;114: 1495-1510. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  7. Binding of ReO4(-) with an engineered MoO4(2-)-binding protein: towards a new approach in radiopharmaceutical applications.

    Science.gov (United States)

    Aryal, Baikuntha P; Brugarolas, Pedro; He, Chuan

    2012-01-01

    Radiolabeled biomolecules are routinely used for clinical diagnostics. (99m)Tc is the most commonly used radioactive tracer in radiopharmaceuticals. (188)Re and (186)Re are also commonly used as radioactive tracers in medicine. However, currently available methods for radiolabeling are lengthy and involve several steps in bioconjugation processes. In this work we present a strategy to engineer proteins that may selectively recognize the perrhenate (ReO(4)(-)) ion as a new way to label proteins. We found that a molybdate (MoO(4)(2-))-binding protein (ModA) from Escherichia coli can bind perrhenate with high affinity. Using fluorescence and isothermal titration calorimetry measurements, we determined the dissociation constant of ModA for ReO(4)(-) to be 541 nM and we solved a crystal structure of ModA with a bound ReO(4)(-). On the basis of the structure we created a mutant protein containing a disulfide linkage, which exhibited increased affinity for perrhenate (K(d) = 104 nM). High-resolution crystal structures of ModA (1.7 Å) and A11C/R153C mutant (2.0 Å) were solved with bound perrhenate. Both structures show that a perrhenate ion occupies the molybdate binding site using the same amino acid residues that are involved in molybdate binding. The overall structure of the perrhenate-bound ModA is unchanged compared with that of the molybdate-bound form. In the mutant protein, the bound perrhenate is further stabilized by the engineered disulfide bond. © SBIC 2011

  8. System analysis of salt and osmotic stress induced proteins in Nostoc muscorum and Bradyrhizobium japonicum

    Directory of Open Access Journals (Sweden)

    Vipin Kaithwas

    2017-06-01

    Full Text Available In this study the proteome response of the two diazotrophic organism’s viz. Nostoc muscorum and Bradyrhizobium japonicum exposed to salt (NaCl and osmotic (sucrose stresses was compared. Out of the total over expressed proteins; we have selected only three over expressed proteins viz. GroEL chaperonin, nitrogenase Mo-Fe protein and argininosuccinate synthase for further analysis, and then we analyzed the amino acid frequencies of all the three over expressed proteins. That led to the conclusion that amino acids e.g. alanine, glycine and valine that were energetically cheaper to produce were showing higher frequencies. This study would help in tracing the phylogenetic relationship between protein families.

  9. Comparative profiling of extractable proteins in extracellular matrices of porcine cholecyst and jejunum intended for preparation of tissue engineering scaffolds.

    Science.gov (United States)

    Muhamed, Jaseer; Rajan, Akhila; Surendran, Arun; Jaleel, Abdul; Anilkumar, Thapasimuthu V

    2017-04-01

    Scaffolds prepared from cholecyst and jejunum have differential immunological potential, despite similar biocompatibility, when used as subcutaneous grafts. The reason for differential immunogenicity is probably due to differences in the nature of protein composition and biomolecules in the extracellular matrices (ECMs) of source organs that are used for preparation of the scaffolds. Against this background, the present study aims to identify the extractable proteins of ECMs derived from porcine cholecyst and jejunum. The proteins were extracted and identified through a conventional database search following sodium dodecyl sulfate-polyacrylamide gel-electrophoresis separation and mass spectroscopy. The resultant protein profile was analyzed and at least 154 proteins in cholecyst-derived extracellular matrix (CDE) and 186 proteins in jejunum-derived extracellular matrix (JDE) were identified. Both the matrices contained several extracelluar proteins including fibronectin, nidogen, decorin, and lumican that are known to participate in wound healing responses. However, the CDE had fewer cellular proteins than JDE, especially the latter contained class-I and class-II histocompatibility antigens which are incriminated as potent immunogens responsible for graft rejection. The results of the study suggested that the ECMs used for the scaffold preparation need not be "acellular" and differences in the protein composition of the ECMs might have caused the differential wound healing responses. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 489-496, 2017. © 2015 Wiley Periodicals, Inc.

  10. Ultra sensitive firefly luciferase-based protein-protein interaction assay (FlimPIA) attained by hinge region engineering and optimized reaction conditions.

    Science.gov (United States)

    Kurihara, Makoto; Ohmuro-Matsuyama, Yuki; Ayabe, Keiichi; Yamashita, Takahiro; Yamaji, Hideki; Ueda, Hiroshi

    2016-01-01

    Detecting and assaying protein-protein interactions are significant research procedures in biology and biotechnology. We recently reported a novel assay to detect protein-protein interaction, i.e. firefly luminescent intermediate-based protein-protein interaction assay (FlimPIA) using two mutant firefly luciferases (Flucs), which complement each other's deficient half reaction. This assay detects neighboring of two mutant Flucs, namely, a "Donor" that catalyzes the adenylation of firefly luciferin to produce a luciferyl-adenylate intermediate, and an "Acceptor" that catalyzes the subsequent light emitting reaction. However, its rather high background signal, derived from the remaining adenylation activity of the Acceptor, has limited its usefulness. To reduce this background signal, we introduced a mutation (R437K) into the hinge region of the Acceptor, while maintaining the oxidative activity. Interestingly, the signal/background (S/B) ratio of the assay was markedly improved by the addition of coenzyme A and reduction of the ATP concentration, probably due to reduced inhibition by dehydroluciferyl-adenylate formed during the catalysis and an increased ATP-based Km value of the Acceptor, respectively. As a result, a significantly improved maximal S/B ratio from 2.5 to ∼40 was attained, which promises wider use of the assay in in vitro diagnostics, drug discovery, and expanding our knowledge of various biological phenomena. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Protein Engineering by Random Mutagenesis and Structure-Guided Consensus of Geobacillus stearothermophilus Lipase T6 for Enhanced Stability in Methanol

    Science.gov (United States)

    Dror, Adi; Shemesh, Einav; Dayan, Natali

    2014-01-01

    The abilities of enzymes to catalyze reactions in nonnatural environments of organic solvents have opened new opportunities for enzyme-based industrial processes. However, the main drawback of such processes is that most enzymes have a limited stability in polar organic solvents. In this study, we employed protein engineering methods to generate a lipase for enhanced stability in methanol, which is important for biodiesel production. Two protein engineering approaches, random mutagenesis (error-prone PCR) and structure-guided consensus, were applied in parallel on an unexplored lipase gene from Geobacillus stearothermophilus T6. A high-throughput colorimetric screening assay was used to evaluate lipase activity after an incubation period in high methanol concentrations. Both protein engineering approaches were successful in producing variants with elevated half-life values in 70% methanol. The best variant of the random mutagenesis library, Q185L, exhibited 23-fold-improved stability, yet its methanolysis activity was decreased by one-half compared to the wild type. The best variant from the consensus library, H86Y/A269T, exhibited 66-fold-improved stability in methanol along with elevated thermostability (+4.3°C) and a 2-fold-higher fatty acid methyl ester yield from soybean oil. Based on in silico modeling, we suggest that the Q185L substitution facilitates a closed lid conformation that limits access for both the methanol and substrate excess into the active site. The enhanced stability of H86Y/A269T was a result of formation of new hydrogen bonds. These improved characteristics make this variant a potential biocatalyst for biodiesel production. PMID:24362426