WorldWideScience

Sample records for single recombinant protein

  1. Dynamic protein assemblies in homologous recombination with single DNA molecules

    NARCIS (Netherlands)

    van der Heijden, A.H.

    2007-01-01

    What happens when your DNA breaks? This thesis describes experimental work on the single-molecule level focusing on the interaction between DNA and DNA-repair proteins, in particular bacterial RecA and human Rad51, involved in homologous recombination. Homologous recombination and its central event

  2. Recombinant protein production technology

    Science.gov (United States)

    Recombinant protein production is an important technology for antibody production, biochemical activity study, and structural determination during the post-genomic era. Limiting factors in recombinant protein production include low-level protein expression, protein precipitation, and loss of protein...

  3. Recombinant Collagenlike Proteins

    Science.gov (United States)

    Fertala, Andzej

    2007-01-01

    A group of collagenlike recombinant proteins containing high densities of biologically active sites has been invented. The method used to express these proteins is similar to a method of expressing recombinant procollagens and collagens described in U. S. Patent 5,593,859, "Synthesis of human procollagens and collagens in recombinant DNA systems." Customized collagenous proteins are needed for biomedical applications. In particular, fibrillar collagens are attractive for production of matrices needed for tissue engineering and drug delivery. Prior to this invention, there was no way of producing customized collagenous proteins for these and other applications. Heretofore, collagenous proteins have been produced by use of such biological systems as yeasts, bacteria, and transgenic animals and plants. These products are normal collagens that can also be extracted from such sources as tendons, bones, and hides. These products cannot be made to consist only of biologically active, specific amino acid sequences that may be needed for specific applications. Prior to this invention, it had been established that fibrillar collagens consist of domains that are responsible for such processes as interaction with cells, binding of growth factors, and interaction with a number of structural proteins present in the extracellular matrix. A normal collagen consists of a sequence of domains that can be represented by a corresponding sequence of labels, e.g., D1D2D3D4. A collagenlike protein of the present invention contains regions of collagen II that contain multiples of a single domain (e.g., D1D1D1D1 or D4D4D4D4) chosen for its specific biological activity. By virtue of the multiplicity of the chosen domain, the density of sites having that specific biological activity is greater than it is in a normal collagen. A collagenlike protein according to this invention can thus be made to have properties that are necessary for tissue engineering.

  4. Comparative study to develop a single method for retrieving wide class of recombinant proteins from classical inclusion bodies.

    Science.gov (United States)

    Padhiar, Arshad Ahmed; Chanda, Warren; Joseph, Thomson Patrick; Guo, Xuefang; Liu, Min; Sha, Li; Batool, Samana; Gao, Yifan; Zhang, Wei; Huang, Min; Zhong, Mintao

    2018-03-01

    The formation of inclusion bodies (IBs) is considered as an Achilles heel of heterologous protein expression in bacterial hosts. Wide array of techniques has been developed to recover biochemically challenging proteins from IBs. However, acquiring the active state even from the same protein family was found to be an independent of single established method. Here, we present a new strategy for the recovery of wide sub-classes of recombinant protein from harsh IBs. We found that numerous methods and their combinations for reducing IB formation and producing soluble proteins were not effective, if the inclusion bodies were harsh in nature. On the other hand, different practices with mild solubilization buffers were able to solubilize IBs completely, yet the recovery of active protein requires large screening of refolding buffers. With the integration of previously reported mild solubilization techniques, we proposed an improved method, which comprised low sarkosyl concentration, ranging from 0.05 to 0.1% coupled with slow freezing (- 1 °C/min) and fast thaw (room temperature), resulting in greater solubility and the integrity of solubilized protein. Dilution method was employed with single buffer to restore activity for every sub-class of recombinant protein. Results showed that the recovered protein's activity was significantly higher compared with traditional solubilization/refolding approach. Solubilization of IBs by the described method was proved milder in nature, which restored native-like conformation of proteins within IBs.

  5. Heterogeneity in recombinant protein production

    DEFF Research Database (Denmark)

    Schalén, Martin; Johanson, Ted; Lundin, Luisa

    2012-01-01

    contribute to make a population in a fermenter heterogeneous, resulting in cell-to-cell variation in physiological parameters of the microbial culture. Our study aims at investigating how population heterogeneity and recombinant protein production is affected by environmental gradients in bioreactors....... For this purpose, a Saccharomyces cerevisiae strain, that functions as a protein production reporter, has been developed. A heterologous protein has been tagged with a fluorescent protein providing a way to measure the amount of heterologous protein produced by the cells on single cell level. Gradients...... are simulated in small bioreactors and the population heterogeneity can be visualised by analysing single cells with flow cytometry. This can give new insights to cell physiology and recombinant protein production at the industrial scale....

  6. Anti-Human Endoglin (hCD105) Immunotoxin-Containing Recombinant Single Chain Ribosome-Inactivating Protein Musarmin 1.

    Science.gov (United States)

    Barriuso, Begoña; Antolín, Pilar; Arias, F Javier; Girotti, Alessandra; Jiménez, Pilar; Cordoba-Diaz, Manuel; Cordoba-Diaz, Damián; Girbés, Tomás

    2016-06-10

    Endoglin (CD105) is an accessory component of the TGF-β receptor complex, which is expressed in a number of tissues and over-expressed in the endothelial cells of tumor neovasculature. Targeting endoglin with immunotoxins containing type 2 ribosome-inactivating proteins has proved an effective tool to reduce blood supply to B16 mice tumor xenografts. We prepared anti-endoglin immunotoxin (IT)-containing recombinant musarmin 1 (single chain ribosome-inactivating proteins) linked to the mouse anti-human CD105 44G4 mouse monoclonal antibody via N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). The immunotoxin specifically killed L929 fibroblast mouse cells transfected with the short form of human endoglin with IC50 values in the range of 5 × 10(-10) to 10(-9) M.

  7. Anti-Human Endoglin (hCD105 Immunotoxin—Containing Recombinant Single Chain Ribosome-Inactivating Protein Musarmin 1

    Directory of Open Access Journals (Sweden)

    Begoña Barriuso

    2016-06-01

    Full Text Available Endoglin (CD105 is an accessory component of the TGF-β receptor complex, which is expressed in a number of tissues and over-expressed in the endothelial cells of tumor neovasculature. Targeting endoglin with immunotoxins containing type 2 ribosome-inactivating proteins has proved an effective tool to reduce blood supply to B16 mice tumor xenografts. We prepared anti-endoglin immunotoxin (IT—containing recombinant musarmin 1 (single chain ribosome-inactivating proteins linked to the mouse anti-human CD105 44G4 mouse monoclonal antibody via N-succinimidyl 3-(2-pyridyldithio propionate (SPDP. The immunotoxin specifically killed L929 fibroblast mouse cells transfected with the short form of human endoglin with IC50 values in the range of 5 × 10−10 to 10−9 M.

  8. Improving recombinant protein purification yield

    Science.gov (United States)

    Production of adequate amounts of recombinant proteins is essential for antibody production, biochemical activity study, and structural determination during the post-genomic era. It’s technologically challenging and a limiting factor for tung oil research because analytical reagents such as high qua...

  9. A recombinant protein expression system

    African Journals Online (AJOL)

    Aghomotsegin

    2015-06-23

    Jun 23, 2015 ... Serum free cultivation of Leishmania is cost-effective and improves large scale production of well- defined parasite material. Moreover, the production of recombinant pharmaceutical proteins requires cultivation of the host in a culture medium free of animal materials, so several culture media for.

  10. The effect of a single recombination event

    DEFF Research Database (Denmark)

    Schierup, Mikkel Heide; Jensen, Thomas Mailund; Wiuf, Carsten

    We investigate the variance in how visible a single recombination event is in a SNP data set as a function of the type of recombination event and its age. Data is simulated under the coalescent with recombination and inference is by the popular composite likelihood methods. The major determinant...... of the effect of a recombination event is the genealogical type of the event and whether SNP variation is present that can reveal the genealogical consequences of the recombination event. Recombination events that only change some branch lengths in the genealogy have a very small, but detectable, effect....... The more lineages left when the recombination event occurs, the larger effect it has, implying that it is mainly young recombination events that we detect when estimating the rate. If the population is growing, though, more lineages are present back in time and relatively more ancient recombination events...

  11. A vaccinia virus recombinant transcribing an alphavirus replicon and expressing alphavirus structural proteins leads to packaging of alphavirus infectious single cycle particles.

    Directory of Open Access Journals (Sweden)

    Juana M Sánchez-Puig

    Full Text Available Poxviruses and Alphaviruses constitute two promising viral vectors that have been used extensively as expression systems, or as vehicles for vaccine purposes. Poxviruses, like vaccinia virus (VV are well-established vaccine vectors having large insertion capacity, excellent stability, and ease of administration. In turn, replicons derived from Alphaviruses like Semliki Forest virus (SFV are potent protein expression and immunization vectors but stocks are difficult to produce and maintain. In an attempt to demonstrate the use of a Poxvirus as a means for the delivery of small vaccine vectors, we have constructed and characterized VV/SFV hybrid vectors. A SFV replicon cDNA was inserted in the VV genome and placed under the control of a VV early promoter. The replicon, transcribed from the VV genome as an early transcript, was functional, and thus capable of initiating its own replication and transcription. Further, we constructed a VV recombinant additionally expressing the SFV structural proteins under the control of a vaccinia synthetic early/late promoter. Infection with this recombinant produced concurrent transcription of the replicon and expression of SFV structural proteins, and led to the generation of replicon-containing SFV particles that were released to the medium and were able to infect additional cells. This combined VV/SFV system in a single virus allows the use of VV as a SFV delivery vehicle in vivo. The combination of two vectors, and the possibility of generating in vivo single-cycle, replicon containing alphavirus particles, may open new strategies in vaccine development or in the design of oncolytic viruses.

  12. A vaccinia virus recombinant transcribing an alphavirus replicon and expressing alphavirus structural proteins leads to packaging of alphavirus infectious single cycle particles.

    Science.gov (United States)

    Sánchez-Puig, Juana M; Lorenzo, María M; Blasco, Rafael

    2013-01-01

    Poxviruses and Alphaviruses constitute two promising viral vectors that have been used extensively as expression systems, or as vehicles for vaccine purposes. Poxviruses, like vaccinia virus (VV) are well-established vaccine vectors having large insertion capacity, excellent stability, and ease of administration. In turn, replicons derived from Alphaviruses like Semliki Forest virus (SFV) are potent protein expression and immunization vectors but stocks are difficult to produce and maintain. In an attempt to demonstrate the use of a Poxvirus as a means for the delivery of small vaccine vectors, we have constructed and characterized VV/SFV hybrid vectors. A SFV replicon cDNA was inserted in the VV genome and placed under the control of a VV early promoter. The replicon, transcribed from the VV genome as an early transcript, was functional, and thus capable of initiating its own replication and transcription. Further, we constructed a VV recombinant additionally expressing the SFV structural proteins under the control of a vaccinia synthetic early/late promoter. Infection with this recombinant produced concurrent transcription of the replicon and expression of SFV structural proteins, and led to the generation of replicon-containing SFV particles that were released to the medium and were able to infect additional cells. This combined VV/SFV system in a single virus allows the use of VV as a SFV delivery vehicle in vivo. The combination of two vectors, and the possibility of generating in vivo single-cycle, replicon containing alphavirus particles, may open new strategies in vaccine development or in the design of oncolytic viruses.

  13. Recombinant human activated protein C (Xigris)

    NARCIS (Netherlands)

    Levi, M. [=Marcel M.; de Jonge, E.; van der Poll, T.

    2002-01-01

    An impaired function of the protein C pathway plays a central role in the pathogenesis of sepsis. Administration of human recombinant activated protein C (Xigris) may restore the dysfunctional anticoagulant mechanism and prevent amplification and propagation of thrombin generation and formation of

  14. A single immunization with a recombinant canine adenovirus expressing the rabies virus G protein confers protective immunity against rabies in mice

    International Nuclear Information System (INIS)

    Li Jianwei; Faber, Milosz; Papaneri, Amy; Faber, Marie-Luise; McGettigan, James P.; Schnell, Matthias J.; Dietzschold, Bernhard

    2006-01-01

    Rabies vaccines based on live attenuated rabies viruses or recombinant pox viruses expressing the rabies virus (RV) glycoprotein (G) hold the greatest promise of safety and efficacy, particularly for oral immunization of wildlife. However, while these vaccines induce protective immunity in foxes, they are less effective in other animals, and safety concerns have been raised for some of these vaccines. Because canine adenovirus 2 (CAV2) is licensed for use as a live vaccine for dogs and has an excellent efficacy and safety record, we used this virus as an expression vector for the RVG. The recombinant CAV2-RV G produces virus titers similar to those produced by wild-type CAV2, indicating that the RVG gene does not affect virus replication. Comparison of RVG expressed by CAV2-RV G with that of vaccinia-RV G recombinant virus (V-RG) revealed similar amounts of RV G on the cell surface. A single intramuscular or intranasal immunization of mice with CAV2-RVG induced protective immunity in a dose-dependent manner, with no clinical signs or discomfort from the virus infection regardless of the route of administration or the amount of virus

  15. Agroinfiltration contributes to VP1 recombinant protein degradation.

    Science.gov (United States)

    Pillay, Priyen; Kunert, Karl J; van Wyk, Stefan; Makgopa, Matome Eugene; Cullis, Christopher A; Vorster, Barend J

    2016-11-01

    There is a growing interest in applying tobacco agroinfiltration for recombinant protein production in a plant based system. However, in such a system, the action of proteases might compromise recombinant protein production. Protease sensitivity of model recombinant foot-and-mouth disease (FMD) virus P1-polyprotein (P1) and VP1 (viral capsid protein 1) as well as E. coli glutathione reductase (GOR) were investigated. Recombinant VP1 was more severely degraded when treated with the serine protease trypsin than when treated with the cysteine protease papain. Cathepsin L- and B-like as well as legumain proteolytic activities were elevated in agroinfiltrated tobacco tissues and recombinant VP1 was degraded when incubated with such a protease-containing tobacco extract. In silico analysis revealed potential protease cleavage sites within the P1, VP1 and GOR sequences. The interaction modeling of the single VP1 protein with the proteases papain and trypsin showed greater proximity to proteolytic active sites compared to modeling with the entire P1-polyprotein fusion complex. Several plant transcripts with differential expression were detected 24 hr post-agroinfiltration when the RNA-seq technology was applied to identify changed protease transcripts using the recently available tobacco draft genome. Three candidate genes were identified coding for proteases which included the Responsive-to-Desiccation-21 (RD21) gene and genes for coding vacuolar processing enzymes 1a (NbVPE1a) and 1b (NbVPE1b). The data demonstrates that the tested recombinant proteins are sensitive to protease action and agroinfiltration induces the expression of potential proteases that can compromise recombinant protein production.

  16. Thermostable exoshells fold and stabilize recombinant proteins.

    Science.gov (United States)

    Deshpande, Siddharth; Masurkar, Nihar D; Girish, Vallerinteavide Mavelli; Desai, Malan; Chakraborty, Goutam; Chan, Juliana M; Drum, Chester L

    2017-11-13

    The expression and stabilization of recombinant proteins is fundamental to basic and applied biology. Here we have engineered a thermostable protein nanoparticle (tES) to improve both expression and stabilization of recombinant proteins using this technology. tES provides steric accommodation and charge complementation to green fluorescent protein (GFPuv), horseradish peroxidase (HRPc), and Renilla luciferase (rLuc), improving the yields of functional in vitro folding by ~100-fold. Encapsulated enzymes retain the ability to metabolize small-molecule substrates, presumably via four 4.5-nm pores present in the tES shell. GFPuv exhibits no spectral shifts in fluorescence compared to a nonencapsulated control. Thermolabile proteins internalized by tES are resistant to thermal, organic, chaotropic, and proteolytic denaturation and can be released from the tES assembly with mild pH titration followed by proteolysis.

  17. Recombinant protein hydrazides: application to site-specific protein PEGylation.

    Science.gov (United States)

    Thom, Jennifer; Anderson, David; McGregor, Joanne; Cotton, Graham

    2011-06-15

    Here, we describe a novel method for the site-specific C-terminal PEGylation of recombinant proteins. This general approach exploits chemical cleavage of precursor intein-fusion proteins with hydrazine to directly produce recombinant protein hydrazides. This unique functionality within the protein sequence then facilitates site-specific C-terminal modification by hydrazone-forming ligation reactions. This approach was used to generate folded, site-specifically C-terminal PEGylated IFNalpha2b and IFNbeta1b, which retained excellent antiviral activity, demonstrating the utility of this technology in the PEGylation of therapeutic proteins. As this methodology is straightforward to perform, is compatible with disulfide bonds, and is exclusively selective for the protein C-terminus, it shows great potential as general technology for the site-specific engineering and labeling of recombinant proteins.

  18. Carbon source feeding strategies for recombinant protein ...

    African Journals Online (AJOL)

    Pichia pastoris and Pichia methanolica have been used as expression systems for the production of recombinant protein. The main problems of the production are the slow hierarchic consumption of ethanol and acetate which cause toxicity problems due to methanol accumulation when this surpasses 0.5 gl-1. In some ...

  19. Improving recombinant protein solubility in Escherichia coli ...

    African Journals Online (AJOL)

    user

    2010-11-22

    Nov 22, 2010 ... capable of improving solubility ratio of human lysozyme. All these studies show that while this approach has been very successful for a large number of unrelated sub- strates, there is no guarantee that chaperones co- overproduction will improve the folding of a recombinant protein. For the first time in this ...

  20. Cultivating Insect Cells To Produce Recombinant Proteins

    Science.gov (United States)

    Spaulding, Glenn; Goodwin, Thomas; Prewett, Tacey; Andrews, Angela; Francis, Karen; O'Connor, Kim

    1996-01-01

    Method of producing recombinant proteins involves growth of insect cells in nutrient solution in cylindrical bioreactor rotating about cylindrical axis, oriented horizontally and infecting cells with viruses into which genes of selected type cloned. Genes in question those encoding production of desired proteins. Horizontal rotating bioreactor preferred for use in method, denoted by acronym "HARV", described in "High-Aspect-Ratio Rotating Cell-Culture Vessel" (MSC-21662).

  1. Protein Crystal Recombinant Human Insulin

    Science.gov (United States)

    1994-01-01

    The comparison of protein crystal, Recombiant Human Insulin; space-grown (left) and earth-grown (right). On STS-60, Spacehab II indicated that space-grown crystals are larger and of greater optical clarity than their earth-grown counterparts. Recombiant Human Insulin facilitates the incorporation of glucose into cells. In diabetics, there is either a decrease in or complete lack of insulin, thereby leading to several harmful complications. Principal Investigator is Larry DeLucas.

  2. Extracellular secretion of recombinant proteins

    Science.gov (United States)

    Linger, Jeffrey G.; Darzins, Aldis

    2014-07-22

    Nucleic acids encoding secretion signals, expression vectors containing the nucleic acids, and host cells containing the expression vectors are disclosed. Also disclosed are polypeptides that contain the secretion signals and methods of producing polypeptides, including methods of directing the extracellular secretion of the polypeptides. Exemplary embodiments include cellulase proteins fused to secretion signals, methods to produce and isolate these polypeptides, and methods to degrade lignocellulosic biomass.

  3. Novel 1:1 labeling and purification process for C-terminal thioester and single cysteine recombinant proteins using generic peptidic toolbox reagents.

    Science.gov (United States)

    Portal, Christophe F; Seifert, Jan-Marcus; Buehler, Christof; Meisner-Kober, Nicole-Claudia; Auer, Manfred

    2014-07-16

    We developed a versatile set of chemical labeling reagents which allow dye ligation to the C-terminus of a protein or a single internal cysteine and target purification in a simple two-step process. This simple process results in a fully 1:1 labeled conjugate suitable for all quantitative fluorescence spectroscopy and imaging experiments. We refer to a "generic labeling toolbox" because of the flexibility to choose one of many available dyes, spacers of different lengths and compositions which increase the target solubility, a variety of affinity purification tags, and different cleavage chemistries to release the 1:1 labeled proteins. Studying protein function in vitro or in the context of live cells and organisms is of vital importance in biological research. Although label free detection technologies gain increasing interest in molecular recognition science, fluorescence spectroscopy is still the most often used detection technique for assays and screens both in academic as well as in industrial groups. For generations, fluorescence spectroscopists have labeled their proteins of interest with small fluorescent dyes by random chemical linking on the proteins' exposed lysines and cysteines. Chemical reactions with a certain excess of activated esters or maleimides of longer wavelength dyes hardly ever result in quantitative labeling of the target protein. Most of the time, more than one exposed amino acid side chain reacts. This results in a mixture of dye-protein complexes of different labeling stoichiometries and labeling sites. Only mass spectrometry allows resolving the precise chemical composition of the conjugates. In "classical" ensemble averaging fluorescent experiments, these labeled proteins are still useful, and quantification of, e.g., ligand binding experiments, is achieved via knowledge of the overall protein concentration and a fluorescent signal change which is proportional to the amount of complex formed. With the development of fluorescence

  4. Characterization of hepatitis C virus recombinants with chimeric E1/E2 envelope proteins and identification of single amino acids in the E2 stem region important for entry

    DEFF Research Database (Denmark)

    Carlsen, Thomas H R; Scheel, Troels K H; Ramirez, Santseharay

    2013-01-01

    The hepatitis C virus (HCV) envelope proteins E1 and E2 play a key role in host cell entry and represent important targets for vaccine and drug development. Here, we characterized HCV recombinants with chimeric E1/E2 complexes in vitro. Using genotype 1a/2a JFH1-based recombinants expressing 1a...... core-NS2, we exchanged E2 with functional isolate sequences of genotypes 1a (alternative isolate), 1b, and 2a. While the 1a-E2 exchange did not impact virus viability, the 2a-E2 recombinant was nonviable. After E2 exchange from three 1b isolates, long delays were observed before spread of infection....... For recovered 1b-E2 recombinants, single E2 stem region amino acid changes were identified at residues 706, 707, and 710. In reverse genetic studies, these mutations increased infectivity titers by ~100-fold, apparently without influencing particle stability or cell binding although introducing slight decrease...

  5. Recombinant protein scaffolds for tissue engineering

    International Nuclear Information System (INIS)

    Werkmeister, Jerome A; Ramshaw, John A M

    2012-01-01

    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. (topical review)

  6. Single Molecule Study of DNA Organization and Recombination

    Science.gov (United States)

    Xiao, Botao

    We have studied five projects related to DNA organization and recombination using mainly single molecule force-spectroscopy and statistical tools. First, HU is one of the most abundant DNA-organizing proteins in bacterial chromosomes and participates in gene regulation. We report experiments that study the dependence of DNA condensation by HU on force, salt and HU concentration. A first important result is that at physiological salt levels, HU only bends DNA, resolving a previous paradox of why a chromosome-compacting protein should have a DNA-stiffening function. A second major result is quantitative demonstration of strong dependencies of HU-DNA dissociation on both salt concentration and force. Second, we have used a thermodynamic Maxwell relation to count proteins driven off large DNAs by tension, an effect important to understanding DNA organization. Our results compare well with estimates of numbers of proteins HU and Fis in previous studies. We have also shown that a semi-flexible polymer model describes our HU experimental data well. The force-dependent binding suggests mechano-chemical mechanisms for gene regulation. Third, the elusive role of protein H1 in chromatin has been clarified with purified H1 and Xenopus extracts. We find that H1 compacts DNA by both bending and looping. Addition of H1 enhances chromatin formation and maintains the plasticity of the chromatin. Fourth, the topology and mechanics of DNA twisting are critical to DNA organization and recombination. We have systematically measured DNA extension as a function of linking number density from 0.08 to -2 with holding forces from 0.2 to 2.4 pN. Unlike previous proposals, the DNA extension decreases with negative linking number. Finally, DNA recombination is a dynamic process starting from enzyme-DNA binding. We report that the Int-DBD domain of lambda integrase binds to DNA without compaction at low Int-DBD concentration. High concentration of Int-DBD loops DNA below a threshold force

  7. Overview of the purification of recombinant proteins.

    Science.gov (United States)

    Wingfield, Paul T

    2015-04-01

    When the first version of this unit was written in 1995, protein purification of recombinant proteins was based on a variety of standard chromatographic methods and approaches, many of which were described and mentioned throughout Current Protocols in Protein Science. In the interim, there has been a shift toward an almost universal usage of the affinity or fusion tag. This may not be the case for biotechnology manufacture where affinity tags can complicate producing proteins under regulatory conditions. Regardless of the protein expression system, questions are asked as to which and how many affinity tags to use, where to attach them in the protein, and whether to engineer a self-cleavage system or simply leave them on. We will briefly address some of these issues. Also, although this overview focuses on E.coli, protein expression and purification, other commonly used expression systems are mentioned and, apart from cell-breakage methods, protein purification methods and strategies are essentially the same. Copyright © 2015 John Wiley & Sons, Inc.

  8. 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.

  9. General introduction: recombinant protein production and purification of insoluble proteins.

    Science.gov (United States)

    Ferrer-Miralles, Neus; Saccardo, Paolo; Corchero, José Luis; Xu, Zhikun; García-Fruitós, Elena

    2015-01-01

    Proteins are synthesized in heterologous systems because of the impossibility to obtain satisfactory yields from natural sources. The production of soluble and functional recombinant proteins is among the main goals in the biotechnological field. In this context, it is important to point out that under stress conditions, protein folding machinery is saturated and this promotes protein misfolding and, consequently, protein aggregation. Thus, the selection of the optimal expression organism and the most appropriate growth conditions to minimize the formation of insoluble proteins should be done according to the protein characteristics and downstream requirements. Escherichia coli is the most popular recombinant protein expression system despite the great development achieved so far by eukaryotic expression systems. Besides, other prokaryotic expression systems, such as lactic acid bacteria and psychrophilic bacteria, are gaining interest in this field. However, it is worth mentioning that prokaryotic expression system poses, in many cases, severe restrictions for a successful heterologous protein production. Thus, eukaryotic systems such as mammalian cells, insect cells, yeast, filamentous fungus, and microalgae are an interesting alternative for the production of these difficult-to-express proteins.

  10. Stress in recombinant protein producing yeasts.

    Science.gov (United States)

    Mattanovich, Diethard; Gasser, Brigitte; Hohenblum, Hubertus; Sauer, Michael

    2004-09-30

    It is well established today that heterologous overexpression of proteins is connected with different stress reactions. The expression of a foreign protein at a high level may either directly limit other cellular processes by competing for their substrates, or indirectly interfere with metabolism, if their manufacture is blocked, thus inducing a stress reaction of the cell. Especially the unfolded protein response (UPR) in Saccharomyces cerevisiae (as well as some other yeasts) is well documented, and its role for the limitation of expression levels is discussed. One potential consequence of endoplasmatic reticulum folding limitations is the ER associated protein degradation (ERAD) involving retrotranslocation and decay in the cytosol. High cell density fermentation, the typical process design for recombinant yeasts, exerts growth conditions that deviate far from the natural environment of the cells. Thus, different environmental stresses may be exerted on the host. High osmolarity, low pH and low temperature are typical stress factors. Whereas the molecular pathways of stress responses are well characterized, there is a lack of knowledge concerning the impact of stress responses on industrial production processes. Accordingly, most metabolic engineering approaches conducted so far target at the improvement of protein folding and secretion, whereas only few examples of cell engineering against general stress sensitivity were published. Apart from discussing well-documented stress reactions of yeasts in the context of heterologous protein production, some more speculative topics like quorum sensing and apoptosis are addressed.

  11. Recombinant Brucella abortus gene expressing immunogenic protein

    Energy Technology Data Exchange (ETDEWEB)

    Mayfield, J.E.; Tabatabai, L.B.

    1991-06-11

    This patent describes a synthetic recombinant DNA molecule containing a DNA sequence. It comprises a gene of Brucella abortus encoding an immunogenic protein having a molecular weight of approximately 31,000 daltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis under denaturing conditions, the protein having an isoelectric point around 4.9, and containing a twenty-five amino acid sequence from its amino terminal end consisting of Gln-Ala-Pro-Thr-Phe-Phe-Arg-Ile-Gly-Thr-Gly-Gly-Thr-Ala-Gly-Thr-Tyr-Tyr-Pro-Ile-Gly-Gly-Leu-Ile-Ala, wherein Gln, Ala, Pro, Thr, Phe, Arg, Ile, Gly, Tyr, and Leu, respectively, represent glutamine, alanine, proline, threonine, phenylalanine, arginine, isolecuine, glycine, tyrosine, and leucine.

  12. Recombinant HT.sub.m4 gene, protein and assays

    Science.gov (United States)

    Lim, Bing; Adra, Chaker N.; Lelias, Jean-Michel

    1996-01-01

    The invention relates to a recombinant DNA molecule which encodes a HT.sub.m4 protein, a transformed host cell which has been stably transfected with a DNA molecule which encodes a HT.sub.m4 protein and a recombinant HT.sub.m4 protein. The invention also relates to a method for detecting the presence of a hereditary atopy.

  13. Co-solute assistance in refolding of recombinant proteins | Gerami ...

    African Journals Online (AJOL)

    Prokaryotic expression system is the most widely used host for the production of recombinant proteins but inclusion body formation is a major bottleneck in the production of recombinant proteins in prokaryotic cells, especially in Escherichia coli. In vitro refolding of inclusion body into the the proteins with native ...

  14. Recombinant tagging system using ribosomal frameshifting to monitor protein expression.

    Science.gov (United States)

    Han, Se Jong; Cho, Sayeon; Lowehhaupt, Ky; Park, So-Young; Sim, Sang Jun; Kim, Yang-Gyun

    2013-03-01

    For rapid and accurate quantitation of recombinant proteins during expression and after purification, we introduce a new tagging strategy that expresses both target proteins and limitedly tagged target proteins together in a single cell at a constant ratio by utilizing cis-elements of programmed -1 ribosomal frameshifting (-1RFS) as an embedded device. -1RFS is an alternative reading mechanism that effectively controls protein expression by many viruses. When a target gene is fused to the enhanced green fluorescent protein (EGFP) gene with a -1RFS element implanted between them, the unfused target and the target-GFP fusion proteins are expressed at a fixed ratio. The expression ratio between these two protein products is adjustable simply by changing -1RFS signals. This limited-tagging system would be valuable for the real-time monitoring of protein expression when optimizing expression condition for a new protein, and in monitoring large-scale bioprocesses without a large metabolic burden on host cells. Furthermore, this strategy allows for the direct measurement of the quantity of a protein on a chip surface and easy application to proteomewide study of gene products. Copyright © 2012 Wiley Periodicals, Inc.

  15. Evaluation of somatic embryos of alfalfa for recombinant protein expression.

    Science.gov (United States)

    Fu, Guohua; Grbic, Vojislava; Ma, Shengwu; Tian, Lining

    2015-02-01

    Somatic embryos of alfalfa can accumulate higher levels of recombinant proteins comparing to vegetative organs. Somatic embryos may be explored as a new system for new protein production for plants. Plants have been explored via genetic engineering as an inexpensive system for recombinant protein production. However, protein expression levels in vegetative tissues have been low, which limits the commercial utilization of plant expression systems. Somatic embryos resemble zygotic embryos in many aspects and may accumulate higher levels of proteins as true seed. In this study, somatic embryo of alfalfa (Medicago sativa L.) was investigated for the expression of recombinant proteins. Three heterologous genes, including the standard scientific reporter uid that codes for β-glucuronidase and two genes of interest: ctb coding for cholera toxin B subunit (CTB), and hIL-13 coding for human interleukin 13, were independently introduced into alfalfa via Agrobacterium-mediated transformation. Somatic embryos were subsequently induced from transgenic plants carrying these genes. Somatic embryos accumulated approximately twofold more recombinant proteins than vegetative organs including roots, stems, and leaves. The recombinant proteins of CTB and hIL-13 accumulated up to 0.15 and 0.18 % of total soluble protein in alfalfa somatic embryos, respectively. The recombinant proteins expressed in somatic embryos also exhibited biological activities. As somatic embryos can be induced in many plant species and their production can be scaled up via different avenues, somatic embryos may be developed as an efficient expression system for recombinant protein production.

  16. 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.......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...

  17. Integrated continuous production of recombinant therapeutic proteins.

    Science.gov (United States)

    Warikoo, Veena; Godawat, Rahul; Brower, Kevin; Jain, Sujit; Cummings, Daniel; Simons, Elizabeth; Johnson, Timothy; Walther, Jason; Yu, Marcella; Wright, Benjamin; McLarty, Jean; Karey, Kenneth P; Hwang, Chris; Zhou, Weichang; Riske, Frank; Konstantinov, Konstantin

    2012-12-01

    In the current environment of diverse product pipelines, rapidly fluctuating market demands and growing competition from biosimilars, biotechnology companies are increasingly driven to develop innovative solutions for highly flexible and cost-effective manufacturing. To address these challenging demands, integrated continuous processing, comprised of high-density perfusion cell culture and a directly coupled continuous capture step, can be used as a universal biomanufacturing platform. This study reports the first successful demonstration of the integration of a perfusion bioreactor and a four-column periodic counter-current chromatography (PCC) system for the continuous capture of candidate protein therapeutics. Two examples are presented: (1) a monoclonal antibody (model of a stable protein) and (2) a recombinant human enzyme (model of a highly complex, less stable protein). In both cases, high-density perfusion CHO cell cultures were operated at a quasi-steady state of 50-60 × 10(6) cells/mL for more than 60 days, achieving volumetric productivities much higher than current perfusion or fed-batch processes. The directly integrated and automated PCC system ran uninterrupted for 30 days without indications of time-based performance decline. The product quality observed for the continuous capture process was comparable to that for a batch-column operation. Furthermore, the integration of perfusion cell culture and PCC led to a dramatic decrease in the equipment footprint and elimination of several non-value-added unit operations, such as clarification and intermediate hold steps. These findings demonstrate the potential of integrated continuous bioprocessing as a universal platform for the manufacture of various kinds of therapeutic proteins. Copyright © 2012 Wiley Periodicals, Inc.

  18. Improved means and methods for expressing recombinant proteins

    NARCIS (Netherlands)

    Poolman, Berend; Martinez Linares, Daniel; Gul, Nadia

    2014-01-01

    The invention relates to the field of genetic engineering and the production of recombinant proteins in microbial host cells. Provided is a method for enhanced expression of a recombinant protein of interest in a microbial host cell, comprising providing a microbial host cell wherein the function of

  19. Companion Protease Inhibitors for the In Situ Protection of Recombinant Proteins in Plants.

    Science.gov (United States)

    Robert, Stéphanie; Jutras, Philippe V; Khalf, Moustafa; D'Aoust, Marc-André; Goulet, Marie-Claire; Sainsbury, Frank; Michaud, Dominique

    2016-01-01

    We previously described a procedure for the use of plant protease inhibitors as "companion" accessory proteins to prevent unwanted proteolysis of clinically useful recombinant proteins in leaf crude protein extracts (Benchabane et al. Methods Mol Biol 483:265-273, 2009). Here we describe the use of these inhibitors for the protection of recombinant proteins in planta, before their extraction from leaf tissues. A procedure is first described involving inhibitors co-expressed along-and co-migrating-with the protein of interest in host plant cells. An alternative, single transgene scheme is then described involving translational fusions of the recombinant protein and companion inhibitor. These approaches may allow for a significant improvement of protein steady-state levels in leaves, comparable to yield improvements observed with protease-deficient strains of less complex protein expression hosts such as E. coli or yeasts.

  20. Data Mining for Expressivity of Recombinant Protein Expression

    Science.gov (United States)

    Kira, Satoshi; Isoai, Atsushi; Yamamura, Masayuki

    We analyzed the expressivity of recombinant proteins by using data mining methods. The expression technique of recombinant protein is a key step towards elucidating the functions of genes discovered through genomic sequence projects. We have studied the productive efficiency of recombinant proteins in fission yeast, Schizosaccharomyces pombe (S.pombe), by mining the expression results. We gathered 57 proteins whose expression levels were known roughly in the host. Correlation analysis, principal component analysis and decision tree analysis were applied to these expression data. Analysis featuring codon usage and amino acid composition clarified that the amino acid composition affected to the expression levels of a recombinant protein strongly than the effect of codon usage. Furthermore, analysis of amino acid composition showed that protein solubility and the metabolism cost of amino acids correlated with a protein expressivity. Codon usage was often interesting in the field of recombinant expressions. However, our analysis found the weak correlation codon features with expressivities. These results indicated that ready-made indices of codon bias were irrelevant ones for modeling the expressivities of recombinant proteins. Our data driven approach was an easy and powerful method to improve recombinant protein expression, and this approach should be concentrated attention with the huge amount of expression data accumulating through the post-genome era.

  1. Recombinant pinoresinol/lariciresinol reductase, recombinant dirigent protein, and methods of use

    Science.gov (United States)

    Lewis, Norman G.; Davin, Laurence B.; Dinkova-Kostova, Albena T.; Fujita, Masayuki; Gang, David R.; Sarkanen, Simo; Ford, Joshua D.

    2001-04-03

    Dirigent proteins and pinoresinol/lariciresinol reductases have been isolated, together with cDNAs encoding dirigent proteins and pinoresinol/lariciresinol reductases. Accordingly, isolated DNA sequences are provided which code for the expression of dirigent proteins and pinoresinol/lariciresinol reductases. In other aspects, replicable recombinant cloning vehicles are provided which code for dirigent proteins or pinoresinol/lariciresinol reductases or for a base sequence sufficiently complementary to at least a portion of dirigent protein or pinoresinol/lariciresinol reductase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding dirigent protein or pinoresinol/lariciresinol reductase. Thus, systems and methods are provided for the recombinant expression of dirigent proteins and/or pinoresinol/lariciresinol reductases.

  2. Metal binding proteins, recombinant host cells and methods

    Science.gov (United States)

    Summers, Anne O.; Caguiat, Jonathan J.

    2004-06-15

    The present disclosure provides artificial heavy metal binding proteins termed chelons by the inventors. These chelons bind cadmium and/or mercuric ions with relatively high affinity. Also disclosed are coding sequences, recombinant DNA molecules and recombinant host cells comprising those recombinant DNA molecules for expression of the chelon proteins. In the recombinant host cells or transgenic plants, the chelons can be used to bind heavy metals taken up from contaminated soil, groundwater or irrigation water and to concentrate and sequester those ions. Recombinant enteric bacteria can be used within the gastrointestinal tracts of animals or humans exposed to toxic metal ions such as mercury and/or cadmium, where the chelon recombinantly expressed in chosen in accordance with the ion to be rededicated. Alternatively, the chelons can be immobilized to solid supports to bind and concentrate heavy metals from a contaminated aqueous medium including biological fluids.

  3. Recombinant HT{sub m4} gene, protein and assays

    Science.gov (United States)

    Lim, B.; Adra, C.N.; Lelias, J.M.

    1996-09-03

    The invention relates to a recombinant DNA molecule which encodes a HT{sub m4} protein, a transformed host cell which has been stably transfected with a DNA molecule which encodes a HT{sub m4} protein and a recombinant HT{sub m4} protein. The invention also relates to a method for detecting the presence of a hereditary atopy. 2 figs.

  4. Effect of the electrostatic surface potential on the oligomerization of full-length human recombinant prion protein at single-molecule level

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Bin; Xu, Bingqian, E-mail: bxu@engr.uga.edu [Single Molecule Study Laboratory, College of Engineering and Nanoscale Science, and Engineering Center, University of Georgia, Athens, Georgia 30605 (United States); Lou, Zhichao [Single Molecule Study Laboratory, College of Engineering and Nanoscale Science, and Engineering Center, University of Georgia, Athens, Georgia 30605 (United States); College of Materials Science and Technology, Nanjing University of Aeronautics and Astronautics, Nanjing 210016 (China); Zhang, Haiqian [College of Materials Science and Technology, Nanjing University of Aeronautics and Astronautics, Nanjing 210016 (China)

    2016-03-21

    The electrostatic surface potential (ESP) of prion oligomers has critical influences on the aggregating processes of the prion molecules. The atomic force microscopy (AFM) and structural simulation were combined to investigate the molecular basis of the full-length human recombinant prion oligomerization on mica surfaces. The high resolution non-intrusive AFM images showed that the prion oligomers formed different patterns on mica surfaces at different buffer pH values. The basic binding units for the large oligomers were determined to be prion momoners (Ms), dimers (Ds), and trimers (Ts). The forming of the D and T units happened through the binding of hydrophobic β-sheets of the M units. In contrast, the α-helices of these M, D, and T units were the binding areas for the formation of large oligomers. At pH 4.5, the binding units M, D, and T showed clear polarized ESP distributions on the surface domains, while at pH 7.0, they showed more evenly distributed ESPs. Based on the conformations of oligomers observed from AFM images, the D and T units were more abundantly on mica surface at pH 4.5 because the ESP re-distribution of M units helped to stabilize these larger oligomers. The amino acid side chains involved in the binding interfaces were stabilized by hydrogen bonds and electrostatic interactions. The detailed analysis of the charged side chains at pH 4.5 indicated that the polarized ESPs induced the aggregations among M, D, and T to form larger oligomers. Therefore, the hydrogen bonds and electrostatic interactions worked together to form the stabilized prion oligomers.

  5. Effect of the electrostatic surface potential on the oligomerization of full-length human recombinant prion protein at single-molecule level

    Science.gov (United States)

    Wang, Bin; Lou, Zhichao; Zhang, Haiqian; Xu, Bingqian

    2016-03-01

    The electrostatic surface potential (ESP) of prion oligomers has critical influences on the aggregating processes of the prion molecules. The atomic force microscopy (AFM) and structural simulation were combined to investigate the molecular basis of the full-length human recombinant prion oligomerization on mica surfaces. The high resolution non-intrusive AFM images showed that the prion oligomers formed different patterns on mica surfaces at different buffer pH values. The basic binding units for the large oligomers were determined to be prion momoners (Ms), dimers (Ds), and trimers (Ts). The forming of the D and T units happened through the binding of hydrophobic β-sheets of the M units. In contrast, the α-helices of these M, D, and T units were the binding areas for the formation of large oligomers. At pH 4.5, the binding units M, D, and T showed clear polarized ESP distributions on the surface domains, while at pH 7.0, they showed more evenly distributed ESPs. Based on the conformations of oligomers observed from AFM images, the D and T units were more abundantly on mica surface at pH 4.5 because the ESP re-distribution of M units helped to stabilize these larger oligomers. The amino acid side chains involved in the binding interfaces were stabilized by hydrogen bonds and electrostatic interactions. The detailed analysis of the charged side chains at pH 4.5 indicated that the polarized ESPs induced the aggregations among M, D, and T to form larger oligomers. Therefore, the hydrogen bonds and electrostatic interactions worked together to form the stabilized prion oligomers.

  6. In vivo production of recombinant proteins using occluded recombinant AcMNPV-derived baculovirus vectors.

    Science.gov (United States)

    Guijarro-Pardo, Eva; Gómez-Sebastián, Silvia; Escribano, José M

    2017-12-01

    Trichoplusia ni insect larvae infected with vectors derived from the Autographa californica multiple nucleopolyhedrovirus (AcMNPV), are an excellent alternative to insect cells cultured in conventional bioreactors to produce recombinant proteins because productivity and cost-efficiency reasons. However, there is still a lot of work to do to reduce the manual procedures commonly required in this production platform that limit its scalability. To increase the scalability of this platform technology, a current bottleneck to be circumvented in the future is the need of injection for the inoculation of larvae with polyhedrin negative baculovirus vectors (Polh-) because of the lack of oral infectivity of these viruses, which are commonly used for production in insect cell cultures. In this work we have developed a straightforward alternative to obtain orally infective vectors derived from AcMNPV and expressing recombinant proteins that can be administered to the insect larvae (Trichoplusia ni) by feeding, formulated in the insect diet. The approach developed was based on the use of a recombinant polyhedrin protein expressed by a recombinant vector (Polh+), able to co-occlude any recombinant Polh- baculovirus vector expressing a recombinant protein. A second alternative was developed by the generation of a dual vector co-expressing the recombinant polyhedrin protein and the foreign gene of interest to obtain the occluded viruses. Additionally, by the incorporation of a reporter gene into the helper Polh+ vector, it was possible the follow-up visualization of the co-occluded viruses infection in insect larvae and will help to homogenize infection conditions. By using these methodologies, the production of recombinant proteins in per os infected larvae, without manual infection procedures, was very similar in yield to that obtained by manual injection of recombinant Polh- AcMNPV-based vectors expressing the same proteins. However, further analyses will be required for a

  7. Overview of the recombinant proteins purification by affinity tags and ...

    African Journals Online (AJOL)

    From protein within isolation process which the same matter increases labor costs further and prevents application of these tags in industrial scale. Therefore proper replacement is emphasized for enzymatic removal of purification tags. Keywords: protein purification; recombinant proteins; self-cleavable tags; Intein tags; ...

  8. Production of recombinant proteins from Plasmodium falciparum in Escherichia coli.

    Science.gov (United States)

    Guerra, Ángela Patricia; Calvo, Eliana Patricia; Wasserman, Moisés; Chaparro-Olaya, Jacqueline

    2016-02-23

    The production of recombinant proteins is essential for the characterization and functional study of proteins from Plasmodium falciparum. However, the proteins of P. falciparum are among the most challenging to express, and when expression is achieved, the recombinant proteins usually fold incorrectly and lead to the formation of inclusion bodies.  To obtain and purify four recombinant proteins and to use them as antigens to produce polyclonal antibodies. The production efficiency and solubility were evaluated as the proteins were expressed in two genetically modified strains of Escherichia coli to favor the production of heterologous proteins (BL21-CodonPlus (DE3)-RIL and BL21-pG-KJE8).  The four recombinant P. falciparum proteins corresponding to partial sequences of PfMyoA (Myosin A) and PfGAP50 (gliding associated protein 50), and the complete sequences of PfMTIP (myosin tail interacting protein) and PfGAP45 (gliding associated protein 45), were produced as glutathione S-transferase-fusion proteins, purified and used for immunizing mice.  The protein expression was much more efficient in BL21-CodonPlus, the strain that contains tRNAs that are rare in wild-type E. coli, compared to the expression in BL21-pG-KJE8. In spite of the fact that BL21-pG-KJE8 overexpresses chaperones, this strain did not minimize the formation of inclusion bodies.  The use of genetically modified strains of E. coli was essential to achieve high expression levels of the four evaluated P. falciparum proteins and lead to improved solubility of two of them. The approach used here allowed us to obtain and purify four P. falciparum proteins in enough quantity to produce polyclonal antibodies in mice, and a fair amount of two pure and soluble recombinant proteins for future assays.

  9. Obtaining classical swine fever virus E2 recombinant protein and DNA-vaccine on the basis of one subunit

    International Nuclear Information System (INIS)

    Deryabin, O.; Deryabina, O.; Verbitskiy, P.; Kordyum, V.

    2005-01-01

    Three forms of E2 recombinant protein were expressed in E. coli. Swine sera obtained against different forms of the recombinant protein were cross-studied with indirect ELISA. Using individual proteins as an antigen, only 15% of sera against other forms of protein reacted positively, while 100% of heterologous sera showed positive reaction with fused protein. Challenge experiments showed the existence of protective action only from the individual protein. Specificity and activity of sera obtained from the animals after control challenge was confirmed in a blocking variant of ELISA. Genetic construction used a eukaryotic vector that contained the E2 protein gene. Immunization of mice with the resulting DNA induced synthesis of specific antibodies, the titre of which increased considerably after additional single immunization with the E2 recombinant protein, expressed in E. coli. This demonstrated the effectiveness of animal priming by DNA vaccine, and the possibility of using the E2 recombinant protein in E. coli for booster vaccination. (author)

  10. Recombinant production of a peroxidase-protein G fusion protein in Pichia pastoris.

    Science.gov (United States)

    Krainer, Florian Wolfgang; Darnhofer, Barbara; Birner-Gruenberger, Ruth; Glieder, Anton

    2016-02-10

    Streptococcal protein G (SpG) binds immunoglobulin G from a broad range of mammalian species with high affinity. Chemical conjugations of SpG to the reporter enzyme horseradish peroxidase (HRP) are commonly used in immunohistochemical applications. However, commercial HRP preparations are typically isolated from horseradish roots as varying mixtures of HRP isoenzymes with different biochemical properties, and chemical conjugation procedures lead to heterogeneous HRP-SpG preparations, partially including inactivated enzyme. A recombinant process allows the production of a well-defined HRP isoenzyme fused to SpG at constant 1:1 stoichiometry in a single step without the need for laborious chemical conjugation. By using state-of-the-art biotechnological tools, we produced a recombinant HRP-SpG fusion protein in Pichia pastoris in bioreactor cultivations. Purified HRP-SpG was tested successfully for functional binding of antibodies from different mammalian serums. Recombinant production of this novel well-defined fusion protein follows quality-by-design principles and facilitates the production of more reliable and cost-effective diagnostic kits. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  11. Synthesis and characterization of recombinant abductin-based proteins.

    Science.gov (United States)

    Su, Renay S-C; Renner, Julie N; Liu, Julie C

    2013-12-09

    Recombinant proteins are promising tools for tissue engineering and drug delivery applications. Protein-based biomaterials have several advantages over natural and synthetic polymers, including precise control over amino acid composition and molecular weight, modular swapping of functional domains, and tunable mechanical and physical properties. In this work, we describe recombinant proteins based on abductin, an elastomeric protein that is found in the inner hinge of bivalves and functions as a coil spring to keep shells open. We illustrate, for the first time, the design, cloning, expression, and purification of a recombinant protein based on consensus abductin sequences derived from Argopecten irradians . The molecular weight of the protein was confirmed by mass spectrometry, and the protein was 94% pure. Circular dichroism studies showed that the dominant structures of abductin-based proteins were polyproline II helix structures in aqueous solution and type II β-turns in trifluoroethanol. Dynamic light scattering studies illustrated that the abductin-based proteins exhibit reversible upper critical solution temperature behavior and irreversible aggregation behavior at high temperatures. A LIVE/DEAD assay revealed that human umbilical vein endothelial cells had a viability of 98 ± 4% after being cultured for two days on the abductin-based protein. Initial cell spreading on the abductin-based protein was similar to that on bovine serum albumin. These studies thus demonstrate the potential of abductin-based proteins in tissue engineering and drug delivery applications due to the cytocompatibility and its response to temperature.

  12. Recombinant protein expression in Escherichia coli: advances and challenges

    Science.gov (United States)

    Rosano, Germán L.; Ceccarelli, Eduardo A.

    2014-01-01

    Escherichia coli is one of the organisms of choice for the production of recombinant proteins. Its use as a cell factory is well-established and it has become the most popular expression platform. For this reason, there are many molecular tools and protocols at hand for the high-level production of heterologous proteins, such as a vast catalog of expression plasmids, a great number of engineered strains and many cultivation strategies. We review the different approaches for the synthesis of recombinant proteins in E. coli and discuss recent progress in this ever-growing field. PMID:24860555

  13. Trends in recombinant protein use in animal production.

    Science.gov (United States)

    Gifre, Laia; Arís, Anna; Bach, Àlex; Garcia-Fruitós, Elena

    2017-03-04

    Recombinant technologies have made possible the production of a broad catalogue of proteins of interest, including those used for animal production. The most widely studied proteins for the animal sector are those with an important role in reproduction, feed efficiency, and health. Nowadays, mammalian cells and fungi are the preferred choice for recombinant production of hormones for reproductive purposes and fibrolytic enzymes to enhance animal performance, respectively. However, the development of low-cost products is a priority, particularly in livestock. The study of cell factories such as yeast and bacteria has notably increased in the last decades to make the new developed reproductive hormones and fibrolytic enzymes a real alternative to the marketed ones. Important efforts have also been invested to developing new recombinant strategies for prevention and therapy, including passive immunization and modulation of the immune system. This offers the possibility to reduce the use of antibiotics by controlling physiological processes and improve the efficacy of preventing infections. Thus, nowadays different recombinant fibrolytic enzymes, hormones, and therapeutic molecules with optimized properties have been successfully produced through cost-effective processes using microbial cell factories. However, despite the important achievements for reducing protein production expenses, alternative strategies to further reduce these costs are still required. In this context, it is necessary to make a giant leap towards the use of novel strategies, such as nanotechnology, that combined with recombinant technology would make recombinant molecules affordable for animal industry.

  14. Capillary gel electrophoresis for the quantification and purity determination of recombinant proteins in inclusion bodies.

    Science.gov (United States)

    Espinosa-de la Garza, Carlos E; Perdomo-Abúndez, Francisco C; Campos-García, Víctor R; Pérez, Néstor O; Flores-Ortiz, Luis F; Medina-Rivero, Emilio

    2013-09-01

    In this work, a high-resolution CGE method for quantification and purity determination of recombinant proteins was developed, involving a single-component inclusion bodies (IBs) solubilization solution. Different recombinant proteins expressed as IBs were used to show method capabilities, using recombinant interferon-β 1b as the model protein for method validation. Method linearity was verified in the range from 0.05 to 0.40 mg/mL and a determination coefficient (r(2) ) of 0.99 was obtained. The LOQs and LODs were 0.018 and 0.006 mg/mL, respectively. RSD for protein content repeatability test was 2.29%. In addition, RSD for protein purity repeatability test was 4.24%. Method accuracy was higher than 90%. Specificity was confirmed, as the method was able to separate recombinant interferon-β 1b monomer from other aggregates and impurities. Sample content and purity was demonstrated to be stable for up to 48 h. Overall, this method is suitable for the analysis of recombinant proteins in IBs according to the attributes established on the International Conference for Harmonization guidelines. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Structural analysis of recombinant human protein QM

    International Nuclear Information System (INIS)

    Gualberto, D.C.H.; Fernandes, J.L.; Silva, F.S.; Saraiva, K.W.; Affonso, R.; Pereira, L.M.; Silva, I.D.C.G.

    2012-01-01

    Full text: The ribosomal protein QM belongs to a family of ribosomal proteins, which is highly conserved from yeast to humans. The presence of the QM protein is necessary for joining the 60S and 40S subunits in a late step of the initiation of mRNA translation. Although the exact extra-ribosomal functions of QM are not yet fully understood, it has been identified as a putative tumor suppressor. This protein was reported to interact with the transcription factor c-Jun and thereby prevent c-Jun actives genes of the cellular growth. In this study, the human QM protein was expressed in bacterial system, in the soluble form and this structure was analyzed by Circular Dichroism and Fluorescence. The results of Circular Dichroism showed that this protein has less alpha helix than beta sheet, as described in the literature. QM protein does not contain a leucine zipper region; however the ion zinc is necessary for binding of QM to c-Jun. Then we analyzed the relationship between the removal of zinc ions and folding of protein. Preliminary results obtained by the technique Fluorescence showed a gradual increase in fluorescence with the addition of increasing concentration of EDTA. This suggests that the zinc is important in the tertiary structure of the protein. More studies are being made for better understand these results. (author)

  16. Production of recombinant proteins in suspension-cultured plant cells.

    Science.gov (United States)

    Plasson, Carole; Michel, Rémy; Lienard, David; Saint-Jore-Dupas, Claude; Sourrouille, Christophe; de March, Ghislaine Grenier; Gomord, Véronique

    2009-01-01

    Plants have emerged in the past decade as a suitable alternative to the current production systems for recombinant pharmaceutical proteins and, today their potential for low-cost production of high quality, much safer and biologically active mammalian proteins is largely documented. Among various plant expression systems being explored, genetically modified suspension-cultured plant cells offer a promising system for production of biopharmaceuticals. Indeed, when compared to other plant-based production platforms that have been explored, suspension-cultured plant cells have the advantage of being totally devoid of problems associated with the vagaries of weather, pest, soil and gene flow in the environment. Because of short growth cycles, the timescale needed for the production of recombinant proteins in plant cell culture can be counted in days or weeks after transformation compared to months needed for the production in transgenic plants. Moreover, recovery and purification of recombinant proteins from plant biomass is an expensive and technically challenging business that may amount to 80-94% of the final product cost. One additional advantage of plant cell culture is that the recombinant protein fused with a signal sequence can be expressed and secreted into the culture medium, and therefore recovered and purified in the absence of large quantities of contaminating proteins. Consequently, the downstream processing of proteins extracted from plant cell culture medium is less expensive, which may/does balance the higher costs of fermentation. When needed for clinical use, recombinant proteins are easily produced in suspension-cultured plant cells under certified, controllable and sterile conditions that offer improved safety and provide advantages for good manufacturing practices and regulatory compliance. In this chapter, we present basic protocols for rapid generation of transgenic suspension-cultured cells of Nicotiana tabacum, Oriza sativa and Arabidopis

  17. Yeast synthetic biology for the production of recombinant therapeutic proteins.

    Science.gov (United States)

    Kim, Hyunah; Yoo, Su Jin; Kang, Hyun Ah

    2015-02-01

    The production of recombinant therapeutic proteins is one of the fast-growing areas of molecular medicine and currently plays an important role in treatment of several diseases. Yeasts are unicellular eukaryotic microbial host cells that offer unique advantages in producing biopharmaceutical proteins. Yeasts are capable of robust growth on simple media, readily accommodate genetic modifications, and incorporate typical eukaryotic post-translational modifications. Saccharomyces cerevisiae is a traditional baker's yeast that has been used as a major host for the production of biopharmaceuticals; however, several nonconventional yeast species including Hansenula polymorpha, Pichia pastoris, and Yarrowia lipolytica have gained increasing attention as alternative hosts for the industrial production of recombinant proteins. In this review, we address the established and emerging genetic tools and host strains suitable for recombinant protein production in various yeast expression systems, particularly focusing on current efforts toward synthetic biology approaches in developing yeast cell factories for the production of therapeutic recombinant proteins. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

  18. Smart sustainable bottle (SSB) system for E. coli based recombinant protein production.

    Science.gov (United States)

    Li, Zhaopeng; Carstensen, Bettina; Rinas, Ursula

    2014-11-05

    Recombinant proteins are usually required in laboratories interested in the protein but not in the production process itself. Thus, technical equipment which is easy to handle and straight forward protein production procedures are of great benefit to those laboratories. Companies selling single use cultivation bags and bioreactors are trying to satisfy at least part of these needs. However, single-use systems can contribute to major costs which might be acceptable when "good manufacturing practices" are required but not acceptable for most laboratories facing tight funding. The assembly and application of a simple self-made "smart sustainable bottle" (SSB) system for E. coli based protein production is presented. The core of the SSB system is a 2-L glass bottle which is operated at constant temperature, air flow, and stirrer speed without measurement and control of pH and dissolved oxygen. Oxygen transfer capacities are in the range as in conventional bioreactors operated at intermediate aeration rates and by far exceed those found in conventional shaking flasks and disposable bioreactors. The SSB system was applied for the production of various recombinant proteins using T7-based expression systems and a defined autoinduction medium. The production performance regarding amount and solubility of proteins with robust and delicate properties was as good as in state-of-the-art stirred tank commercial bioreactors. The SSB system represents a low cost protein production device applicable for easy, effective, and reproducible recombinant protein production.

  19. Cellular Reprogramming Employing Recombinant Sox2 Protein

    Directory of Open Access Journals (Sweden)

    Marc Thier

    2012-01-01

    Full Text Available Induced pluripotent stem (iPS cells represent an attractive option for the derivation of patient-specific pluripotent cells for cell replacement therapies as well as disease modeling. To become clinically meaningful, safe iPS cells need to be generated exhibiting no permanent genetic modifications that are caused by viral integrations of the reprogramming transgenes. Recently, various experimental strategies have been applied to accomplish transgene-free derivation of iPS cells, including the use of nonintegrating viruses, episomal expression, or excision of transgenes after reprogramming by site-specific recombinases or transposases. A straightforward approach to induce reprogramming factors is the direct delivery of either synthetic mRNA or biologically active proteins. We previously reported the generation of cell-permeant versions of Oct4 (Oct4-TAT and Sox2 (Sox2-TAT proteins and showed that Oct4-TAT is reprogramming-competent, that is, it can substitute for Oct4-encoding virus. Here, we explore conditions for enhanced Sox2-TAT protein stabilization and functional delivery into somatic cells. We show that cell-permeant Sox2 protein can be stabilized by lipid-rich albumin supplements in serum replacement or low-serum-supplemented media. Employing optimized conditions for protein delivery, we demonstrate that Sox2-TAT protein is able to substitute for viral Sox2. Sox2-piPS cells express pluripotency-associated markers and differentiate into all three germ layers.

  20. Single transverse mode protein laser

    Science.gov (United States)

    Dogru, Itir Bakis; Min, Kyungtaek; Umar, Muhammad; Bahmani Jalali, Houman; Begar, Efe; Conkar, Deniz; Firat Karalar, Elif Nur; Kim, Sunghwan; Nizamoglu, Sedat

    2017-12-01

    Here, we report a single transverse mode distributed feedback (DFB) protein laser. The gain medium that is composed of enhanced green fluorescent protein in a silk fibroin matrix yields a waveguiding gain layer on a DFB resonator. The thin TiO2 layer on the quartz grating improves optical feedback due to the increased effective refractive index. The protein laser shows a single transverse mode lasing at the wavelength of 520 nm with the threshold level of 92.1 μJ/ mm2.

  1. RecO protein initiates DNA recombination and strand annealing through two alternative DNA binding mechanisms.

    Science.gov (United States)

    Ryzhikov, Mikhail; Gupta, Richa; Glickman, Michael; Korolev, Sergey

    2014-10-17

    Recombination mediator proteins (RMPs) are important for genome stability in all organisms. Several RMPs support two alternative reactions: initiation of homologous recombination and DNA annealing. We examined mechanisms of RMPs in both reactions with Mycobacterium smegmatis RecO (MsRecO) and demonstrated that MsRecO interacts with ssDNA by two distinct mechanisms. Zinc stimulates MsRecO binding to ssDNA during annealing, whereas the recombination function is zinc-independent and is regulated by interaction with MsRecR. Thus, different structural motifs or conformations of MsRecO are responsible for interaction with ssDNA during annealing and recombination. Neither annealing nor recombinase loading depends on MsRecO interaction with the conserved C-terminal tail of single-stranded (ss) DNA-binding protein (SSB), which is known to bind Escherichia coli RecO. However, similarly to E. coli proteins, MsRecO and MsRecOR do not dismiss SSB from ssDNA, suggesting that RMPs form a complex with SSB-ssDNA even in the absence of binding to the major protein interaction motif. We propose that alternative conformations of such complexes define the mechanism by which RMPs initiate the repair of stalled replication and support two different functions during recombinational repair of DNA breaks. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Isolation of recombinant cysteine dioxygenase protein from Trichophyton mentagrophytes

    Czech Academy of Sciences Publication Activity Database

    Kašperová, A.; Kunert, J.; Horynová, M.; Weigl, E.; Sebela, M.; Lenobel, René; Raška, M.

    2011-01-01

    Roč. 54, č. 5 (2011), E456-E462 ISSN 0933-7407 R&D Projects: GA ČR GA301/08/1649 Institutional research plan: CEZ:AV0Z50380511 Keywords : Cysteine dioxygenase * dermatophytes * recombinant protein * keratinolytic fungi * cDNA Subject RIV: CE - Biochemistry Impact factor: 2.247, year: 2011

  3. The effect of the unfolded protein response on the production of recombinant proteins in plants.

    Science.gov (United States)

    Thomas, David Rhys; Walmsley, Amanda Maree

    2015-02-01

    Recombinant proteins are currently produced through a wide variety of host systems, including yeast, E. coli, insect and mammalian cells. One of the most recent systems developed uses plant cells. While considerable advances have been made in the yields and fidelity of plant-made recombinant proteins, many of these gains have arisen from the development of recombinant factors. This includes elements such as highly effective promoters and untranslated regions, deconstructed viral vectors, silencing inhibitors, and improved DNA delivery techniques. However, unlike other host systems, much of the work on recombinant protein production in plants uses wild-type hosts that have not been modified to facilitate recombinant protein expression. As such, there are still endogenous mechanisms functioning to maintain the health of the cell. The result is that these pathways, such as the unfolded protein response, can actively work to reduce recombinant protein production to maintain the integrity of the cell. This review examines how issues arising from the unfolded protein response have been addressed in other systems, and how these methods may be transferable to plant systems. We further identify several areas of host plant biology that present attractive targets for modification to facilitate recombinant protein production.

  4. Green factory: plants as bioproduction platforms for recombinant proteins.

    Science.gov (United States)

    Xu, Jianfeng; Dolan, Maureen C; Medrano, Giuliana; Cramer, Carole L; Weathers, Pamela J

    2012-01-01

    Molecular farming, long considered a promising strategy to produce valuable recombinant proteins not only for human and veterinary medicine, but also for agriculture and industry, now has some commercially available products. Various plant-based production platforms including whole-plants, aquatic plants, plant cell suspensions, and plant tissues (hairy roots) have been compared in terms of their advantages and limits. Effective recombinant strategies are summarized along with descriptions of scalable culture systems and examples of commercial progress and success. Copyright © 2011 Elsevier Inc. All rights reserved.

  5. Impact of protein uptake and degradation on recombinant protein secretion in yeast

    DEFF Research Database (Denmark)

    Tyo, Keith E. J.; Liu, Zihe; Magnusson, Ylva

    2014-01-01

    Protein titers, a key bioprocessing metric, depend both on the synthesis of protein and the degradation of protein. Secreted recombinant protein production in Saccharomyces cerevisiae is an attractive platform as minimal media can be used for cultivation, thus reducing fermentation costs and simp...

  6. Recombinant protein-based nanoscale biomemory devices.

    Science.gov (United States)

    Yagati, A K; Min, J; Choi, J W

    2014-01-01

    Biomolecular computing devices that are based on the properties of biomolecular activities offer a unique possibility for constructing new computing structures. A new concept of using various biomolecules has been proposed in order to develop a protein-based memory device that is capable of switching physical properties when electrical input signals are applied to perform memory switching. To clarify the proposed concept, redox protein is immobilized on Au nanoelectrodes to catalyze reversible reactions of redox-active molecules, which is controlled electrochemically and reversibly converted between its ON/OFF states. In this review, we summarize recent research towards developing nanoscale biomemory devices including design, synthesis, fabrication, and functionalization based on the proposed concept. At first we analyze the memory function properties of the proposed device at bulk material level and then explain the WORM (write-once-read-many times) nature of the device, later we extend the analysis to multi-bit and multi-level storage functions, and then we focus the developments in nanoscale biomemory devices based on the electron transport of redox molecules to the underlying Au patterned surface. The developed device operates at very low voltages and has good stability and excellent reversibility, proving to be a promising platform for future memory devices.

  7. Production and Purification of Recombinant SUMOylated Proteins Using Engineered Bacteria.

    Science.gov (United States)

    Brockly, Frédérique; Piechaczyk, Marc; Bossis, Guillaume

    2016-01-01

    SUMO is a ubiquitin-like protein that is covalently conjugated to numerous cellular proteins to modify their function and fate. Although large progresses have been made in the identification of SUMOylated proteins, the molecular consequences of their SUMOylation are generally unknown. This is, most often, due to the low abundance of SUMOylated proteins in the cell, usually less than 1 % of a given protein being modified at steady state. To gain insights into the role of specific SUMOylation targets, SUMO conjugation can be reconstituted in vitro using purified proteins. However, for most substrates, the efficiency of in vitro SUMOylation is too low to obtain sufficient amounts of their SUMOylated forms for biochemical studies. Here, we describe a detailed protocol to purify large amounts of recombinant SUMOylated proteins using bacteria modified to express His-tagged SUMO as well as the SUMO-activating and -conjugating enzymes.

  8. Development of a lectin binding assay to differentiate between recombinant and endogenous proteins in pharmacokinetic studies of protein-biopharmaceuticals.

    Science.gov (United States)

    Weber, Alfred; Minibeck, Eva; Scheiflinger, Friedrich; Turecek, Peter L

    2015-04-10

    Human glycoproteins, expressed in hamster cell lines, show similar glycosylation patterns to naturally occurring human molecules except for a minute difference in the linkage of terminal sialic acid: both cell types lack α2,6-galactosyl-sialyltransferase, abundantly expressed in human hepatocytes and responsible for the α2,6-sialylation of circulating glycoproteins. This minute difference, which is currently not known to have any physiological relevance, was the basis for the selective measurement of recombinant glycoproteins in the presence of their endogenous counterparts. The assay is based on using the lectin Sambucus nigra agglutinin (SNA), selectively binding to α2,6-sialylated N-glycans. Using von Willebrand factor (VWF), factor IX (FIX), and factor VIIa (FVIIa), it was demonstrated that (i) the plasma-derived proteins, but not the corresponding recombinant proteins, specifically bind to SNA and (ii) this binding can be used to deplete the plasma-derived proteins. The feasibility of this approach was confirmed in spike-recovery studies for all three recombinant coagulation proteins in human plasma and for recombinant VWF (rVWF) in macaque plasma. Analysis of plasma samples from macaques after administration of recombinant and a plasma-derived VWF demonstrated the suitability and robustness of this approach. Data showed that rVWF could be selectively measured without changing the ELISAs and furthermore revealed the limitations of baseline adjustment using a single measurement of the predose concentration only. The SNA gel-based depletion procedure can easily be integrated in existing procedures as a specific sample pre-treatment step. While ELISA-based methods were used to measure the recombinant coagulation proteins in the supernatants obtained by depletion, this procedure is applicable for all biochemical analyses. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Antibodies to a recombinant glutamate-rich Plasmodium falciparum protein

    DEFF Research Database (Denmark)

    Hogh, B; Petersen, E; Dziegiel, Morten Hanefeld

    1992-01-01

    A Plasmodium falciparum antigen gene coding for a 220-kD glutamate-rich protein (GLURP) has been cloned, and the 783 C-terminal amino acids of this protein (GLURP489-1271) have been expressed as a beta-galactosidase fusion protein in Escherichia coli. The encoded 783 amino acid residues contain two...... areas of repeated amino acid sequences. Antibodies against recombinant GLURP489-1271, as well as against a synthetic peptide corresponding to GLURP899-916, and against a synthetic peptide representing the major glutamate rich repeat sequence from the P. falciparum ring erythrocyte surface antigen (Pf155...

  10. A Bacillus megaterium System for the Production of Recombinant Proteins and Protein Complexes.

    Science.gov (United States)

    Biedendieck, Rebekka

    2016-01-01

    For many years the Gram-positive bacterium Bacillus megaterium has been used for the production and secretion of recombinant proteins. For this purpose it was systematically optimized. Plasmids with different inducible promoter systems, with different compatible origins, with small tags for protein purification and with various specific signals for protein secretion were combined with genetically improved host strains. Finally, the development of appropriate cultivation conditions for the production strains established this organism as a bacterial cell factory even for large proteins. Along with the overproduction of individual proteins the organism is now also used for the simultaneous coproduction of up to 14 recombinant proteins, multiple subsequently interacting or forming protein complexes. Some of these recombinant strains are successfully used for bioconversion or the biosynthesis of valuable components including vitamins. The titers in the g per liter scale for the intra- and extracellular recombinant protein production prove the high potential of B. megaterium for industrial applications. It is currently further enhanced for the production of recombinant proteins and multi-subunit protein complexes using directed genetic engineering approaches based on transcriptome, proteome, metabolome and fluxome data.

  11. Intracellular transport of recombinant coronavirus spike proteins: implications for virus assembly

    NARCIS (Netherlands)

    Horzinek, M.C.; Vennema, H.; Heijnen, L.; Zijderveld, A.; Spaan, W.J.M.

    1990-01-01

    Coronavirus spike protein genes were expressed in vitro by using the recombinant vaccinia virus expression system. Recombinant spike proteins were expressed at the cell surface and induced cell fusion in a host-cell-dependent fashion. The intracellular transport of recombinant spike proteins was

  12. Recombinant protein expression by targeting pre-selected chromosomal loci

    Directory of Open Access Journals (Sweden)

    Krömer Wolfgang

    2009-12-01

    Full Text Available Abstract Background Recombinant protein expression in mammalian cells is mostly achieved by stable integration of transgenes into the chromosomal DNA of established cell lines. The chromosomal surroundings have strong influences on the expression of transgenes. The exploitation of defined loci by targeting expression constructs with different regulatory elements is an approach to design high level expression systems. Further, this allows to evaluate the impact of chromosomal surroundings on distinct vector constructs. Results We explored antibody expression upon targeting diverse expression constructs into previously tagged loci in CHO-K1 and HEK293 cells that exhibit high reporter gene expression. These loci were selected by random transfer of reporter cassettes and subsequent screening. Both, retroviral infection and plasmid transfection with eGFP or antibody expression cassettes were employed for tagging. The tagged cell clones were screened for expression and single copy integration. Cell clones producing > 20 pg/cell in 24 hours could be identified. Selected integration sites that had been flanked with heterologous recombinase target sites (FRTs were targeted by Flp recombinase mediated cassette exchange (RMCE. The results give proof of principle for consistent protein expression upon RMCE. Upon targeting antibody expression cassettes 90-100% of all resulting cell clones showed correct integration. Antibody production was found to be highly consistent within the individual cell clones as expected from their isogenic nature. However, the nature and orientation of expression control elements revealed to be critical. The impact of different promoters was examined with the tag-and-targeting approach. For each of the chosen promoters high expression sites were identified. However, each site supported the chosen promoters to a different extent, indicating that the strength of a particular promoter is dominantly defined by its chromosomal context

  13. An insight into fusion technology aiding efficient recombinant protein production for functional proteomics.

    Science.gov (United States)

    Yadav, Dinesh K; Yadav, Neelam; Yadav, Sarika; Haque, Shafiul; Tuteja, Narendra

    2016-12-15

    Advancements in peptide fusion technologies to maximize the protein production has taken a big leap to fulfill the demands of post-genomics era targeting elucidation of structure/function of the proteome and its therapeutic applications, by over-expression in heterologous expression systems. Despite being most preferred protein expression system armed with variety of cardinal fusion tags, expression of the functionally active recombinant protein in E. coli remains plagued. The present review critically analyses the aptness of well-characterized fusion tags utilized for over-expression of recombinant proteins with improved solubility and their compatibility with downstream purification procedures. The combinatorial tandem affinity strategies have shown to provide more versatile options. Solubility decreasing fusion tags have proved to facilitate the overproduction of antimicrobial peptides. Efficient removal of fusion tags prior to final usage is of utmost importance and has been summarized discussing the efficiency of various enzymatic and chemical methods of tag removal. Unfortunately, no single fusion tag works as a magic bullet to completely fulfill the requirements of protein expression and purification in active form. The information provided might help in selection and development of a successful protocol for efficient recombinant protein production for functional proteomics. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Specific Genetic Immunotherapy Induced by Recombinant Vaccine Alpha-Fetoprotein-Heat Shock Protein 70 Complex

    Science.gov (United States)

    Wang, Xiaoping; Lin, Huanping; Wang, Qiaoxia

    Purposes: To construct a recombinant vaccine alpha-fetoprotein (AFP)-heat shock protein (HSP70) complex, and study its ability to induce specific CTL response and its protective effect against AFP-producing tumor. Material/Methods: A recombinant vaccine was constructed by conjugating mouse alpha-fetoprotein to heat shock protein 70. By way of intracutaneous injection, mice were primed and boosted with recombinant vaccine mAFP/HSP70, whereas single mAFP or HSP70 injection as controls. The ELISPOT and ELISA were used to measure the frequency of cells producing the cytokine IFN-γ in splenocytes and the level of anti-AFP antibody of serum from immunized mice respectively. In vivo tumor challenge were carried out to assess the immune effect of the recombinant vaccine. Results: By recombinant mAFP/HSP70 vaccine immunization, the results of ELISPOT and ELISA showed that the number of splenic cells producing IFN-γ and the level of anti-AFP antibody of serum were significantly higher in mAFP/HSP70 group than those in mAFP and HSP70 groups (108.50±11.70 IFN-γ spots/106 cells vs 41.60±10.40 IFN-γ spots/106 cells, 7.32±3.14 IFN-γ spots/106 cells, Pimmune adjuvant. Sequential immunization with recombinant mAFP/HSP70 vaccine could generate effective antitumor immunity on AFP-producing tumor. The recombined mAFP/HSP70 vaccine may be suitable for serving as an immunotherapy for hepatocellular carcinoma.

  15. Selection and characterization of a promoter for expression of single-copy recombinant genes in Gram-positive bacteria

    Directory of Open Access Journals (Sweden)

    Manganelli Riccardo

    2005-01-01

    Full Text Available Abstract Background In the past ten years there has been a growing interest in engineering Gram-positive bacteria for biotechnological applications, including vaccine delivery and production of recombinant proteins. Usually, bacteria are manipulated using plasmid expression vectors. The major limitation of this approach is due to the fact that recombinant plasmids are often lost from the bacterial culture upon removal of antibiotic selection. We have developed a genetic system based on suicide vectors on conjugative transposons allowing stable integration of recombinant DNA into the chromosome of transformable and non-transformable Gram-positive bacteria. Results The aim of this work was to select a strong chromosomal promoter from Streptococcus gordonii to improve this genetic system making it suitable for expression of single-copy recombinant genes. To achieve this task, a promoterless gene encoding a chloramphenicol acetyltransferase (cat, was randomly integrated into the S. gordonii chromosome and transformants were selected for chloramphenicol resistance. Three out of eighteen chloramphenicol resistant transformants selected exhibited 100% stability of the phenotype and only one of them, GP215, carried the cat gene integrated as a single copy. A DNA fragment of 600 base pairs exhibiting promoter activity was isolated from GP215 and sequenced. The 5' end of its corresponding mRNA was determined by primer extention analysis and the putative -10 and a -35 regions were identified. To study the possibility of using this promoter (PP for single copy heterologous gene expression, we created transcriptional fusions of PP with genes encoding surface recombinant proteins in a vector capable of integrating into the conjugative transposon Tn916. Surface recombinant proteins whose expression was controlled by the PP promoter were detected in Tn916-containing strains of S. gordonii and Bacillus subtilis after single copy chromosomal integration of the

  16. High-throughput purification of recombinant proteins using self-cleaving intein tags.

    Science.gov (United States)

    Coolbaugh, M J; Shakalli Tang, M J; Wood, D W

    2017-01-01

    High throughput methods for recombinant protein production using E. coli typically involve the use of affinity tags for simple purification of the protein of interest. One drawback of these techniques is the occasional need for tag removal before study, which can be hard to predict. In this work, we demonstrate two high throughput purification methods for untagged protein targets based on simple and cost-effective self-cleaving intein tags. Two model proteins, E. coli beta-galactosidase (βGal) and superfolder green fluorescent protein (sfGFP), were purified using self-cleaving versions of the conventional chitin-binding domain (CBD) affinity tag and the nonchromatographic elastin-like-polypeptide (ELP) precipitation tag in a 96-well filter plate format. Initial tests with shake flask cultures confirmed that the intein purification scheme could be scaled down, with >90% pure product generated in a single step using both methods. The scheme was then validated in a high throughput expression platform using 24-well plate cultures followed by purification in 96-well plates. For both tags and with both target proteins, the purified product was consistently obtained in a single-step, with low well-to-well and plate-to-plate variability. This simple method thus allows the reproducible production of highly pure untagged recombinant proteins in a convenient microtiter plate format. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Physicochemical characterisation of rVIII-SingleChain, a novel recombinant single-chain factor VIII.

    Science.gov (United States)

    Schmidbauer, Stefan; Witzel, Reinhild; Robbel, Lars; Sebastian, Petra; Grammel, Nicolas; Metzner, Hubert J; Schulte, Stefan

    2015-08-01

    rVIII-SingleChain is a novel recombinant single-chain factor VIII (FVIII) construct, comprising covalently bonded heavy and light chains. Post-translational modifications of FVIII affect physicochemical parameters, including hydrophobicity and charge. The most relevant post-translational modifications of FVIII products are N-glycosylation of asparagine residues and tyrosine sulphations. Here, the physicochemical properties, thrombin cleavage products and post-translational modifications of rVIII-SingleChain were investigated and compared against commercially available recombinant FVIII (rFVIII) products with a predominant two-chain structure (B-domain deleted rFVIII and full-length rFVIII). rVIII-SingleChain was expressed in Chinese hamster ovary (CHO) cells and purified by chromatographic methods. Physicochemical properties of rVIII-SingleChain or thrombin-derived cleavage products were assessed using size-exclusion chromatography, reversed-phase chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis. Analysis of the respective carbohydrate structures was performed after release of N-glycans by PNGase F followed by fluorescence labelling and high-performance liquid chromatography. Proteolysis by trypsin generated the corresponding peptides, which were analysed for sulphated tyrosines by liquid chromatography-electrospray ionisation time of flight-mass spectrometry. rVIII-SingleChain was shown to be of high purity and homogeneity, and presented a well-defined single-chain molecule with predominant β-sheet conformation. The coagulation-relevant thrombin-activation products of rVIII-SingleChain were comparable with those obtained by activation of commercially available rFVIII products. rVIII-SingleChain post-translational modifications were similar to other CHO cell-derived rFVIII products for N-glycopattern and tyrosine sulphation. In conclusion, rVIII-SingleChain is of high homogeneity and purity, and provides an expected cleavage pattern on

  18. Tailoring recombinant protein quality by rational media design.

    Science.gov (United States)

    Brühlmann, David; Jordan, Martin; Hemberger, Jürgen; Sauer, Markus; Stettler, Matthieu; Broly, Hervé

    2015-01-01

    Clinical efficacy and safety of recombinant proteins are closely associated with their structural characteristics. The major quality attributes comprise glycosylation, charge variants (oxidation, deamidation, and C- & N-terminal modifications), aggregates, low-molecular-weight species (LMW), and misincorporation of amino acids in the protein backbone. Cell culture media design has a great potential to modulate these quality attributes due to the vital role of medium in mammalian cell culture. The purpose of this review is to provide an overview of the way both classical cell culture medium components and novel supplements affect the quality attributes of recombinant therapeutic proteins expressed in mammalian hosts, allowing rational and high-throughput optimization of mammalian cell culture media. A selection of specific and/or potent inhibitors and activators of oligosaccharide processing as well as components affecting multiple quality attributes are presented. Extensive research efforts in this field show the feasibility of quality engineering through media design, allowing to significantly modulate the protein function. © 2015 American Institute of Chemical Engineers.

  19. High Resolution QC Analysis of Intact Recombinant Proteins

    Science.gov (United States)

    Wunderlich, D.; Barsch, A.; Baessmann, C.; Jabs, W.

    2010-01-01

    RP-89 Connected with the increased interest in new biopharma products is the need for quality control to ensure the use of proper batches in the production of such recombinant proteins. This includes knowledge about the correct amino acid sequence as well as modifications like glycosylation, phosphorylation etc. Using a high resolution instrument is preferred here due to the heterogeneity of those samples as well as the advantage of having an exact mass on the intact compound as well as chemical or enzymatic fragments. With the possibility of running the intact proteins directly, a high-throughput analysis is possible. In this study, the maXis QTOF was applied for the LC/MS analysis of various proteins, including recombinant IgG (MW∼149 kDa) and its component light chains (MW∼24.4 kDa) as well as proteins in monomer and dimeric forms ranging from 11 up to 177 kDa. Proteins were separated with a Zorbax SBC8, Rapid Resolution Cartridge (2.1 × 30 mm, 3.5 μm) within 15 min. and directly MS analyzed. The resolution of this instrument is able to discriminate discrete changes in the glyco­sylation patterns of those large molecules. When the ecombinant IgG1 from Chinese Hamster Ovary (CHO) cells was further analyzed by reduction and alkylation, it was possible to measure the resulting light chains at 0.2 ppm mass accuracy. Fractions from intact e.coli protein separations were injected by infusion into the maxis. Here, various proteins of ∼11, 20.8, and 177 kDa could be mass-separated and analyzed with high mass accuracy. The data is analyzed by specially designed software modules which compare mass patterns and accuracies with expected values ensuring a fast and reliable information retrieval.

  20. Mandibular defect reconstruction using three-dimensional polycaprolactone scaffold in combination with platelet-rich plasma and recombinant human bone morphogenetic protein-2: de novo synthesis of bone in a single case.

    Science.gov (United States)

    Schuckert, Karl-Heinz; Jopp, Stefan; Teoh, Swee-Hin

    2009-03-01

    This publication describes the clinical case of a 71-year-old female patient. Using polycaprolactone (PCL) scaffold, platelet-rich plasma (PRP) and recombinant human bone morphogenetic protein-2 (rhBMP-2), a critical-sized defect in the anterior mandible was regenerated using de novo-grown bone. A bacterial infection had caused a periimplantitis in two dental implants leading to a large destruction in the anterior mandible. Both implants were removed under antibiotic prophylaxis. A PCL scaffold was prepared especially for this clinical case. In a second procedure with antibiotic prophylaxis, the bony defect was reopened. The PCL scaffold was fitted and charged with PRP and rhBMP-2 (1.2 mg). After complication-free wound healing, the radiological control demonstrated de novo-grown bone in the anterior mandible 6 months postoperatively. Dental implants were inserted in a third operation. A bone biopsy of the newly grown bone, as well as of the bordering local bone, was taken and histologically examined. The bone samples were identical and presented vital laminar bone.

  1. Expression and biochemical characterization of recombinant human epididymis protein 4.

    Science.gov (United States)

    Hua, Ling; Liu, Yunhui; Zhen, Shuai; Wan, Deyou; Cao, Jiyue; Gao, Xin

    2014-10-01

    Whey acidic proteins (WAP) belong to a large gene family of antibacterial peptides that perform critical immune system functions. The function of human epididymis protein 4 (HE4), a 124-amino acid long polypeptide that has two whey acidic protein four-disulfide core (WFDC) domains, is not well studied. Here, a fusion gene encoding the HE4 protein fused to an IgG1 Fc domain was constructed. The recombinant HE4 protein was expressed as a secretory protein in Pichia pastoris and mammalian HEK293-F cells and was subsequently purified. Our data suggested that the HE4 protein produced by these two expression systems bound to both gram-negative and gram-positive bacteria, but demonstrated slightly inhibitory activity towards the growth of Staphylococcus aureus. Moreover, HE4 exhibited proteinase inhibitory activity towards trypsin, elastase, matrix metallopeptidase 9, and the secretory proteinases from Bacillus subtilis. The effects of glycosylation on the biochemical characterization of HE4 were also investigated. LC-ESI-MS glycosylation analysis showed that the high-mannose glycosylated form of HE4 expressed by P. pastoris has lower biological activity when compared to its complex-glycosylated form produced from HEK293-F cells. The implications of this are discussed, which may be provide theoretical basis for its important role in the development of cancer and innate immune system. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Comparison of Immunoprotection of Leptospira Recombinant Proteins with conventional vaccine in experimental animals.

    Science.gov (United States)

    Parthiban, M; Kumar, S Senthil; Balachandran, C; Kumanan, K; Aarthi, K S; Nireesha, G

    2015-12-01

    Leptospirosis is a bacterial disease caused by bacteria of the genus Leptospira affecting humans and animals. Untreated leptospirosis may result in severe kidney damage, meningitis, liver failure, respiratory distress, and even death. Virulent leptospirosis can rapidly enter kidney fibroblasts and induce a programmed cell death. Thus, it is a challenge for immunologists to develop an effective and safe leptospirosis vaccine. Here, we compared the commercial canine leptospira vaccine and recombinant proteins (OmpL1 and LipL41) with and without adjuvant in terms of immune response and challenge studies in hamsters and immune response studies alone in experimental dogs. The outer membrane proteins viz., lipL41 and OmpL1 of leptospira interrogans serovars icterohaemorrhagiae were amplified. The primers were designed in such a way that amplified products of OmpL1 and lipL41 were ligated and cloned simultaneously into a single vector. The cloned products were expressed in E. coli BL21 cells. The immunoprotection studies were conducted for both recombinant proteins and commercial vaccine. The challenge experiment studies revealed that combination of both rLip41 and rOmpL1 and commercial vaccine gave 83% and 87% protection, respectively. Histopathological investigation revealed mild sub lethal changes were noticed in liver and kidney in commercially vaccinated group alone. The immune responses against recombinant leptospiral proteins were also demonstrated in dogs.

  3. Multi-host expression system for recombinant production of challenging proteins.

    Science.gov (United States)

    Meyer, Steffen; Lorenz, Carmen; Baser, Bahar; Wördehoff, Mona; Jäger, Volker; van den Heuvel, Joop

    2013-01-01

    Recombinant production of complex eukaryotic proteins for structural analyses typically requires a profound screening process to identify suitable constructs for the expression of ample amounts of properly folded protein. Furthermore, the evaluation of an optimal expression host has a major impact on protein yield and quality as well as on actual cost of the production process. Here we present a novel fast expression system for multiple hosts based on a single donor vector termed pFlp-Bac-to-Mam. The range of applications of pFlp-Bac-to-Mam comprises highly efficient transient transfection of HEK293-6E in serum-free suspension culture and subsequent large-scale production of challenging proteins expressing in mg per Liter level using either the baculoviral expression vector system or stable CHO production cell lines generated by Flp-mediated cassette exchange. The success of the multi-host expression vector to identify the optimal expression strategy for efficient production of high quality protein is demonstrated in a comparative expression study of three model proteins representing different protein classes: intracellular expression using a fluorescent protein, secretion of a single-chain-Fv-hIgG1Fc fusion construct and production of a large amount of highly homogeneous protein sample of the extracellular domain of a Toll-like receptor. The evaluation of the production efficiency shows that the pFlp-Bac-to-Mam system allows a fast and individual optimization of the expression strategy for each protein class.

  4. Immunogenicity of recombinant proteins consisting of Plasmodium vivax circumsporozoite protein allelic variant-derived epitopes fused with Salmonella enterica Serovar Typhimurium flagellin.

    Science.gov (United States)

    Leal, Monica Teixeira Andrade; Camacho, Ariane Guglielmi Ariza; Teixeira, Laís Helena; Bargieri, Daniel Youssef; Soares, Irene Silva; Tararam, Cibele Aparecida; Rodrigues, Mauricio M

    2013-09-01

    A Plasmodium falciparum circumsporozoite protein (CSP)-based recombinant fusion vaccine is the first malaria vaccine to reach phase III clinical trials. Resistance to infection correlated with the production of antibodies to the immunodominant central repeat region of the CSP. In contrast to P. falciparum, vaccine development against the CSP of Plasmodium vivax malaria is far behind. Based on this gap in our knowledge, we generated a recombinant chimeric protein containing the immunodominant central repeat regions of the P. vivax CSP fused to Salmonella enterica serovar Typhimurium-derived flagellin (FliC) to activate the innate immune system. The recombinant proteins that were generated contained repeat regions derived from each of the 3 different allelic variants of the P. vivax CSP or a fusion of regions derived from each of the 3 allelic forms. Mice were subcutaneously immunized with the fusion proteins alone or in combination with the Toll-like receptor 3 (TLR-3) agonist poly(I·C), and the anti-CSP serum IgG response was measured. Immunization with a mixture of the 3 recombinant proteins, each containing immunodominant epitopes derived from a single allelic variant, rather than a single recombinant protein carrying a fusion of regions derived from each of 3 allelic forms elicited a stronger immune response. This response was independent of TLR-4 but required TLR-5/MyD88 activation. Antibody titers significantly increased when poly(I·C) was used as an adjuvant with a mixture of the 3 recombinant proteins. These recombinant fusion proteins are novel candidates for the development of an effective malaria vaccine against P. vivax.

  5. Expression of recombinant green fluorescent protein in Bacillus methanolicus.

    Science.gov (United States)

    Nilasari, Dewi; Dover, Nir; Rech, Sabine; Komives, Claire

    2012-01-01

    Microbial biocatalysts are used in a wide range of industries to produce large scale quantities of proteins, amino acids, and commodity chemicals. While the majority of these processes use glucose or other low-cost sugars as the substrate, Bacillus methanolicus is one example of a biocatalyst that has shown sustained growth on methanol as a carbon source at elevated temperature (50-53°C optimum) resulting in reduced feed and utility costs. Specifically, the complete chemical process enabled by this approach takes methane from natural gas, and following a low-cost conversion to methanol, can be used for the production of high value products. In this study, production of recombinant green fluorescent protein (GFPuv) by B. methanolicus is explored. A plasmid was constructed that incorporates the methanol dehydrogenase (mdh) promoter of B. methanolicus MGA3 together with the GFPuv gene. The plasmid, pNW33N, was shown to be effective for expression in other Bacillus strains, although not previously in B. methanolicus. A published electroporation protocol for transformation of B. methanolicus was modified to result in expression of GFP using plasmid pNW33N-mdh-GFPuv (pNmG). Transformation was confirmed by both agarose gel electrophoresis and by observation of green fluorescence under UV light exposure. The mass yield of cells and protein were measured in shake flask experiments. The optimum concentration of methanol for protein production was found to be at 200 mM. Higher concentrations than 200 mM resulted in slightly higher biomass production but lower amounts of recombinant protein. Copyright © 2012 American Institute of Chemical Engineers (AIChE).

  6. Construction of an oral recombinant DNA vaccine from H pylori neutrophil activating protein and its immunogenicity.

    Science.gov (United States)

    Sun, Bo; Li, Zhao-Shen; Tu, Zhen-Xing; Xu, Guo-Ming; Du, Yi-Qi

    2006-11-21

    To construct a live attenuated Salmonella typhimurium (S. typhimurium) strain harboring the H pylori neutrophil activating protein (HP-NAP) gene as an oral recombinant DNA vaccine, and to evaluate its immunogenicity. By genetic engineering methods, the genomic DNA of H pylori was extracted as a template. The total length of the HP-NAP gene was amplified by polymerase chain reaction (PCR) and cloned into pBT vector for sequencing and BLAST analysis, then subcloned into a eukaryotic expression vector pIRES followed by PCR identification and restriction enzyme digestion. The identified recombinant plasmid pIRES-NAP was transfected into COS-7 cells for target fusion protein expression, and its antigenicity was detected by Western blotting. Then the recombinant plasmid was transformed into a live attenuated S. typhimurium strain SL7207 as an oral vaccine strain, and its immunogenicity was evaluated with animal experiments. A 435 bp product was cloned using high homology with HP-NAP gene in GenBank (more than 98%). With identification by PCR and restriction enzyme digestion, a recombinant eukaryotic expression plasmid pIRES-NAP containing the HP-NAP gene of H pylori was successfully constructed. The expressed target protein had a specific reaction with H pylorii whole cell antibody and showed a single strip result detected by Western blotting. Oral immunization of mice with recombinant DNA vaccine strain SL7207 (pIRES-NAP) also induced a specific immune response. The successful construction of HP-NAP oral DNA vaccine with good immunogenicity may help to further investigate its immunoprotection effects and develop vaccine against H pylori infection.

  7. Utilizing protein-lean coproducts from corn containing recombinant pharmaceutical proteins for ethanol production.

    Science.gov (United States)

    Paraman, Ilankovan; Moeller, Lorena; Scott, M Paul; Wang, Kan; Glatz, Charles E; Johnson, Lawrence A

    2010-10-13

    Protein-lean fractions of corn (maize) containing recombinant (r) pharmaceutical proteins were evaluated as a potential feedstock to produce fuel ethanol. The levels of residual r-proteins in the coproduct, distillers dry grains with solubles (DDGS), were determined. Transgenic corn lines containing recombinant green fluorescence protein (r-GFP) and a recombinant subunit vaccine of Escherichia coli enterotoxin (r-LTB), primarily expressed in endosperm, and another two corn lines containing recombinant human collagen (r-CIα1) and r-GFP, primarily expressed in germ, were used as model systems. The kernels were either ground and used for fermentation or dry fractionated to recover germ-rich fractions prior to grinding for fermentation. The finished beers of whole ground kernels and r-protein-spent endosperm solids contained 127-139 and 138-155 g/L ethanol concentrations, respectively. The ethanol levels did not differ among transgenic and normal corn feedstocks, indicating the residual r-proteins did not negatively affect ethanol production. r-Protein extraction and germ removal also did not negatively affect fermentation of the remaining mass. Most r-proteins were inactivated during the mashing process used to prepare corn for fermentation. No functionally active r-GFP or r-LTB proteins were found after fermentation of the r-protein-spent solids; however, a small quantity of residual r-CIα1 was detected in DDGS, indicating that the safety of DDGS produced from transgenic grain for r-protein production needs to be evaluated for each event. Protease treatment during fermentation completely hydrolyzed the residual r-CIα1, and no residual r-proteins were detectable in DDGS.

  8. The RAG1 and RAG2 proteins establish the 12/23 rule in V(D)J recombination

    NARCIS (Netherlands)

    D.C. van Gent (Dik)

    1996-01-01

    textabstractV(D)J recombination requires a pair of signal sequences with spacer lengths of 12 and 23 base pairs. Cleavage by the RAG1 AND RAG2 proteins was previously shown to demand only a single signal sequence. Here, we established conditions where 12- and 23-spacer signal

  9. Protein solubility and differential proteomic profiling of recombinant Escherichia coli overexpressing double-tagged fusion proteins.

    Science.gov (United States)

    Cheng, Chung-Hsien; Lee, Wen-Chien

    2010-08-28

    Overexpression of recombinant proteins usually triggers the induction of heat shock proteins that regulate aggregation and solubility of the overexpressed protein. The two-dimensional gel electrophoresis (2-DE)-mass spectrometry approach was used to profile the proteome of Escherichia coli overexpressing N-acetyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase) and N-acetyl-D-neuraminic acid aldolase (Neu5Ac aldolase), both fused to glutathione S-transferase (GST) and polyionic peptide (5D or 5R). Overexpression of fusion proteins by IPTG induction caused significant differential expression of numerous cellular proteins; most of these proteins were down-regulated, including enzymes connected to the pentose phosphate pathway and the enzyme LuxS that could lead to an inhibition of tRNA synthesis. Interestingly, when plasmid-harboring cells were cultured in LB medium, gluconeogenesis occurred mainly through MaeB, while in the host strain, gluconeogenesis occurred by a different pathway (by Mdh and PckA). Significant up-regulation of the chaperones ClpB, HslU and GroEL and high-level expression of two protective small heat shock proteins (IbpA and IbpB) were found in cells overexpressing GST-GlcNAc 2-epimerase-5D but not in GST-Neu5Ac aldolase-5R-expressing E. coli. Although most of the recombinant protein was present in insoluble aggregates, the soluble fraction of GST-GlcNAc 2-epimerase-5D was higher than that of GST-Neu5Ac aldolase-5R. Also, in cells overexpressing recombinant GST-GlcNAc 2-epimerase-5D, the expression of σ32 was maintained at a higher level following induction. Differential expression of metabolically functional proteins, especially those in the gluconeogenesis pathway, was found between host and recombinant cells. Also, the expression patterns of chaperones/heat shock proteins differed among the plasmid-harboring bacteria in response to overproduction of recombinant proteins. In conclusion, the solubility of overexpressed recombinant proteins could

  10. Cloning and expression of the recombinant NP24I protein from ...

    African Journals Online (AJOL)

    Jane

    2011-10-24

    Oct 24, 2011 ... The recombinant NP24I protein was purified using Ni-NTA resin, and then the antimicrobial activity of the purified recombinant NP24I protein was tested. The aims of this work were to study the cloning and expression of NP24 protein from local tomato cultivar in a prokaryotic system and to test the activity of.

  11. A Generic Protocol for Intracellular Expression of Recombinant Proteins in Bacillus subtilis.

    Science.gov (United States)

    Phan, Trang; Huynh, Phuong; Truong, Tuom; Nguyen, Hoang

    2017-01-01

    Bacillus subtilis (B. subtilis) is a potential and attractive host for the production of recombinant proteins. Different expression systems for B. subtilis have been developed recently, and various target proteins have been recombinantly synthesized and purified using this host. In this chapter, we introduce a generic protocol to express a recombinant protein in B. subtilis. It includes protocols for (1) using our typical expression vector (plasmid pHT254) to introduce a target gene, (2) transformation of the target vector into B. subtilis, and (3) evaluation of the actual expression of a recombinant protein.

  12. Recombinant human albumin supports single cell cloning of CHO cells in chemically defined media.

    Science.gov (United States)

    Zhu, Jiang; Wooh, Jong Wei; Hou, Jeff Jia Cheng; Hughes, Benjamin S; Gray, Peter P; Munro, Trent P

    2012-01-01

    Biologic drugs, such as monoclonal antibodies, are commonly made using mammalian cells in culture. The cell lines used for manufacturing should ideally be clonal, meaning derived from a single cell, which represents a technically challenging process. Fetal bovine serum is often used to support low cell density cultures, however, from a regulatory perspective, it is preferable to avoid animal-derived components to increase process consistency and reduce the risk of contamination from adventitious agents. Chinese hamster ovary (CHO) cells are the most widely used cell line in industry and a large number of serum-free, protein-free, and fully chemically defined growth media are commercially available, although these media alone do not readily support efficient single cell cloning. In this work, we have developed a simple, fully defined, single-cell cloning media, specifically for CHO cells, using commercially available reagents. Our results show that a 1:1 mixture of CD-CHO™ and DMEM/F12 supplemented with 1.5 g/L of recombinant albumin (Albucult®) supports single cell cloning. This formulation can support recovery of single cells in 43% of cultures compared to 62% in the presence of serum. Copyright © 2012 American Institute of Chemical Engineers (AIChE).

  13. Efficient purification of recombinant proteins fused to maltose-binding protein by mixed-mode chromatography.

    Science.gov (United States)

    Cabanne, Charlotte; Pezzini, Jérôme; Joucla, Gilles; Hocquellet, Agnès; Barbot, Caroline; Garbay, Bertrand; Santarelli, Xavier

    2009-05-15

    Two mixed-mode resins were evaluated as an alternative to conventional affinity resins for the purification of recombinant proteins fused to maltose-binding protein (MPB). We purified recombinant MBP, MBP-LacZ and MBP-Leap2 from crude Escherichia coli extracts. Mixed-mode resins allowed the efficient purification of MBP-fused proteins. Indeed, the quantity of purified proteins was significantly higher with mixed-mode resins, and their purity was equivalent to that obtained with affinity resins. By using purified MBP, MBP-LacZ and MBP-Leap2, the dynamic binding capacity of mixed-mode resins was 5-fold higher than that of affinity resins. Moreover, the recovery for the three proteins studied was in the 50-60% range for affinity resins, and in the 80-85% range for mixed-mode resins. Mixed-mode resins thus represent a powerful alternative to the classical amylose or dextrin resins for the purification of recombinant proteins fused to maltose-binding protein.

  14. Immunocontraception of Eastern Grey kangaroos (Macropus giganteus) with recombinant brushtail possum (Trichosurus vulpecula) ZP3 protein.

    Science.gov (United States)

    Kitchener, Anne L; Harman, Amanda; Kay, David J; McCartney, Carmen A; Mate, Karen E; Rodger, John C

    2009-01-01

    This study examined the potential of a recombinant marsupial zona pellucida 3 protein as a contraceptive vaccine for the Eastern Grey kangaroo, a marsupial that is locally overabundant in several regions of eastern Australia. First, a pilot study using porcine zona pellucidae (PZP) demonstrated that ZP proteins, primarily the ZP3 component of PZP, are highly immunogenic in the grey kangaroo and produce a long-lasting humoral response to a single immunisation, as found in other marsupials. Immunisation with 300 microg of a non-glycosylated recombinant brushtail possum ZP3 (recBP-ZP3) protein in complete Freund's adjuvant produced a similar, significant and sustained antibody response, and none of the immunised kangaroos (n=7) produced offspring during the following breeding season compared with four out of the six control animals. An epitope analysis of the B-cell response to recBP-ZP3 using a brushtail possum ZP3 identified numerous B-cell epitope regions clustered around the N- and C-terminal regions of the protein. Two regions of interest for further fertility vaccine development based on their immunogenicity and fertility trials and functional studies in other species were found to be immunogenic. These results suggest that immunocontraception based on targeting the ZP3 protein within the zona pellucida may be an effective strategy for fertility reduction in Eastern Grey kangaroos.

  15. Selective blockade of trypanosomatid protein synthesis by a recombinant antibody anti-Trypanosoma cruzi P2β protein.

    Science.gov (United States)

    Ayub, Maximiliano Juri; Nyambega, Benson; Simonetti, Leandro; Duffy, Tomas; Longhi, Silvia A; Gómez, Karina A; Hoebeke, Johan; Levin, Mariano J; Smulski, Cristian R

    2012-01-01

    The ribosomal P proteins are located on the stalk of the ribosomal large subunit and play a critical role during the elongation step of protein synthesis. The single chain recombinant antibody C5 (scFv C5) directed against the C-terminal region of the Trypanosoma cruzi P2β protein (TcP2β) recognizes the conserved C-terminal end of all T. cruzi ribosomal P proteins. Although this region is highly conserved among different species, surface plasmon resonance analysis showed that the scFv C5 possesses very low affinity for the corresponding mammalian epitope, despite having only one single amino-acid change. Crystallographic analysis, in silico modelization and NMR assays support the analysis, increasing our understanding on the structural basis of epitope specificity. In vitro protein synthesis experiments showed that scFv C5 was able to specifically block translation by T. cruzi and Crithidia fasciculata ribosomes, but virtually had no effect on Rattus norvegicus ribosomes. Therefore, we used the scFv C5 coding sequence to make inducible intrabodies in Trypanosoma brucei. Transgenic parasites showed a strong decrease in their growth rate after induction. These results strengthen the importance of the P protein C terminal regions for ribosomal translation activity and suggest that trypanosomatid ribosomal P proteins could be a possible target for selective therapeutic agents that could be derived from structural analysis of the scFv C5 antibody paratope.

  16. Selective blockade of trypanosomatid protein synthesis by a recombinant antibody anti-Trypanosoma cruzi P2β protein.

    Directory of Open Access Journals (Sweden)

    Maximiliano Juri Ayub

    Full Text Available The ribosomal P proteins are located on the stalk of the ribosomal large subunit and play a critical role during the elongation step of protein synthesis. The single chain recombinant antibody C5 (scFv C5 directed against the C-terminal region of the Trypanosoma cruzi P2β protein (TcP2β recognizes the conserved C-terminal end of all T. cruzi ribosomal P proteins. Although this region is highly conserved among different species, surface plasmon resonance analysis showed that the scFv C5 possesses very low affinity for the corresponding mammalian epitope, despite having only one single amino-acid change. Crystallographic analysis, in silico modelization and NMR assays support the analysis, increasing our understanding on the structural basis of epitope specificity. In vitro protein synthesis experiments showed that scFv C5 was able to specifically block translation by T. cruzi and Crithidia fasciculata ribosomes, but virtually had no effect on Rattus norvegicus ribosomes. Therefore, we used the scFv C5 coding sequence to make inducible intrabodies in Trypanosoma brucei. Transgenic parasites showed a strong decrease in their growth rate after induction. These results strengthen the importance of the P protein C terminal regions for ribosomal translation activity and suggest that trypanosomatid ribosomal P proteins could be a possible target for selective therapeutic agents that could be derived from structural analysis of the scFv C5 antibody paratope.

  17. Nickel-Salen supported paramagnetic nanoparticles for 6-His-target recombinant protein affinity purification.

    Science.gov (United States)

    Rashid, Zahra; Ghahremanzadeh, Ramin; Nejadmoghaddam, Mohammad-Reza; Nazari, Mahboobeh; Shokri, Mohammad-Reza; Naeimi, Hossein; Zarnani, Amir-Hassan

    2017-03-24

    In this research, a simple, efficient, inexpensive, rapid and high yield method for the purification of 6×histidine-tagged recombinant protein was developed. For this purpose, manganese ferrite magnetic nanoparticles (MNPs) were synthesized through a co-precipitation method and then they were conveniently surface-modified with tetraethyl orthosilicate (TEOS) in order to prevent oxidation and form high density of hydroxyl groups. Next, the salen ligand was prepared from condensation reaction of salicylaldehyde and 3-aminopropyl (trimethoxy) silane (APTMS) in 1:1 molar ratio; followed by complexation with Ni(OAc) 2 .4H 2 O. Finally, the prepared Ni(II)-salen complex conjugated to silica coated MNPs and MnFe 2 O 4 @SiO 2 @Ni-Salen complex nanoparticles were obtained. The functionalized nanoparticles were spherical with an average diameter around 70nm. The obtained MNPs had a saturation magnetization about 54 emu/g and had super paramagnetic character. These MNPs were used efficiently to enrich recombinant histidine-tagged (His-tagged) protein-A from bacterial cell lysate. In about 45min, highly pure His-tagged recombinant protein was obtained, as judged by SDS-PAGE analysis and silver staining. The amount of target protein in flow through and washing fractions was minimal denoting the high efficiency of purification process. The average capacity of the matrix was found to be high and about 180±15mgg -1 (protein/MnFe 2 O 4 @SiO 2 @Ni-Salen complex). Collectively, purification process with MnFe 2 O 4 @SiO 2 @Ni-Salen complex nanoparticles is rapid, efficient, selective and whole purification can be carried out in only a single tube without the need for expensive systems. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Extensive Lysine Methylation in Hyperthermophilic Crenarchaea: Potential Implications for Protein Stability and Recombinant Enzymes

    Directory of Open Access Journals (Sweden)

    Catherine H. Botting

    2010-01-01

    Full Text Available In eukarya and bacteria, lysine methylation is relatively rare and is catalysed by sequence-specific lysine methyltransferases that typically have only a single-protein target. Using RNA polymerase purified from the thermophilic crenarchaeum Sulfolobus solfataricus, we identified 21 methyllysines distributed across 9 subunits of the enzyme. The modified lysines were predominantly in α-helices and showed no conserved sequence context. A limited survey of the Thermoproteus tenax proteome revealed widespread modification with 52 methyllysines in 30 different proteins. These observations suggest the presence of an unusual lysine methyltransferase with relaxed specificity in the crenarchaea. Since lysine methylation is known to enhance protein thermostability, this may be an adaptation to a thermophilic lifestyle. The implications of this modification for studies and applications of recombinant crenarchaeal enzymes are discussed.

  19. Purification of recombinant tissue plasminogen activator (rtPA) protein from transplastomic tobacco plants.

    Science.gov (United States)

    Abdoli Nasab, Maryam; Jalali Javaran, Mokhtar; Cusido, Rosa M; Palazon, Javier

    2016-11-01

    Plants are low cost platforms for the production of recombinant proteins, but their complexity renders the purification of plant recombinant proteins more difficult than proteins expressed in yeast or bacteria. Plastid transformation enables high-level expression of foreign genes and the accumulation of recombinant proteins in plastid organelles. Histidine (His) tags are widely used for affinity purification of recombinant proteins in a nickel column. The human tissue-type plasminogen activator (tPA) is one of the most important pharmaceutical recombinant proteins involved in the breakdown of blood clots in different parts of the body. The truncated form of the tissue plasminogen activator (K2S) has a longer plasma half-life, better diffusion into the clot, and higher fibrinolytic activity. In a construct designed to insert the K2S gene in the tobacco chloroplast, the sequence of six histidines and a factor Xa protease site was fused to the C-terminus of the K2S protein. The presence and amount of tPA recombinant protein in transplastomic tobacco plants was estimated by ELISA analysis using a specific antibody. The protein was purified from total soluble protein, insoluble protein aggregates and the protein was extracted from the isolated chloroplast using nickel resin and a chromatography column. After digestion of the purified protein with factor Xa, the presence of the purified tPA protein was confirmed by western blot analysis. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  20. Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells.

    Science.gov (United States)

    Fabrick, Jeffrey A; Hull, J Joe

    2017-04-20

    Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

  1. Stable Plastid Transformation for High-Level Recombinant Protein Expression: Promises and Challenges

    Directory of Open Access Journals (Sweden)

    Meili Gao

    2012-01-01

    Full Text Available Plants are a promising expression system for the production of recombinant proteins. However, low protein productivity remains a major obstacle that limits extensive commercialization of whole plant and plant cell bioproduction platform. Plastid genetic engineering offers several advantages, including high levels of transgenic expression, transgenic containment via maternal inheritance, and multigene expression in a single transformation event. In recent years, the development of optimized expression strategies has given a huge boost to the exploitation of plastids in molecular farming. The driving forces behind the high expression level of plastid bioreactors include codon optimization, promoters and UTRs, genotypic modifications, endogenous enhancer and regulatory elements, posttranslational modification, and proteolysis. Exciting progress of the high expression level has been made with the plastid-based production of two particularly important classes of pharmaceuticals: vaccine antigens, therapeutic proteins, and antibiotics and enzymes. Approaches to overcome and solve the associated challenges of this culture system that include low transformation frequencies, the formation of inclusion bodies, and purification of recombinant proteins will also be discussed.

  2. Limiting factors in Escherichia colifed-batch production of recombinant proteins

    DEFF Research Database (Denmark)

    Sanden, A.M.; Prytz, I.; Tubelekas, I.

    2003-01-01

    recombinant protein production, fed-batch, specific growth rate, feed profile, induction, mRNA, transcription, translation, acetic acid formation......recombinant protein production, fed-batch, specific growth rate, feed profile, induction, mRNA, transcription, translation, acetic acid formation...

  3. Recombinant proteins from plants: production and isolation of clinically useful compounds

    National Research Council Canada - National Science Library

    Cunningham, Charles; Porter, Andrew J. R

    1998-01-01

    ... of recombinant proteins for use as specialist industrial or therapeutic biomolecules. The intention of Recombinant Proteins from Plants is to provide comprehensive and detailed protocols covering all the latest molecular approaches. Because the production oftransgenic plants has become routine in many laboratories, coverage is also given to some of the more "...

  4. Heparin-binding peptide as a novel affinity tag for purification of recombinant proteins.

    Science.gov (United States)

    Morris, Jacqueline; Jayanthi, Srinivas; Langston, Rebekah; Daily, Anna; Kight, Alicia; McNabb, David S; Henry, Ralph; Kumar, Thallapuranam Krishnaswamy Suresh

    2016-10-01

    Purification of recombinant proteins constitutes a significant part of the downstream processing in biopharmaceutical industries. Major costs involved in the production of bio-therapeutics mainly depend on the number of purification steps used during the downstream process. Affinity chromatography is a widely used method for the purification of recombinant proteins expressed in different expression host platforms. Recombinant protein purification is achieved by fusing appropriate affinity tags to either N- or C- terminus of the target recombinant proteins. Currently available protein/peptide affinity tags have proved quite useful in the purification of recombinant proteins. However, these affinity tags suffer from specific limitations in their use under different conditions of purification. In this study, we have designed a novel 34-amino acid heparin-binding affinity tag (HB-tag) for the purification of recombinant proteins expressed in Escherichia coli (E. coli) cells. HB-tag fused recombinant proteins were overexpressed in E. coli in high yields. A one-step heparin-Sepharose-based affinity chromatography protocol was developed to purify HB-fused recombinant proteins to homogeneity using a simple sodium chloride step gradient elution. The HB-tag has also been shown to facilitate the purification of target recombinant proteins from their 8 M urea denatured state(s). The HB-tag has been demonstrated to be successfully released from the fusion protein by an appropriate protease treatment to obtain the recombinant target protein(s) in high yields. Results of the two-dimensional NMR spectroscopy experiments indicate that the purified recombinant target protein(s) exist in the native conformation. Polyclonal antibodies raised against the HB-peptide sequence, exhibited high binding specificity and sensitivity to the HB-fused recombinant proteins (∼10 ng) in different crude cell extracts obtained from diverse expression hosts. In our opinion, the HB-tag provides a

  5. Accelerators of Osteogenesis by Recombinant Human Bone Morphogenetic Protein-2

    Directory of Open Access Journals (Sweden)

    Yasunori Okubo

    2007-01-01

    Full Text Available Bone morphogenetic protein (BMP appears to be one of the most promising cytokine and for clinical use in reconstructive surgery for bony defects and augmentation. To evaluate the effect of basic fibroblast growth factor (bFGF, FK506, elcatonin, and hyperbaric oxygenation (HBO on osteoinduction by recombinant human bone morphogenetic protein-2 (rhBMP-2, 2 or 5 μg of rhBMP-2 was implanted into intramuscular sites of rats. At 21 days after implantation, the osteoinductive activity in the treatment group and control group was compared radiographically, biochemically, and histologically. The amount of new bone in the treatment group was significantly greater than that in the control group. The alkaline phosphatase activity and calcium content in the treatment group were significantly higher than those in the control group. These results suggest that bFGF, FK506, elcatonin, and HBO accelerated the activity and rate of osteoinduction by rhBMP2. These results may be useful when BMP is applied clinically in near future.

  6. Accelerators of Osteogenesis by Recombinant Human Bone Morphogenetic Protein-2

    Directory of Open Access Journals (Sweden)

    Yasunori Okubo

    2007-01-01

    Full Text Available Bone morphogenetic protein (BMP appears to be one of the most promising cytokine and for clinical use in reconstructive surgery for bony defects and augmentation. To evaluate the effect of basic fibroblast growth factor (bFGF, FK506, elcatonin, and hyperbaric oxygenation (HBO on osteoinduction by recombinant human bone morphogenetic protein-2 (rhBMP-2, 2 or 5 μg of rhBMP-2 was implanted into intramuscular sites of rats. At 21 days after implantation, the osteoinductive activity in the treatment group and control group was compared radiographically, biochemically, and histologically. The amount of new bone in the treatment group was signifi cantly greater than that in the control group. The alkaline phosphatase activity and calcium content in the treatment group were signifi cantly higher than those in the control group. These results suggest that bFGF, FK506, elcatonin, and HBO accelerated the activity and rate of osteoinduction by rhBMP2. These results may be useful when BMP is applied clinically in near future.

  7. Human Rad51 filaments on double- and single-stranded DNA : Correlating regular and irregular forms with recombination function

    NARCIS (Netherlands)

    Ristic, D.; Modesti, M.; Van der Heijden, T.; Van Noort, J.; Dekker, C.; Kanaar, R.; Wyman, C.

    Recombinase proteins assembled into helical filaments on DNA are believed to be the catalytic core of homologous recombination. The assembly, disassembly and dynamic rearrangements of this structure must drive the DNA strand exchange reactions of homologous recombination. The sensitivity of

  8. The Effects of the Recombinant CCR5 T4 Lysozyme Fusion Protein on HIV-1 Infection.

    Directory of Open Access Journals (Sweden)

    Qingwen Jin

    Full Text Available Insertion of T4 lysozyme (T4L into the GPCR successfully enhanced GPCR protein stability and solubilization. However, the biological functions of the recombinant GPCR protein have not been analyzed.We engineered the CCR5-T4L mutant and expressed and purified the soluble recombinant protein using an E.coli expression system. The antiviral effects of this recombinant protein in THP-1 cell lines, primary human macrophages, and PBMCs from different donors were investigated. We also explored the possible mechanisms underlying the observed antiviral effects.Our data showed the biphasic inhibitory and promotion effects of different concentrations of soluble recombinant CCR5-T4L protein on R5 tropic human immunodeficiency virus-1 (HIV-1 infection in THP-1 cell lines, human macrophages, and PBMCs from clinical isolates. We demonstrated that soluble recombinant CCR5-T4L acts as a HIV-1 co-receptor, interacts with wild type CCR5, down-regulates the surface CCR5 expression in human macrophages, and interacts with CCL5 to inhibit macrophage migration. Using binding assays, we further determined that recombinant CCR5-T4L and [125I]-CCL5 compete for the same binding site on wild type CCR5.Our results suggest that recombinant CCR5-T4L protein marginally promotes HIV-1 infection at low concentrations and markedly inhibits infection at higher concentrations. This recombinant protein may be helpful in the future development of anti-HIV-1 therapeutic agents.

  9. The Effects of the Recombinant CCR5 T4 Lysozyme Fusion Protein on HIV-1 Infection.

    Science.gov (United States)

    Jin, Qingwen; Chen, Hong; Wang, Xingxia; Zhao, Liandong; Xu, Qingchen; Wang, Huijuan; Li, Guanyu; Yang, Xiaofan; Ma, Hongming; Wu, Haoquan; Ji, Xiaohui

    2015-01-01

    Insertion of T4 lysozyme (T4L) into the GPCR successfully enhanced GPCR protein stability and solubilization. However, the biological functions of the recombinant GPCR protein have not been analyzed. We engineered the CCR5-T4L mutant and expressed and purified the soluble recombinant protein using an E.coli expression system. The antiviral effects of this recombinant protein in THP-1 cell lines, primary human macrophages, and PBMCs from different donors were investigated. We also explored the possible mechanisms underlying the observed antiviral effects. Our data showed the biphasic inhibitory and promotion effects of different concentrations of soluble recombinant CCR5-T4L protein on R5 tropic human immunodeficiency virus-1 (HIV-1) infection in THP-1 cell lines, human macrophages, and PBMCs from clinical isolates. We demonstrated that soluble recombinant CCR5-T4L acts as a HIV-1 co-receptor, interacts with wild type CCR5, down-regulates the surface CCR5 expression in human macrophages, and interacts with CCL5 to inhibit macrophage migration. Using binding assays, we further determined that recombinant CCR5-T4L and [125I]-CCL5 compete for the same binding site on wild type CCR5. Our results suggest that recombinant CCR5-T4L protein marginally promotes HIV-1 infection at low concentrations and markedly inhibits infection at higher concentrations. This recombinant protein may be helpful in the future development of anti-HIV-1 therapeutic agents.

  10. Comparison of the protective efficacy between single and combination of recombinant adenoviruses expressing complete and truncated glycoprotein, and nucleoprotein of the pathogenic street rabies virus in mice.

    Science.gov (United States)

    Kim, Ha-Hyun; Yang, Dong-Kun; Nah, Jin-Ju; Song, Jae-Young; Cho, In-Soo

    2017-06-24

    Rabies is an important viral zoonosis that causes acute encephalitis and death in mammals. To date, several recombinant vaccines have been developed based on G protein, which is considered to be the main antigen, and these vaccines are used for rabies control in many countries. Most recombinant viruses expressing RABV G protein retain the G gene from attenuated RABV. Not enough is currently known about the protective effect against RABV of a combination of recombinant adenoviruses expressing the G and N proteins of pathogenic street RABV. We constructed a recombinant adenovirus (Ad-0910Gsped) expressing the signal peptide and ectodomain (sped) of G protein of the Korean street strain, and evaluated the immunological protection conferred by a single and combination of three kinds of recombinant adenoviruses (Ad-0910Gsped and Ad-0910G with or without Ad-0910 N) in mice. A combination of Ad-0910G and Ad-0910 N conferred improved immunity against intracranial challenge compared to single administration of Ad-0910G. The Ad-0910G virus, expressing the complete G protein, was more immunogenic than Ad-0910Gsped, which expressed a truncated G protein with the transmembrane and cytoplasmic domains removed. Additionally, oral vaccination using a combination of viruses led to complete protection. Our results suggest that this combination of viruses is a viable new intramuscular and oral vaccine candidate.

  11. Short-chain fluorescent tryptophan tags for on-line detection of functional recombinant proteins

    Directory of Open Access Journals (Sweden)

    Siepert Eva-Maria

    2012-09-01

    Full Text Available Abstract Background Conventional fluorescent proteins, such as GFP, its derivatives and flavin mononucleotide based fluorescent proteins (FbFPs are often used as fusion tags for detecting recombinant proteins during cultivation. These reporter tags are state-of-the-art; however, they have some drawbacks, which can make on-line monitoring challenging. It is discussed in the literature that the large molecular size of proteins of the GFP family may stress the host cell metabolism during production. In addition, fluorophore formation of GFP derivatives is oxygen-dependent resulting in a lag-time between expression and fluorescence detection and the maturation of the protein is suppressed under oxygen-limited conditions. On the contrary, FbFPs are also applicable in an oxygen-limited or even anaerobic environment but are still quite large (58% of the size of GFP. Results As an alternative to common fluorescent tags we developed five novel tags based on clustered tryptophan residues, called W-tags. They are only 5-11% of the size of GFP. Based on the property of tryptophan to fluoresce in absence of oxygen it is reasonable to assume that the functionality of our W-tags is also given under anaerobic conditions. We fused these W-tags to a recombinant protein model, the anti-CD30 receptor single-chain fragment variable antibody (scFv Ki-4(scFv and the anti-MucI single-chain fragment variable M12(scFv. During cultivation in Microtiter plates, the overall tryptophan fluorescence intensity of all cultures was measured on-line for monitoring product formation via the different W-tags. After correlation of the scattered light signal representing biomass concentration and tryptophan fluorescence for the uninduced cultures, the fluorescence originating from the biomass was subtracted from the overall tryptophan signal. The resulting signal, thus, represents the product fluorescence of the tagged and untagged antibody fragments. The product fluorescence signal

  12. Recombinant expression and purification of a MAP30-cell penetrating peptide fusion protein with higher anti-tumor bioactivity.

    Science.gov (United States)

    Lv, Qiang; Yang, Xu-Zhong; Fu, Long-Yun; Lu, Yv-Ting; Lu, Yan-Hua; Zhao, Jian; Wang, Fu-Jun

    2015-07-01

    MAP30 (Momordica Antiviral Protein 30 Kd), a single-stranded type-I ribosome inactivating protein, possesses versatile biological activities including anti-tumor abilities. However, the low efficiency penetrating into tumor cells hampers the tumoricidal effect of MAP30. This paper describes MAP30 fused with a human-derived cell penetrating peptide HBD which overcome the low uptake efficiency by tumor cells and exhibits higher anti-tumor bioactivity. MAP30 gene was cloned from the genomic DNA of Momordica charantia and the recombinant plasmid pET28b-MAP30-HBD was established and transferred into Escherichia coli BL21 (DE3). The recombinant MAP30-HBD protein (rMAP30-HBD) was expressed in a soluble form after being induced by 0.5mM IPTG for 14h at 15°C. The recombinant protein was purified to greater than 95% purity with Ni-NTA affinity chromatography. The rMAP30-HBD protein not only has topological inactivation and protein translation inhibition activity but also showed significant improvements in cytotoxic activity compared to that of the rMAP30 protein without HBD in the tested tumor cell lines, and induced higher apoptosis rates in HeLa cells analyzed by Annexin V-FITC with FACS. This paper demonstrated a new method for improving MAP30 protein anti-tumor activity and might have potential applications in cancer therapy area. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. In vivo recombineering of bacteriophage λ by PCR fragments and single-strand oligonucleotides

    International Nuclear Information System (INIS)

    Oppenheim, Amos B.; Rattray, Alison J.; Bubunenko, Mikhail; Thomason, Lynn C.; Court, Donald L.

    2004-01-01

    We demonstrate that the bacteriophage λ Red functions efficiently recombine linear DNA or single-strand oligonucleotides (ss-oligos) into bacteriophage λ to create specific changes in the viral genome. Point mutations, deletions, and gene replacements have been created. While recombineering with oligonucleotides, we encountered other mutations accompanying the desired point mutational change. DNA sequence analysis suggests that these unwanted mutations are mainly frameshift deletions introduced during oligonucleotide synthesis

  14. Induction of antibodies protecting against transmissible gastroenteritis coronavirus (TGEV) by recombinant adenovirus expressing TGEV spike protein.

    Science.gov (United States)

    Torres, J M; Sánchez, C; Suñé, C; Smerdou, C; Prevec, L; Graham, F; Enjuanes, L

    1995-11-10

    Ten recombinant adenoviruses expressing either fragments of 1135, 1587, or 3329 nt or the full-length spike gene of transmissible gastroenteritis coronavirus (TGEV) have been constructed. These recombinants produce S polypeptides with apparent molecular masses of 68, 86, 135, and 200 kDa, respectively. Expression of the recombinant antigen driven by Ad5 promoters was inhibited by the insertion of an exogenous SV-40 promoter. Most of the recombinant antigens remain intracytoplasmic in infected cells. All the recombinant-directed expression products contain functional antigenic sites C and B (Gebauer et al., 1991, Virology 183, 225-238). The recombinant antigen of 135 kDa and that of 200 kDa, which represents the whole spike protein, also contain antigenic sites D and A, which have previously been shown to be the major inducers of TGEV-neutralizing antibodies. Interestingly, here we show that recombinant S protein fragments expressing only sites C and B also induced TGEV-neutralizing antibodies. The chimeric Ad5-TGEV recombinants elicited lactogenic immunity in hamsters, including the production of TGEV-neutralizing antibodies. The antisera induced in swine by the Ad5 recombinants expressing the amino-terminal 26% of the spike protein (containing sites C and B) or the full-length spike protein, when mixed with a lethal dose of virus prior to administration to susceptible piglets, delayed or completely prevented the induction of symptoms of disease, respectively.

  15. Constraints from protein structure and intra-molecular coevolution influence the fitness of HIV-1 recombinants.

    Science.gov (United States)

    Woo, Jeongmin; Robertson, David L; Lovell, Simon C

    2014-04-01

    A major challenge for developing effective treatments for HIV-1 is the viruses' ability to generate new variants. Inter-strain recombination is a major contributor to this high evolutionary rate, since at least 20% of viruses are observed to be recombinant. However, the patterns of recombination vary across the viral genome. A number of factors influence recombination, including sequence identity and secondary RNA structure. In addition the recombinant genome must code for a functional virus, and expressed proteins must fold to stable and functional structures. Any intragenic recombination that disrupts internal residue contacts may therefore produce an unfolded protein. Here we find that contact maps based on protein structures predict recombination breakpoints observed in the HIV-1 pandemic. Moreover, many pairs of contacting residues that are unlikely to be disrupted by recombination are coevolving. We conclude that purifying selection arising from protein structure and intramolecular coevolutionary changes shapes the observed patterns of recombination in HIV-1. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. Intraclonal protein expression heterogeneity in recombinant CHO cells.

    Directory of Open Access Journals (Sweden)

    Warren Pilbrough

    2009-12-01

    Full Text Available Therapeutic glycoproteins have played a major role in the commercial success of biotechnology in the post-genomic era. But isolating recombinant mammalian cell lines for large-scale production remains costly and time-consuming, due to substantial variation and unpredictable stability of expression amongst transfected cells, requiring extensive clone screening to identify suitable high producers. Streamlining this process is of considerable interest to industry yet the underlying phenomena are still not well understood. Here we examine an antibody-expressing Chinese hamster ovary (CHO clone at single-cell resolution using flow cytometry and vectors, which couple light and heavy chain transcription to fluorescent markers. Expression variation has traditionally been attributed to genetic heterogeneity arising from random genomic integration of vector DNA. It follows that single cell cloning should yield a homogeneous cell population. We show, in fact, that expression in a clone can be surprisingly heterogeneous (standard deviation 50 to 70% of the mean, approaching the level of variation in mixed transfectant pools, and each antibody chain varies in tandem. Phenotypic variation is fully developed within just 18 days of cloning, yet is not entirely explained by measurement noise, cell size, or the cell cycle. By monitoring the dynamic response of subpopulations and subclones, we show that cells also undergo slow stochastic fluctuations in expression (half-life 2 to 11 generations. Non-genetic diversity may therefore play a greater role in clonal variation than previously thought. This also has unexpected implications for expression stability. Stochastic gene expression noise and selection bias lead to perturbations from steady state at the time of cloning. The resulting transient response as clones reestablish their expression distribution is not ordinarily accounted for but can contribute to declines in median expression over timescales of up to 50

  17. Construction of leaky strains and extracellular production of exogenous proteins in recombinant Escherichia coli.

    Science.gov (United States)

    Chen, Zhao-Yuan; Cao, Jie; Xie, Li; Li, Xiao-Fei; Yu, Zhen-Hai; Tong, Wang-Yu

    2014-07-01

    In this study, a strategy of the construction of leaky strains for the extracellular production of target proteins was exploited, in which the genes mrcA, mrcB, pal and lpp (as a control) from Escherichia coli were knocked out by using single- and/or double-gene deletion methods. Then the recombinant strains for the expression of exogenous target proteins including Trx-hPTH (human parathyroid hormone 1-84 coupled with thioredoxin as a fusion partner) and reteplase were reconstructed to test the secretory efficiency of the leaky strains. Finally, the fermentation experiments of the target proteins from these recombinant leaky strains were carried out in basic media (Modified R media) and complex media (Terrific Broth media) in flasks or fermenters. The results demonstrated that the resultant leaky strains were genetically stable and had a similar growth profile in the complex media as compared with the original strain, and the secretory levels of target proteins into Modified R media from the strains with double-gene deletion (up to 88.9%/mrcA lpp-pth) are higher than the excretory levels from the strains with single-gene deletion (up to 71.1%/lpp-pth) and the host E. coli JM109 (DE3) (near zero). The highest level of extracellular production of Trx-hPTH in fermenters is up to 680 mg l(-1). © 2014 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  18. Protein expression of Myt272-3 recombinant clone and in silico ...

    African Journals Online (AJOL)

    Purpose: To investigate the expression of Myt272-3 recombinant protein and also to predict a possible protein vaccine candidate against Mycobacterium tuberculosis. Methods: Myt272-3 protein was expressed in pET30a+-Myt272-3 clone. The purity of the protein was determined using Dynabeads® His-Tag Isolation ...

  19. A new potential secretion pathway for recombinant proteins in Bacillus subtilis.

    Science.gov (United States)

    Wang, Guangqiang; Xia, Yongjun; Gu, Zhennan; Zhang, Hao; Chen, Yong Q; Chen, Haiqin; Ai, Lianzhong; Chen, Wei

    2015-11-10

    Secretion of cytoplasmic expressed proteins into growth media has significant advantages. Due to the lack of an outer membrane, Bacillus subtilis is considered as a desirable 'cell factory' for the secretion of recombinant proteins. However, bottlenecks in the classical pathway for the secretion of recombinant proteins limit its use on a wide scale. In this study, we attempted to use four typical non-classically secreted proteins as signals to export three recombinant model proteins to the culture medium. All four non-classically secreted proteins can direct the export of the intrinsically disordered nucleoskeletal-like protein (Nsp). Two of them can guide the secretion of alkaline phosphatase (PhoA). One can lead the secretion of the thermostable β-galactosidase BgaB, which cannot be secreted with the aid of typical Sec-dependent signal peptides. Our results show that the non-classically secreted proteins lead the recombinant proteins to the culture medium, and thus non-classical protein secretion pathways can be exploited as a novel secretion pathway for recombinant proteins.

  20. Protein trafficking, ergosterol biosynthesis and membrane physics impact recombinant protein secretion in Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Mattanovich Diethard

    2011-11-01

    Full Text Available Abstract Background The increasing availability of 'omics' databases provide important platforms for yeast engineering strategies since they offer a lot of information on the physiology of the cells under diverse growth conditions, including environmental stresses. Notably, only a few of these approaches have considered a performance under recombinant protein production conditions. Recently, we have identified a beneficial effect of low oxygen availability on the expression of a human Fab fragment in Pichia pastoris. Transcriptional analysis and data mining allowed for the selection of potential targets for strain improvement. A first selection of these candidates has been evaluated as recombinant protein secretion enhancers. Results Based on previous transcriptomics analyses, we selected 8 genes for co-expression in the P. pastoris strain already secreting a recombinant Fab fragment. Notably, WSC4 (which is involved in trafficking through the ER has been identified as a novel potential target gene for strain improvement, with up to a 1.2-fold increase of product yield in shake flask cultures. A further transcriptomics-based strategy to modify the yeast secretion system was focused on the ergosterol pathway, an aerobic process strongly affected by oxygen depletion. By specifically partially inhibiting ergosterol synthesis with the antifungal agent fluconazole (inhibiting Erg11p, we tried to mimic the hypoxic conditions, in which the cellular ergosterol content was significantly decreased. This strategy led to an improved Fab yield (2-fold without impairing cellular growth. Since ergosterol shortage provokes alterations in the plasma membrane composition, an important role of this cellular structure in protein secretion is suggested. This hypothesis was additionally supported by the fact that the addition of non-ionic surfactants also enhanced Fab secretion. Conclusions The current study presents a systems biotechnology-based strategy for the

  1. New insights into the evolutionary origins of the recombination-activating gene proteins and V(D)J recombination.

    Science.gov (United States)

    Carmona, Lina Marcela; Schatz, David G

    2017-06-01

    The adaptive immune system of jawed vertebrates relies on V(D)J recombination as one of the main processes to generate the diverse array of receptors necessary for the recognition of a wide range of pathogens. The DNA cleavage reaction necessary for the assembly of the antigen receptor genes from an array of potential gene segments is mediated by the recombination-activating gene proteins RAG1 and RAG2. The RAG proteins have been proposed to originate from a transposable element (TE) as they share mechanistic and structural similarities with several families of transposases and are themselves capable of mediating transposition. A number of RAG-like proteins and TEs with sequence similarity to RAG1 and RAG2 have been identified, but only recently has their function begun to be characterized, revealing mechanistic links to the vertebrate RAGs. Of particular significance is the discovery of ProtoRAG, a transposon superfamily found in the genome of the basal chordate amphioxus. ProtoRAG has many of the sequence and mechanistic features predicted for the ancestral RAG transposon and is likely to be an evolutionary relative of RAG1 and RAG2. In addition, early observations suggesting that RAG1 is able to mediate V(D)J recombination in the absence of RAG2 have been confirmed, implying independent evolutionary origins for the two RAG genes. Here, recent progress in identifying and characterizing RAG-like proteins and the TEs that encode them is summarized and a refined model for the evolution of V(D)J recombination and the RAG proteins is presented. © 2016 Federation of European Biochemical Societies.

  2. Recombinant Protein Expression in Escherichia coli (E.coli): What We Need to Know.

    Science.gov (United States)

    Hayat, Seyed Mohammad Gheibi; Farahani, Najmeh; Golichenari, Behrouz; Sahebkar, Amir Hosein

    2018-01-31

    Host, vector, and culture conditions (including cultivation media) are considered among the three main elements contributing to a successful production of recombinant proteins. Accordingly, one of the most common hosts to produce recombinant therapeutic proteins is Escherichia coli. A comprehensive literature review was performed to identify important factors affecting production of recombinant proteins in Escherichia coli. Escherichia coli is taken into account as the easiest, quickest, and cheapest host with a fully known genome. Thus, numerous modifications have been carried out on Escherichia coli to optimize it as a good candidate for protein expression and; as a result, several engineered strains of Escherichia coli have been designed. In general; host strain, vector, and cultivation parameters are recognized as crucial ones determining success of recombinant protein expression in Escherichia coli. In this review, the role of host, vector, and culture conditions along with current pros and cons of different types of these factors leading to success of recombinant protein expression in Escherichia coli were discussed. Successful protein expression in Escherichia coli necessitates a broad knowledge about physicochemical properties of recombinant proteins, selection among common strains of Escherichia coli and vectors, as well as factors related to media including time, temperature, and inducer. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  3. Investigating the dynamics of recombinant protein secretion from a microalgal host.

    Science.gov (United States)

    Lauersen, Kyle J; Huber, Isabel; Wichmann, Julian; Baier, Thomas; Leiter, Andreas; Gaukel, Volker; Kartushin, Viktor; Rattenholl, Anke; Steinweg, Christian; von Riesen, Lena; Posten, Clemens; Gudermann, Frank; Lütkemeyer, Dirk; Mussgnug, Jan H; Kruse, Olaf

    2015-12-10

    Production of recombinant proteins with microalgae represents an alternative platform over plant- or bacterial-based expression systems for certain target proteins. Secretion of recombinant proteins allows accumulation of the target product physically separate from the valuable algal biomass. To date, there has been little investigation into the dynamics of recombinant protein secretion from microalgal hosts-the culture parameters that encourage secreted product accumulation and stability, while encouraging biomass production. In this work, the efficiency of recombinant protein production was optimized by adjusting cultivation parameters for a strain of Chlamydomonas reinhardtii previously engineered to secrete a functional recombinant Lolium perenne ice binding protein (LpIBP), which has applications as a frozen food texturing and cryopreservation additive, into its culture medium. Three media and several cultivation styles were investigated for effects on secreted LpIBP titres and culture growth. A combination of acetate and carbon dioxide feeding with illumination resulted in the highest overall biomass and recombinant protein titres up to 10mgL(-1) in the culture medium. Pure photoautotrophic production was possible using two media types, with recombinant protein accumulation in all cultivations correlating to culture cell density. Two different cultivation systems were used for scale-up to 10L cultivations, one of which produced yields of secreted recombinant protein up to 12mgL(-1) within six cultivation days. Functional ice recrystallization inhibition (IRI) of the LpIBP from total concentrated extracellular protein extracts was demonstrated in a sucrose solution used as a simplified ice cream model. IRI lasted up to 7 days, demonstrating the potential of secreted products from microalgae for use as food additives. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Expression and characterization of recombinant single-chain salmon class I MHC fused with beta2-microglobulin with biological activity

    DEFF Research Database (Denmark)

    Zhao, Heng; Stet, René J M; Skjødt, Karsten

    2008-01-01

    Heterodimeric class I major histocompatibility complex (MHC) molecules consist of a putative 45-kDa heavy chain and a 12-kDa beta2-microglobulin (beta2m) light chain. The knowledge about MHC genes in Atlantic salmon accumulated during the last decade has allowed us to generate soluble and stable ...... MHC class I molecules with biological activity. We report here the use of a bacterial expression system to produce the recombinant single-chain MHC molecules based on a specific allele Sasa-UBA*0301. This particular allele was selected because previous work has shown its association...... antibodies were successfully produced against both the MHC class I heavy chain and beta(2)m, and showed binding to the recombinant molecule. The recombinant complex Sasabeta2mUBA*0301 was expressed and isolated; the production was scaled up by adjusting to its optimal conditions. Subsequently......, the recombinant proteins were purified by affinity chromatography using mAb against beta2m and alpha3. Eluates were analyzed by Western blot and refolded by the removal of denaturant. The correct folding was confirmed by measuring its binding capacity against mAb produced to recognize the native form of MHC...

  5. [Adenovirus mediated expression of recombinant human single chain interleukin-27(rhscIL-27) fusion gene in hepatoma cells].

    Science.gov (United States)

    Cheng, Xiao-gang; Zhang, Ya-qing; Ye, Wan; Zou, Qiang; Chen, Wei; Jin, Hong; Xu, Yan; Zhang, Shao-lan

    2010-06-01

    To explore the adenovirus mediated expression of recombinant human single chain interleukin-27(rhscIL-27) fusion gene in hepatoma cells. The rhscIL-27 fusion gene was subcloned into the shuttle plasmid pAdTrack-CMV and then clone the homologous recombinant adenovirus genomic plasmid pAdEasy in bacteria. The identified recombinant plasmid AdIL-27 was tranfected into 293 cells, and then the adenovirus did the package and amplification. The HepG2 cells were infected with AdIL-27 and the target gene expression was determined by RT-PCR and ELISA. The biological activity of rhscIL-27 was detected by IFN-gamma inducing assay. Restriction endonuclease and gene sequencing confirmed that the recombinant adenovirus vector of rhscIL-27 fusion gene was successfully constructed. The expression of rhscIL-27 fusion gene was observed at 48 h after the transfection of the HepG2 cells with AdIL-27. The IFN-gamma inducing assay showed that the rhscIL-27 protein has the ability inducing IFN-gamma secretion. By using adenovirus expression system, rhscIL-27 fusion gene with biological activity is expressed successfully in hepatoma cells. This experiment laid a foundation for gene therapy of hepatoma with IL-27.

  6. Infectious mutants of cassava latent virus generated in vivo from intact recombinant DNA clones containing single copies of the genome.

    Science.gov (United States)

    Stanley, J; Townsend, R

    1986-08-11

    Intact recombinant DNAs containing single copies of either component of the cassava latent virus genome can elicit infection when mechanically inoculated to host plants in the presence of the appropriate second component. Characterisation of infectious mutant progeny viruses, by analysis of virus-specific supercoiled DNA intermediates, indicates that most if not all of the cloning vector has been deleted, achieved at least in some cases by intermolecular recombination in vivo between DNAs 1 and 2. Significant rearrangements within the intergenic region of DNA 2, predominantly external to the common region, can be tolerated without loss of infectivity suggesting a somewhat passive role in virus multiplication for the sequences in question. Although packaging constraints might impose limits on the amount of DNA within geminate particles, isolation of an infectious coat protein mutant defective in virion production suggests that packaging is not essential for systemic spread of the viral DNA.

  7. Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF.

    Science.gov (United States)

    Cantu-Bustos, J Enrique; Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Galbraith, David W; McEvoy, Megan M; Zarate, Xristo

    2016-05-01

    Production of recombinant proteins in Escherichia coli has been improved considerably through the use of fusion proteins, because they increase protein solubility and facilitate purification via affinity chromatography. In this article, we propose the use of CusF as a new fusion partner for expression and purification of recombinant proteins in E. coli. Using a cell-free protein expression system, based on the E. coli S30 extract, Green Fluorescent Protein (GFP) was expressed with a series of different N-terminal tags, immobilized on self-assembled protein microarrays, and its fluorescence quantified. GFP tagged with CusF showed the highest fluorescence intensity, and this was greater than the intensities from corresponding GFP constructs that contained MBP or GST tags. Analysis of protein production in vivo showed that CusF produces large amounts of soluble protein with low levels of inclusion bodies. Furthermore, fusion proteins can be exported to the cellular periplasm, if CusF contains the signal sequence. Taking advantage of its ability to bind copper ions, recombinant proteins can be purified with readily available IMAC resins charged with this metal ion, producing pure proteins after purification and tag removal. We therefore recommend the use of CusF as a viable alternative to MBP or GST as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Genetic Structures and Conditions of their Expression, which Allow Receiving Native Recombinant Proteins with High Output

    OpenAIRE

    Michael M. Shavlovsky, PhD, ScD¹; Irina V. Morozova¹; Dmitry S. Polyakov, PhD¹; Tatyana D. Aleynikova, PhD¹; Anna M. Kern²; Natalya A. Grudinina, PhD¹; Kirill V. Solovyov, PhD¹

    2012-01-01

    We investigated the possibility of obtaining native recombinant amyloidogenic proteins by creating genetic constructs encoding fusion proteins of target proteins with Super Folder Green Fluorescent Protein (sfGFP). In this study, we show that the structures, containing the sfGFP gene, provide a synthesis, within a bacterial system, of fusion proteins with minimal formation of inclusion bodies. Constructs containing genes of the target proteins in the 3'-terminal region of the sfGFP gene follo...

  9. Recombination-stable multimeric green fluorescent protein for characterization of weak promoter outputs in Saccharomyces cerevisiae.

    Science.gov (United States)

    Rugbjerg, Peter; Knuf, Christoph; Förster, Jochen; Sommer, Morten O A

    2015-12-01

    Green fluorescent proteins (GFPs) are widely used for visualization of proteins to track localization and expression dynamics. However, phenotypically important processes can operate at too low expression levels for routine detection, i.e. be overshadowed by autofluorescence noise. While GFP functions well in translational fusions, the use of tandem GFPs to amplify fluorescence signals is currently avoided in Saccharomyces cerevisiae and many other microorganisms due to the risk of loop-out by direct-repeat recombination. We increased GFP fluorescence by translationally fusing three different GFP variants, yeast-enhanced GFP, GFP+ and superfolder GFP to yield a sequence-diverged triple GFP molecule 3vGFP with 74-84% internal repeat identity. Unlike a single GFP, the brightness of 3vGFP allowed characterization of a weak promoter in S. cerevisiae. Utilizing 3vGFP, we further engineered a less leaky Cu(2+)-inducible promoter based on CUP1. The basal expression level of the new promoter was approximately 61% below the wild-type CUP1 promoter, thus expanding the absolute range of Cu(2+)-based gene control. The stability of 3vGFP towards direct-repeat recombination was assayed in S. cerevisiae cultured for 25 generations under strong and slightly toxic expression after which only limited reduction in fluorescence was detectable. Such non-recombinogenic GFPs can help quantify intracellular responses operating a low copy number in recombination-prone organisms. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  10. Recombinant Protein Production and Insect Cell Culture and Process

    Science.gov (United States)

    Spaulding, Glenn F. (Inventor); Goodwin, Thomas J. (Inventor); OConnor, Kim C. (Inventor); Francis, Karen M. (Inventor); Andrews, Angela D. (Inventor); Prewett, Tracey L. (Inventor)

    1997-01-01

    A process has been developed for recombinant production of selected polypeptides using transformed insect cells cultured in a horizontally rotating culture vessel modulated to create low shear conditions. A metabolically transformed insect cell line is produced using the culture procedure regardless of genetic transformation. The recombinant polypeptide can be produced by an alternative process using virtually infected or stably transformed insect cells containing a gene encoding the described polypeptide. The insect cells can also be a host for viral production.

  11. Stabilizing Additives Added during Cell Lysis Aid in the Solubilization of Recombinant Proteins

    Science.gov (United States)

    Leibly, David J.; Nguyen, Trang Nhu; Kao, Louis T.; Hewitt, Stephen N.; Barrett, Lynn K.; Van Voorhis, Wesley C.

    2012-01-01

    Insoluble recombinant proteins are a major issue for both structural genomics and enzymology research. Greater than 30% of recombinant proteins expressed in Escherichia coli (E. coli) appear to be insoluble. The prevailing view is that insolubly expressed proteins cannot be easily solubilized, and are usually sequestered into inclusion bodies. However, we hypothesize that small molecules added during the cell lysis stage can yield soluble protein from insoluble protein previously screened without additives or ligands. We present a novel screening method that utilized 144 additive conditions to increase the solubility of recombinant proteins expressed in E. coli. These selected additives are natural ligands, detergents, salts, buffers, and chemicals that have been shown to increase the stability of proteins in vivo. We present the methods used for this additive solubility screen and detailed results for 41 potential drug target recombinant proteins from infectious organisms. Increased solubility was observed for 80% of the recombinant proteins during the primary and secondary screening of lysis with the additives; that is 33 of 41 target proteins had increased solubility compared with no additive controls. Eleven additives (trehalose, glycine betaine, mannitol, L-Arginine, potassium citrate, CuCl2, proline, xylitol, NDSB 201, CTAB and K2PO4) solubilized more than one of the 41 proteins; these additives can be easily screened to increase protein solubility. Large-scale purifications were attempted for 15 of the proteins using the additives identified and eight (40%) were prepared for crystallization trials during the first purification attempt. Thus, this protocol allowed us to recover about a third of seemingly insoluble proteins for crystallography and structure determination. If recombinant proteins are required in smaller quantities or less purity, the final success rate may be even higher. PMID:23285060

  12. Stabilizing additives added during cell lysis aid in the solubilization of recombinant proteins.

    Directory of Open Access Journals (Sweden)

    David J Leibly

    Full Text Available Insoluble recombinant proteins are a major issue for both structural genomics and enzymology research. Greater than 30% of recombinant proteins expressed in Escherichia coli (E. coli appear to be insoluble. The prevailing view is that insolubly expressed proteins cannot be easily solubilized, and are usually sequestered into inclusion bodies. However, we hypothesize that small molecules added during the cell lysis stage can yield soluble protein from insoluble protein previously screened without additives or ligands. We present a novel screening method that utilized 144 additive conditions to increase the solubility of recombinant proteins expressed in E. coli. These selected additives are natural ligands, detergents, salts, buffers, and chemicals that have been shown to increase the stability of proteins in vivo. We present the methods used for this additive solubility screen and detailed results for 41 potential drug target recombinant proteins from infectious organisms. Increased solubility was observed for 80% of the recombinant proteins during the primary and secondary screening of lysis with the additives; that is 33 of 41 target proteins had increased solubility compared with no additive controls. Eleven additives (trehalose, glycine betaine, mannitol, L-Arginine, potassium citrate, CuCl(2, proline, xylitol, NDSB 201, CTAB and K(2PO(4 solubilized more than one of the 41 proteins; these additives can be easily screened to increase protein solubility. Large-scale purifications were attempted for 15 of the proteins using the additives identified and eight (40% were prepared for crystallization trials during the first purification attempt. Thus, this protocol allowed us to recover about a third of seemingly insoluble proteins for crystallography and structure determination. If recombinant proteins are required in smaller quantities or less purity, the final success rate may be even higher.

  13. Escherichia coli fusion carrier proteins act as solubilizing agents for recombinant uncoupling protein 1 through interactions with GroEL

    International Nuclear Information System (INIS)

    Douette, Pierre; Navet, Rachel; Gerkens, Pascal; Galleni, Moreno; Levy, Daniel; Sluse, Francis E.

    2005-01-01

    Fusing recombinant proteins to highly soluble partners is frequently used to prevent aggregation of recombinant proteins in Escherichia coli. Moreover, co-overexpression of prokaryotic chaperones can increase the amount of properly folded recombinant proteins. To understand the solubility enhancement of fusion proteins, we designed two recombinant proteins composed of uncoupling protein 1 (UCP1), a mitochondrial membrane protein, in fusion with MBP or NusA. We were able to express soluble forms of MBP-UCP1 and NusA-UCP1 despite the high hydrophobicity of UCP1. Furthermore, the yield of soluble fusion proteins depended on co-overexpression of GroEL that catalyzes folding of polypeptides. MBP-UCP1 was expressed in the form of a non-covalent complex with GroEL. MBP-UCP1/GroEL was purified and characterized by dynamic light scattering, gel filtration, and electron microscopy. Our findings suggest that MBP and NusA act as solubilizing agents by forcing the recombinant protein to pass through the bacterial chaperone pathway in the context of fusion protein

  14. Side effects of chaperone gene co-expression in recombinant protein production

    Directory of Open Access Journals (Sweden)

    Martínez-Alonso Mónica

    2010-09-01

    Full Text Available Abstract Insufficient availability of molecular chaperones is observed as a major bottleneck for proper protein folding in recombinant protein production. Therefore, co-production of selected sets of cell chaperones along with foreign polypeptides is a common approach to increase the yield of properly folded, recombinant proteins in bacterial cell factories. However, unbalanced amounts of folding modulators handling folding-reluctant protein species might instead trigger undesired proteolytic activities, detrimental regarding recombinant protein stability, quality and yield. This minireview summarizes the most recent observations of chaperone-linked negative side effects, mostly focusing on DnaK and GroEL sets, when using these proteins as folding assistant agents. These events are discussed in the context of the complexity of the cell quality network and the consequent intricacy of the physiological responses triggered by protein misfolding.

  15. Microalgae as platforms for production of recombinant proteins and valuable compounds: progress and prospects.

    Science.gov (United States)

    Gong, Yangmin; Hu, Hanhua; Gao, Yuan; Xu, Xudong; Gao, Hong

    2011-12-01

    Over the last few years microalgae have gained increasing interest as a natural source of valuable compounds and as bioreactors for recombinant protein production. Natural high-value compounds including pigments, long-chain polyunsaturated fatty acids, and polysaccharides, which have a wide range of applications in the food, feed, cosmetics, and pharmaceutical industries, are currently produced with nontransgenic microalgae. However, transgenic microalgae can be used as bioreactors for the production of therapeutic and industrially relevant recombinant proteins. This technology shows great promise to simplify the production process and significantly decrease the production costs. To date, a variety of recombinant proteins have been produced experimentally from the nuclear or chloroplast genome of transgenic Chlamydomonas reinhardtii. These include monoclonal antibodies, vaccines, hormones, pharmaceutical proteins, and others. In this review, we outline recent progress in the production of recombinant proteins with transgenic microalgae as bioreactors, methods for genetic transformation of microalgae, and strategies for highly efficient expression of heterologous genes. In particular, we highlight the importance of maximizing the value of transgenic microalgae through producing recombinant proteins together with recovery of natural high-value compounds. Finally, we outline some important issues that need to be addressed before commercial-scale production of high-value recombinant proteins and compounds from transgenic microalgae can be realized.

  16. High-yield secretion of recombinant proteins from the microalga Chlamydomonas reinhardtii

    DEFF Research Database (Denmark)

    Ramos Martinez, Erick Miguel; Fimognari, Lorenzo; Sakuragi, Yumiko

    2017-01-01

    Microalga-based biomanufacturing of recombinant proteins is attracting growing attention due to its advantages in safety, metabolic diversity, scalability and sustainability. Secretion of recombinant proteins can accelerate the use of microalgal platforms by allowing post-translational modificati......Microalga-based biomanufacturing of recombinant proteins is attracting growing attention due to its advantages in safety, metabolic diversity, scalability and sustainability. Secretion of recombinant proteins can accelerate the use of microalgal platforms by allowing post......-translational modifications and easy recovery of products from the culture media. However, currently, the yields of secreted recombinant proteins are low, which hampers the commercial application of this strategy. This study aimed at expanding the genetic tools for enhancing secretion of recombinant proteins in Chlamydomonas...... reinhardtii, a widely used green microalga as a model organism and a potential industrial biotechnology platform. We demonstrated that the putative signal sequence from C. reinhardtii gametolysin can assist the secretion of the yellow fluorescent protein Venus into the culture media. To increase the secretion...

  17. Refolding techniques for recovering biologically active recombinant proteins from inclusion bodies.

    Science.gov (United States)

    Yamaguchi, Hiroshi; Miyazaki, Masaya

    2014-02-20

    Biologically active proteins are useful for studying the biological functions of genes and for the development of therapeutic drugs and biomaterials in a biotechnology industry. Overexpression of recombinant proteins in bacteria, such as Escherichia coli, often results in the formation of inclusion bodies, which are protein aggregates with non-native conformations. As inclusion bodies contain relatively pure and intact proteins, protein refolding is an important process to obtain active recombinant proteins from inclusion bodies. However, conventional refolding methods, such as dialysis and dilution, are time consuming and, often, recovered yields of active proteins are low, and a trial-and-error process is required to achieve success. Recently, several approaches have been reported to refold these aggregated proteins into an active form. The strategies largely aim at reducing protein aggregation during the refolding procedure. This review focuses on protein refolding techniques using chemical additives and laminar flow in microfluidic chips for the efficient recovery of active proteins from inclusion bodies.

  18. Recombinant human CIS2 (SOCS2) protein: subcloning, expression, purification, and characterization.

    Science.gov (United States)

    Biener, Eva; Maurice, Sarah; Sandowski, Yael; Cohen, Yael; Gusakowsky, Eugene E; Hooghe, Robert; Yoshimura, Akihiko; Livnah, Oded; Gertler, Arieh

    2002-07-01

    The 1x myc-tagged cDNA encoding for human CIS2 protein was subcloned into a pET-29a+ vector in order to express and produce a recombinant S-peptide tagged and 1x myc-tagged protein in Escherichia coli BL21(DE3). The constitutively expressed protein was isolated from inclusion bodies by a simple solubilization-renaturation procedure and purified by anion-exchange chromatography on Q-Sepharose. The recombinant form was found to be pure and monomeric as judged by both SDS-PAGE and gel-filtration chromatography and its biological activity was proven by its ability to bind to the tyrosine-phosphorylated cytosolic fragment of human growth hormone receptor fused to glutathione-S-transferase. Recombinant CIS2 was compared by biochemical, immunological, and molecular methods to the CIS2 protein expressed in eukaryotic cells. This report describes the first substantial production of biologically active recombinant human CIS2.

  19. Analysis of the ligand binding properties of recombinant bovine liver-type fatty acid binding protein

    DEFF Research Database (Denmark)

    Rolf, B; Oudenampsen-Krüger, E; Börchers, T

    1995-01-01

    The coding part of the cDNA for bovine liver-type fatty acid binding protein (L-FABP) has been amplified by RT-PCR, cloned and used for the construction of an Escherichia coli (E. coli) expression system. The recombinant protein made up to 25% of the soluble E. coli proteins and could be isolated...

  20. Combinations of SPR and MS for Characterizations of Native and Recombinant Proteins in Cell Lysates

    DEFF Research Database (Denmark)

    Borch, Jonas; Roepstorff, Peter

    2006-01-01

    of 14-3-3 proteins in order to test their activity. Specific binding of recombinant and native 14-3-3 proteins in complex mixtures to immobilized phosphopeptides and subsequent elution was also tested by SPR-MS. Ammonium sulfate precipitate fractions from lysates of E. coli expressing 14-3-3 protein...

  1. Systems and methods for the secretion of recombinant proteins in gram negative bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Withers, III, Sydnor T.; Dominguez, Miguel A.; DeLisa, Matthew P.; Haitjema, Charles H.

    2017-02-21

    Disclosed herein are systems and methods for producing recombinant proteins utilizing mutant E. coli strains containing expression vectors carrying nucleic acids encoding the proteins, and secretory signal sequences to direct the secretion of the proteins to the culture medium. Host cells transformed with the expression vectors are also provided.

  2. Recombinant protein production data after expression in the bacterium Escherichia coli

    Directory of Open Access Journals (Sweden)

    J. Enrique Cantu-Bustos

    2016-06-01

    Full Text Available Fusion proteins have become essential for the expression and purification of recombinant proteins in Escherichia coli. The metal-binding protein CusF has shown several features that make it an attractive fusion protein and affinity tag: "Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF" (Cantu-Bustos et al., 2016 [1]. Here we present accompanying data from protein expression experiments; we tested different protein tags, temperatures, expression times, cellular compartments, and concentrations of inducer in order to obtain soluble protein and low formation of inclusion bodies. Additionally, we present data from the purification of the green fluorescent protein (GFP tagged with CusF, using Ag(I metal affinity chromatography.

  3. Effect of recombinant Ganoderma lucidum immunoregulatory protein on cyclophosphamide-induced leukopenia in mice.

    Science.gov (United States)

    Zhou, Hui; Sun, Fei; Li, Hongrui; Zhang, Shuqin; Liu, Zhiyi; Pei, Jin; Liang, Chongyang

    2013-06-01

    The aim of the study is to investigate the effect of recombinant Ganoderma lucidum immunoregulatory protein (rLZ-8) on mouse models of cyclophosphamide-induced leukopenia, which we have established with both single-phase and multi-phase administration methods. Treatment with rLZ-8 had a strong effect on both models of cyclophosphamide-induced leukopenia. In particular, it increased the number of neutrophils, lymphocytes and monocytes. rLZ-8 treatment also increased the percentage of CD4(+) T cells and the levels of secreted IL-3 and IL-4, which contributed to the cyclophosphamide-induced immune dysfunction and immune system imbalance. In conclusion, rLZ-8 treatment benefitted mice with cyclophosphamide-induced leukopenia by improving overall immune function and by specifically increasing the number of white blood cells.

  4. 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

  5. Improving recombinant protein production in the Chlamydomonas reinhardtii chloroplast using vivid Verde Fluorescent Protein as a reporter.

    Science.gov (United States)

    Braun-Galleani, Stephanie; Baganz, Frank; Purton, Saul

    2015-08-01

    Microalgae have potential as platforms for the synthesis of high-value recombinant proteins due to their many beneficial attributes including ease of cultivation, lack of pathogenic agents, and low-cost downstream processing. However, current recombinant protein levels are low compared to other microbial platforms and stable insertion of transgenes is available in only a few microalgal species. We have explored different strategies aimed at increasing growth rate and recombinant protein production in the Chlamydomonas reinhardtii chloroplast. A novel fluorescent protein (vivid Verde Fluorescent Protein, VFP) was expressed under the control of the native atpA promoter/5'UTR element. VFP levels were detected by western blotting, with increased protein levels observed when co-expressed with a gene encoding the Escherichia coli Spy chaperone. We used these transformant lines to study the effect of temperature, light and media on recombinant protein production and cell growth. VFP levels and fluorescence, assessed by flow cytometry, allowed a determination of improved cultivation conditions as 30°C under mixotrophic mode. These conditions were tested for the accumulation of an antimicrobial endolysin (Cpl-1) of potential commercial interest, observing that the outcome obtained for VFP could not be easily replicated for Cpl-1. This study suggests that recombinant protein expression is product-specific and needs to be optimized individually. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Exploiting translational coupling for the selection of cells producing toxic recombinant proteins from expression vectors.

    Science.gov (United States)

    Tagliavia, Marcello; Cuttitta, Angela

    2016-01-01

    High rates of plasmid instability are associated with the use of some expression vectors in Escherichia coli, resulting in the loss of recombinant protein expression. This is due to sequence alterations in vector promoter elements caused by the background expression of the cloned gene, which leads to the selection of fast-growing, plasmid-containing cells that do not express the target protein. This phenomenon, which is worsened when expressing toxic proteins, results in preparations containing very little or no recombinant protein, or even in clone loss; however, no methods to prevent loss of recombinant protein expression are currently available. We have exploited the phenomenon of translational coupling, a mechanism of prokaryotic gene expression regulation, in order to select cells containing plasmids still able to express recombinant proteins. Here we designed an expression vector in which the cloned gene and selection marker are co-expressed. Our approach allowed for the selection of the recombinant protein-expressing cells and proved effective even for clones encoding toxic proteins.

  7. Evaluation of antibody response against recombinant domain III proteins of dengue virus type 1 and 2

    Directory of Open Access Journals (Sweden)

    Nagesh K Tripathi

    2017-04-01

    Full Text Available Dengue, a mosquito borne viral disease caused by dengue virus has emerged as a major health problem during the last few decades. The envelope domain III (DIII protein of dengue virus is highly immunogenic and capable of inducing neutralizing antibodies against wild-type dengue virus. The envelope domain III protein is a potential subunit vaccine candidate as well as a diagnostic reagent for dengue. This report describes the high yield production and immunogenicity of recombinant DIII proteins of dengue virus type 1 and 2. The subunit DIII proteins were produced in Escherichia coli using batch and fed-batch fermentation process. Immobilized metal affinity chromatography was used to capture DIII proteins of dengue virus type 1 and 2. The purified proteins were refolded by diafiltration to achieve biologically active proteins. After fed-batch fermentation, the recombinant E. coli resulted in purified DIII proteins of about 10.06 mg and 47.70 mg per gram of dry cell weight for recombinant dengue virus type 1 and 2 respectively with more than 95% purity. Biological function of the purified DIII proteins were confirmed by their ability to generate DIII specific antibodies in mice. The DIII antigens in combination with adjuvant resulted antibody endpoint titers of 1:64,000 and 1:1,28,000 for recombinant dengue virus type 1 and 2 respectively. These findings establish that the DIII proteins in combination with adjuvant are immunogenic, which suggests that refolded and purified DIII proteins can be a potential vaccine candidates.

  8. The synthesis of recombinant membrane proteins in yeast for structural studies.

    Science.gov (United States)

    Routledge, Sarah J; Mikaliunaite, Lina; Patel, Anjana; Clare, Michelle; Cartwright, Stephanie P; Bawa, Zharain; Wilks, Martin D B; Low, Floren; Hardy, David; Rothnie, Alice J; Bill, Roslyn M

    2016-02-15

    Historically, recombinant membrane protein production has been a major challenge meaning that many fewer membrane protein structures have been published than those of soluble proteins. However, there has been a recent, almost exponential increase in the number of membrane protein structures being deposited in the Protein Data Bank. This suggests that empirical methods are now available that can ensure the required protein supply for these difficult targets. This review focuses on methods that are available for protein production in yeast, which is an important source of recombinant eukaryotic membrane proteins. We provide an overview of approaches to optimize the expression plasmid, host cell and culture conditions, as well as the extraction and purification of functional protein for crystallization trials in preparation for structural studies. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  9. Cloning and Expression of Soluble Recombinant HIV-1 CRF35 Protease-HP Thioredoxin Fusion Protein.

    Science.gov (United States)

    Azarnezhad, Asaad; Sharifi, Zohreh; Seyedabadi, Rahmatollah; Hosseini, Arshad; Johari, Behrooz; Sobhani Fard, Mahsa

    2016-01-01

    As a drug target and an antigenic agent, HIV-1 protease (HIV-1 PR) is at the center of attention for designing anti-AIDS inhibitors and diagnostic tests. In previous studies, the production of the recombinant protease has been faced with several difficulties; therefore, the aims of this study were the easy production, purification of the soluble form of protease in E. coli and investigation of its immunoreactivity. Protease coding region was isolated from the serum of an infected individual, amplified by RT-PCR and cloned into PTZ57R using TA-cloning. Protease coding frame was isolated by PCR and cloned in pET102/D. TOPO expression vector and cloned protease was expressed in Escherichia coli ( E. coli) BL21 . Produced recombinant protein was purified by affinity Ni-NTA column and protein concentration was checked by BCA protein assay kit. Subsequently, immunoreactivity of recombinant protease (rPR) was assayed by Western blotting and ELISA. Cloning of the HIV protease by TOPO cloning system in pET102/D.TOPO was confirmed with PCR and sequencing. The concentration range of purified recombinant protein was 85 to 100 μg/ml . Immunogenicity of rPR was confirmed by Western blotting and ELISA. Soluble production of recombinant HIV-1 protease (HIV-1 rPR) was performed successfully. This recombinant protein disclosed 86% specificity and 90% sensitivity in immunoassay tests.

  10. Design and characterization of novel recombinant listeriolysin O-protamine fusion proteins for enhanced gene delivery.

    Science.gov (United States)

    Kim, Na Hyung; Provoda, Chester; Lee, Kyung-Dall

    2015-02-02

    To improve the efficiency of gene delivery for effective gene therapy, it is essential that the vector carries functional components that can promote overcoming barriers in various steps leading to the transport of DNA from extracellular to ultimately nuclear compartment. In this study, we designed genetically engineered fusion proteins as a platform to incorporate multiple functionalities in one chimeric protein. Prototypes of such a chimera tested here contain two domains: one that binds to DNA; the other that can facilitate endosomal escape of DNA. The fusion proteins are composed of listeriolysin O (LLO), the endosomolytic pore-forming protein from Listeria monocytogenes, and a 22 amino acid sequence of the DNA-condensing polypeptide protamine (PN), singly or as a pair: LLO-PN and LLO-PNPN. We demonstrate dramatic enhancement of the gene delivery efficiency of protamine-condensed DNA upon incorporation of a small amount of LLO-PN fusion protein and further improvement with LLO-PNPN in vitro using cultured cells. Additionally, the association of anionic liposomes with cationic LLO-PNPN/protamine/DNA complexes, yielding a net negative surface charge, resulted in better in vitro transfection efficiency in the presence of serum. An initial, small set of data in mice indicated that the observed enhancement in gene expression could also be applicable to in vivo gene delivery. This study suggests that incorporation of a recombinant fusion protein with multiple functional components, such as LLO-protamine fusion protein, in a nonviral vector is a promising strategy for various nonviral gene delivery systems.

  11. High-level expression of Staphylococcal Protein A in Pichia pastoris and purification and characterization of the recombinant protein.

    Science.gov (United States)

    Hao, Jing; Xu, Li; He, Hongde; Du, Xiaojun; Jia, Lingyun

    2013-08-01

    Staphylococcal Protein A (SPA), a cell wall protein of Staphylococcus aureus, is in high demand because of its ability to bind immunoglobulins. Much of the SPA that we use today is recombinant SPA (rSPA), which is produced in Escherichia coli. As rSPA is obtained by expressing SPA as an intracellular protein, its purification is tedious and time consuming. In order to obtain a large amount of highly purified rSPA with relative ease, we expressed SPA as a secretory form in the yeast Pichia pastoris. To increase the expression level of SPA and repress its proteolysis during fermentation, the cell density (OD600), temperature and pH at which SPA expression was induced as well as the induction time were optimized. The final yield of SPA obtained was about 8.8 g per liter of culture, which under the optimized fermentation condition, accounted for 80% of the total protein in the culture supernatant. The expressed SPA was purified from the culture supernatant by DEAE ion-exchange chromatography (IEC) after the supernatant was subjected to a desalting step. The purified SPA was resolved as a single band by SDS-PAGE and as a single peak by HPLC. Its identity was confirmed by MALDI-TOF MS and western-blot. Moreover, the protein also exhibited excellent affinity for IgG when tested with human IgG. The production and purification of SPA described in this study offers a new method for obtaining high level of SPA in relatively pure form that is suitable for practical application. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. Production of Recombinant CCN Proteins by Brevibacillus choshinensis.

    Science.gov (United States)

    Hanagata, Hiroshi; Mizukami, Makoto

    2017-01-01

    Brevibacillus choshinensis is an excellent host for the production of secretory proteins. This host has also been applied successfully to efficient production of CCN proteins. Described herein are methods of constructing plasmids for CCN protein production (IGFBP-, VWC-, TSP-, and CT-domain) with Brevibacillus as a host, cultivation methods for protein production, and methods of purification for domain proteins using his-tag.

  13. Watching Single Proteins Using Engineered Nanopores

    Science.gov (United States)

    Movileanu, Liviu

    2014-01-01

    Recent studies in the area of single-molecule detection of proteins with nanopores show a great promise in fundamental science, bionanotechnology and proteomics. In this mini-review, I discuss a comprehensive array of examinations of protein detection and characterization using protein and solid-state nanopores. These investigations demonstrate the power of the single-molecule nanopore measurements to reveal a broad range of functional, structural, biochemical and biophysical features of proteins, such as their backbone flexibility, enzymatic activity, binding affinity as well as their concentration, size and folding state. Engineered nanopores in organic materials and in inorganic membranes coupled with surface modification and protein engineering might provide a new generation of sensing devices for molecular biomedical diagnosis. PMID:24370252

  14. Production of recombinant dengue non-structural 1 (NS1) proteins from clinical virus isolates.

    Science.gov (United States)

    Yohan, Benediktus; Wardhani, Puspa; Aryati; Trimarsanto, Hidayat; Sasmono, R Tedjo

    2017-01-01

    Dengue is a febrile disease caused by infection of dengue virus (DENV). Early diagnosis of dengue infection is important for better management of the disease. The DENV Non-Structural Protein 1 (NS1) antigen has been routinely used for the early dengue detection. In dengue epidemic countries such as Indonesia, clinicians are increasingly relying on the NS1 detection for confirmation of dengue infection. Various NS1 diagnostic tests are commercially available, however different sensitivities and specificities were observed in various settings. This study was aimed to generate dengue NS1 recombinant protein for the development of dengue diagnostic tests. Four Indonesian DENV isolates were used as the source of the NS1 gene cloning, expression, and purification in bacterial expression system. Recombinant NS1 proteins were successfully purified and their antigenicities were assessed. Immunization of mice with recombinant proteins observed the immunogenicity of the NS1 protein. The generated recombinant proteins can be potentially used in the development of NS1 diagnostic test. With minimal modifications, this method can be used for producing NS1 recombinant proteins from isolates obtained from other geographical regions. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Single-Step Purification and Characterization of A Recombinant Serine Proteinase Inhibitor from Transgenic Plants.

    Science.gov (United States)

    Jha, Shweta; Agarwal, Saurabh; Sanyal, Indraneel; Amla, D V

    2016-05-01

    Expression of recombinant therapeutic proteins in transgenic plants has a tremendous impact on safe and economical production of biomolecules for biopharmaceutical industry. The major limitation in their production is downstream processing of recombinant protein to obtain higher yield and purity of the final product. In this study, a simple and rapid process has been developed for purification of therapeutic recombinant α1-proteinase inhibitor (rα1-PI) from transgenic tomato plants, which is an abundant serine protease inhibitor in human serum and chiefly inhibits the activity of neutrophil elastase in lungs. We have expressed rα1-PI with modified synthetic gene in transgenic tomato plants at a very high level (≃3.2 % of total soluble protein). The heterologous protein was extracted with (NH4)2SO4 precipitation, followed by chromatographic separation on different matrices. However, only immunoaffinity chromatography resulted into homogenous preparation of rα1-PI with 54 % recovery. The plant-purified rα1-PI showed molecular mass and structural conformation comparable to native serum α1-PI, as shown by mass spectrometry and optical spectroscopy. The results of elastase inhibition assay revealed biological activity of the purified rα1-PI protein. This work demonstrates a simple and efficient one-step purification of rα1-PI from transgenic plants, which is an essential prerequisite for further therapeutic development.

  16. [Construction and Fluorescence Analysis of the RecombinantListeria ivanoviiStrain Expressing Green Fluorescent Protein].

    Science.gov (United States)

    Zhang, Xiang; Su, Lin; Liu, Si-Jing; Li, Yong-Yu; Jiang, Ming-Juan; Huang, Huan; Wang, Chuan

    2017-11-01

    Constructing the recombinant Listeria ivanovii strain expressing green fluorescent protein to provide an important tool for study of Listeria ivanovii. The promoter of Listeria monocytogenes Listeriolysin O ( phly ) and the green fluorescent protein (GFP) gene were fused by SOEing PCR,and then ligated the fusion gene into plasmid pCW to result in recombinant plasmid pCW- phly-GFP. Recombinant plasmid was electroporated into Listeria ivanovii ,and fluorescence microscope was used to analyze the expression of GFP. To observe the stability of recombinant plasmid and the stable expression of GFP in Listeria ivanovii ,bacteria were cultured in the BHI broth with or without erythromycin for several generations. The stability of recombinant plasmid pCW- phly-GFP and fluorescent protein in each generation of bacteriawas studied by extracting plasmids and observing fluorescence. The exactness of recombinant plasmid pCW- phly-GFP was confirmed with restrictive endonuclease assay and sequence analysis. Under the fluorescence microscope,the green fluorescence was obvious in Listeria ivanovii carried with pCW- phly-GFP. The recombinant plasmid pCW- phly-GFP was stable in Listeria ivanovii and the GFP kept expressing in a high level under the pressure of erythromycin. The prokaryotic expression plasmid pCW- phly-GFP containing GFP gene was successfully constructed. Listeria ivanovii carried with the plasmid efficiently expressed GFP. This research provides an important tool for further study of Listeria ivanovii as a vaccine carrier.

  17. Expression and purification of recombinant proteins in Escherichia coli tagged with a small metal-binding protein from Nitrosomonas europaea.

    Science.gov (United States)

    Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Zarate, Xristo

    2016-02-01

    Escherichia coli is still the preferred organism for large-scale production of recombinant proteins. The use of fusion proteins has helped considerably in enhancing the solubility of heterologous proteins and their purification with affinity chromatography. Here, the use of a small metal-binding protein (SmbP) from Nitrosomonas europaea is described as a new fusion protein for protein expression and purification in E. coli. Fluorescent proteins tagged at the N-terminal with SmbP showed high levels of solubility, compared with those of maltose-binding protein and glutathione S-transferase, and low formation of inclusion bodies. Using commercially available IMAC resins charged with Ni(II), highly pure recombinant proteins were obtained after just one chromatography step. Proteins may be purified from the periplasm of E. coli if SmbP contains the signal sequence at the N-terminal. After removal of the SmbP tag from the protein of interest, high-yields are obtained since SmbP is a protein of just 9.9 kDa. The results here obtained suggest that SmbP is a good alternative as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Enhancing recombinant protein solubility with ubiquitin-like small archeal modifying protein fusion partners.

    Science.gov (United States)

    Varga, Sándor; Pathare, Ganesh Ramnath; Baka, Erzsébet; Boicu, Marius; Kriszt, Balázs; Székács, András; Zinzula, Luca; Kukolya, József; Nagy, István

    2015-11-01

    A variety of protein expression tags with different biochemical properties has been used to enhance the yield and solubility of recombinant proteins. Ubiquitin, SUMO (small ubiquitin-like modifier) and prokaryotic ubiquitin like MoaD (molybdopterin synthase, small subunit) fusion tags are getting more popular because of their small size. In this paper we report on the use of ubiquitin-like small archaeal modifier proteins (SAMPs) as fusion tags since they proved to increase expression yield, stability and solubility in our experiments. Equally important, they did not co-purify with proteins of the expression host and there was information that their specific JAB1/MPN/Mov34 metalloenzyme (JAMM) protease can recognize the C-terminal VSGG sequence when SAMPs fused, either branched or linearly to target proteins, and cleave it specifically. SAMPs and JAMM proteases from Haloferax volcanii, Thermoplasma acidophilum, Methanococcoides burtonii and Nitrosopumilus maritimus were selected, cloned, expressed heterologously in Escherichia coli and tested as fusion tags and cleaving proteases, respectively. Investigated SAMPs enhanced protein expression and solubility on a wide scale. T. acidophilum SAMPs Ta0895 and Ta01019 were the best performing tags and their effect was comparable to the widely used maltose binding protein (MBP) and N utilization substance protein A (NusA) tags. Moreover, H. volcanii SAMP Hvo_2619 contribution was mediocre, whereas M. burtonii Mbur_1415 could not be expressed. Out of four investigated JAMM proteases, only Hvo_2505 could cleave fusion tags. Interestingly, it was found active not only on its own partner substrate Hvo_2619, but it also cleaved off Ta0895. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Plastoglobules: a new address for targeting recombinant proteins in the chloroplast

    Directory of Open Access Journals (Sweden)

    Kessler Felix

    2007-01-01

    Full Text Available Abstract Background The potential of transgenic plants for cost-effective production of pharmaceutical molecules is now becoming apparent. Plants have the advantage over established fermentation systems (bacterial, yeast or animal cell cultures to circumvent the risk of pathogen contamination, to be amenable to large scaling up and to necessitate only established farming procedures. Chloroplasts have proven a useful cellular compartment for protein accumulation owing to their large size and number, as well as the possibility for organellar transformation. They therefore represent the targeting destination of choice for recombinant proteins in leaf crops such as tobacco. Extraction and purification of recombinant proteins from leaf material contribute to a large extent to the production costs. Developing new strategies facilitating these processes is therefore necessary. Results Here, we evaluated plastoglobule lipoprotein particles as a new subchloroplastic destination for recombinant proteins. The yellow fluorescent protein as a trackable cargo was targeted to plastoglobules when fused to plastoglobulin 34 (PGL34 as the carrier. Similar to adipocyte differentiation related protein (ADRP in animal cells, most of the protein sequence of PGL34 was necessary for targeting to lipid bodies. The recombinant protein was efficiently enriched in plastoglobules isolated by simple flotation centrifugation. The viability of plants overproducing the recombinant protein was not affected, indicating that plastoglobule targeting did not significantly impair photosynthesis or sugar metabolism. Conclusion Our data identify plastoglobules as a new targeting destination for recombinant protein in leaf crops. The wide-spread presence of plastoglobules and plastoglobulins in crop species promises applications comparable to those of transgenic oilbody-oleosin technology in molecular farming.

  20. Generation of monoclonal antibodies for the assessment of protein purification by recombinant ribosomal coupling

    DEFF Research Database (Denmark)

    Kristensen, Janni; Sperling-Petersen, Hans Uffe; Mortensen, Kim Kusk

    2005-01-01

    -His is among the fusion proteins used in our previous study for ribosomal coupling of C-terminally His-tagged green fluorescent protein. To assess the efficiency of separation of target protein from ribosomes, by site-specific proteolysis, we required monoclonal antibodies directed against rpL23 and GFP. We......We recently described a conceptually novel method for the purification of recombinant proteins with a propensity to form inclusion bodies in the cytoplasm of Escherichia coli. Recombinant proteins were covalently coupled to the E. coli ribosome by fusing them to ribosomal protein 23 (rpL23......) followed by expression in an rpL23 deficient strain of E. coli. This allowed for the isolation of ribsomes with covalently coupled target proteins which could be efficiently purified by centrifugation after in vitro proteolysis at a specific site incorporated between rpL23 and the target protein. rpL23-GFP...

  1. Increasing the production yield of recombinant protein in transgenic seeds by expanding the deposition space within the intracellular compartment

    OpenAIRE

    Takaiwa, Fumio

    2013-01-01

    Seeds must maintain a constant level of nitrogen in order to germinate. When recombinant proteins are produced while endogenous seed protein expression is suppressed, the production levels of the foreign proteins increase to compensate for the decreased synthesis of endogenous proteins. Thus, exchanging the production of endogenous seed proteins for that of foreign proteins is a promising approach to increase the yield of foreign recombinant proteins. Providing a space for the deposition of r...

  2. Photosynthetic biomanufacturing in green algae; production of recombinant proteins for industrial, nutritional, and medical uses.

    Science.gov (United States)

    Rasala, Beth A; Mayfield, Stephen P

    2015-03-01

    Recombinant proteins are widely used for industrial, nutritional, and medical applications. Green microalgae have attracted considerable attention recently as a biomanufacturing platform for the production of recombinant proteins for a number of reasons. These photosynthetic eukaryotic microorganisms are safe, scalable, easy to genetically modify through transformation, mutagenesis, or breeding, and inexpensive to grow. Many microalgae species are genetically transformable, but the green alga Chlamydomonas reinhardtii is the most widely used host for recombinant protein expression. An extensive suite of molecular genetic tools has been developed for C. reinhardtii over the last 25 years, including a fully sequenced genome, well-established methods for transformation, mutagenesis and breeding, and transformation vectors for high levels of recombinant protein accumulation and secretion. Here, we review recent successes in the development of C. reinhardtii as a biomanufacturing host for recombinant proteins, including antibodies and immunotoxins, hormones, industrial enzymes, an orally-active colostral protein for gastrointestinal health, and subunit vaccines. In addition, we review the biomanufacturing potential of other green algae from the genera Dunaliella and Chlorella.

  3. A complete approach for recombinant protein expression training: From gene cloning to assessment of protein functionality*.

    Science.gov (United States)

    Novo, M Teresa Marques; Soares-Costa, Andrea; de Souza, Antonia Q L; Figueira, Ana Carolina M; Molina, Gustavo C; Palacios, Carlos A; Kull, Claudia R; Monteiro, Izabel F; Baldan-Pineda, Paulo H; Henrique-Silva, Flavio

    2005-01-01

    A practical course was given to undergraduate biology students enrolled in the elective course "Introduction to Genetic Engineering" at the Federal University of São Carlos (UFSCar), São Paulo, Brazil. The goal of the course was to teach current molecular biology tools applied to a real research situation that could be reported by the students themselves. The purpose was to produce a plant recombinant protein and demonstrate a heretofore unreported biological activity. Cystatins, natural inhibitors of cysteine proteases, were proposed for these studies. Initially, the students searched for plant cystatin cDNA sequences in the NCBI databases and selected the Oryzacystatin I gene (ocI) from rice, Oriza sativa, as the target gene for this study. Total RNA was extracted from rice-germinating seeds and primers containing restriction sites for NdeI and EcoRI were designed based on the ocI cDNA sequence and then used to amplify the open reading frame (ORF). RT-PCR amplification provided a band of the expected size for ocI ORF (309 bp). The PCR product was cut with NdeI and EcoRI restriction enzymes and cloned directly in the pET28a expression vector digested with the same enzymes. A pET28-ocI recombinant clone was selected, checked by sequencing, and used to transform Escherichia coli BL21 (DE3) expression strain. After induction of the bacteria with isopropylthiogalactoside and cellular disruption, the His-tagged OCI protein, present mainly in the soluble fraction, was purified by affinity chromatography in a nickel column. The purified protein was successfully used to inhibit fungal growth (Trichoderma reesei). The results were discussed extensively and the students contributed to the writing of this article, of which they are co-authors. Copyright © 2005 International Union of Biochemistry and Molecular Biology, Inc.

  4. Watching single protein molecules in action

    DEFF Research Database (Denmark)

    Heiðarsson, Pétur Orri

    (NCS1). The NMR solution structure of NCS1, in combination with fluorescence spectroscopy and mutational analysis, suggested a novel role for the C-terminal tail in regulating conformational stability. On the single-molecule level, the C-domain folded through a partially folded intermediate state....... This knowledge-gap is partly due to our inability to unveil the details of folding mechanisms that can be buried in the ensemble-averaged output of traditional bulk methods. Single-molecule techniques have provided a perspective beyond the ensemble average and enable studying the folding trajectories of protein...... molecules in unprecedented detail. These methods can, in principle, detect rare folding or misfolding events, and ultimately lead to a reconstruction of the free energy landscape. In this thesis, the folding mechanism of both single- and double-domain proteins is unraveled using single-molecule optical...

  5. Small-Scale Production of Recombinant Proteins Using the Baculovirus Expression Vector System.

    Science.gov (United States)

    Yang, Jian-Ping

    2016-01-01

    Numerous technological improvements, including progress in vector design, simplification of virus isolation techniques, and advancements in molecular biology and cell culture technologies, have greatly facilitated the use of the baculovirus-insect cell system for routine production of recombinant proteins. This chapter outlines the basic techniques for small-scale protein production using the Baculovirus Expression Vector System (BEVS), including protocols for titer estimation in 96-well plates, expression optimization in 24-well plates, and recombinant protein expression from adherent and suspension cultures in six-well plates and in 50 mL insect cell cultures.

  6. Interactive effects of microbial transglutaminase and recombinant cystatin on the mackerel and hairtail muscle protein.

    Science.gov (United States)

    Jiang, Shann-Tzong; Hsieh, Jung-Feng; Tsai, Guo-Jane

    2004-06-02

    Interactive effects of microbial transglutaminase (MTGase) and recombinant cystatin on the mackerel and hairtail water soluble protein (WSP), salt soluble protein (SSP), and muscle protein (MP) were investigated. According to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enzymic activity analyses, cross-linking of mackerel and hairtail myosin heavy chain and low molecular mass compounds and formation of epsilon-(gamma-glutamyl)lysine cross-links were observed on samples with MTGase, while the recombinant cystatin could effectively inhibit the cathepsins and subsequently prevent degradation of proteins during setting. The cathepsins and MTGase activities in WSP, SSP, and MP solutions decreased, but the recombinant cystatin activity increased during setting at 45 degrees C.

  7. [Expression of recombinant rubella virus E1 protein and initial application for detecting of antibody].

    Science.gov (United States)

    Yi, Yao; Guo, Min-zhuo; Yu, Tao; Xu, Wen-bo; Yang, Jin-ye; Chen, Si-yong

    2008-10-01

    To apply recombinant rubella virus envelope protein-1 (E1) to detect human rubella virus IgG antibody. Rubella virus E1 protein was expressed in E. coli, purified E1 protein was used as the antigen for the detecting of anti rubella in human sera in the way of enzyme linked Immunosorbant assay (ELISA). The antigenicity of the recombinant protein was checked by WHO rubella sera panel. We detected 200 sera samples, which came from Guangxi Guilin. 93% of these samples were positive. The antigenicity of recombinant E1 is a satisfied candidate antigen for the detecting of human rubella virus antibody. The prevalence of anti rubella virus IgG in Guangxi is 93%. It is at the some level compared with other provinces in China.

  8. Transphosphorylation of E. coli proteins during production of recombinant protein kinases provides a robust system to characterize kinase specificity

    Science.gov (United States)

    Protein kinase specificity is of fundamental importance to pathway regulation and signal transduction. Here, we report a convenient system to monitor the activity and specificity of recombinant protein kinases expressed in E.coli. We apply this to the study of the cytoplasmic domain of the plant rec...

  9. Evolved Lactococcus lactis Strains for Enhanced Expression of Recombinant Membrane Proteins

    NARCIS (Netherlands)

    Martinez Linares, Daniel; Geertsma, Eric R.; Poolman, Bert

    2010-01-01

    The production of complex multidomain (membrane) proteins is a major hurdle in structural genomics and a generic approach for optimizing membrane protein expression is still lacking. We have devised a selection method to isolate mutant strains with improved functional expression of recombinant

  10. Recombinant human activated protein C: current insights into its mechanism of action

    NARCIS (Netherlands)

    Levi, Marcel; van der Poll, Tom

    2007-01-01

    Impairment of the protein C pathway plays a central role in the pathogenesis of sepsis. Administration of recombinant human activated protein C (rhAPC) may correct the dysregulated anticoagulant mechanism and prevent propagation of thrombin generation and formation of microvascular thrombosis.

  11. Chapter 15. transforming lepidopteran insect cells for continuous recombinant protein expression

    Science.gov (United States)

    The baculovirus expression vector system (BEVS) is widely used to produce large quantities of recombinant proteins. However, yields of extracellular and membrane-bound proteins obtained with this system often are very low, possibly due to the adverse effects of baculovirus infection on the host ins...

  12. Gene and process level modulation to overcome the bottlenecks of recombinant proteins expression in Pichia pastoris.

    Science.gov (United States)

    Prabhu, Ashish A; Boro, Bibari; Bharali, Biju; Chakraborty, Shuchishloka; Dasu, V Venkata

    2018-03-28

    Process development involving system metabolic engineering and bioprocess engineering has become one of the major thrust for the development of therapeutic proteins or enzymes. Pichia pastoris has emerged as a prominent host for the production of therapeutic protein or enzymes. Despite of producing high protein titers, various cellular and process level bottlenecks hinders the expression of recombinant proteins in P. pastoris. In the present review, we have summarized the recent developments in the expression of foreign proteins in P. pastoris. Further, we have discussed various cellular engineering strategies which include codon optimization, pathway engineering, signal peptide processing, development of protease deficient strain and glyco-engineered strains for the high yield protein secretion of recombinant protein. Bioprocess development of recombinant proteins in large scale bioreactor including medium optimization, optimum feeding strategy and co-substrate feeding in fed batch as well as continuous cultivation have been described. The recent advances in system and synthetic biology studies including metabolic flux analysis in understanding the phenotypic characteristics of recombinant Pichia and genome editing with CRISPR-CAS system have also been summarized. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  13. Genome-scale metabolic model of Pichia pastoris with native and humanized glycosylation of recombinant proteins

    DEFF Research Database (Denmark)

    Irani, Zahra Azimzadeh; Kerkhoven, Eduard J.; Shojaosadati, Seyed Abbas

    2016-01-01

    Pichia pastoris is used for commercial production of human therapeutic proteins, and genome-scale models of P. pastoris metabolism have been generated in the past to study the metabolism and associated protein production by this yeast. A major challenge with clinical usage of recombinant proteins...... native nor humanized N-glycosylation, and we therefore developed ihGlycopastoris, an extension to the iLC915 model with both native and humanized N-glycosylation for recombinant protein production, but also an estimation of N-glycosylation of P. pastoris native proteins. This new model gives a better...... produced by P. pastoris is the difference in N-glycosylation of proteins produced by humans and this yeast. However, through metabolic engineering, a P. pastoris strain capable of producing humanized N-glycosylated proteins was constructed. The current genome-scale models of P. pastoris do not address...

  14. Genetic Structures and Conditions of their Expression, which Allow Receiving Native Recombinant Proteins with High Output

    Directory of Open Access Journals (Sweden)

    Michael M. Shavlovsky, PhD, ScD¹

    2012-03-01

    Full Text Available We investigated the possibility of obtaining native recombinant amyloidogenic proteins by creating genetic constructs encoding fusion proteins of target proteins with Super Folder Green Fluorescent Protein (sfGFP. In this study, we show that the structures, containing the sfGFP gene, provide a synthesis, within a bacterial system, of fusion proteins with minimal formation of inclusion bodies. Constructs containing genes of the target proteins in the 3'-terminal region of the sfGFP gene followed by a polynucleotide sequence, which allows for affinity purification fusion proteins, are optimal. Heating bacterial cultures before the induction of the expression of recombinant genes in 42°С for 30 min (heat shock was found to increase the output of the desired products, thus practically avoiding the formation of insoluble aggregates

  15. Prediction of recombinant protein overexpression in Escherichia coli using a machine learning based model (RPOLP).

    Science.gov (United States)

    Habibi, Narjeskhatoon; Norouzi, Alireza; Mohd Hashim, Siti Z; Shamsir, Mohd Shahir; Samian, Razip

    2015-11-01

    Recombinant protein overexpression, an important biotechnological process, is ruled by complex biological rules which are mostly unknown, is in need of an intelligent algorithm so as to avoid resource-intensive lab-based trial and error experiments in order to determine the expression level of the recombinant protein. The purpose of this study is to propose a predictive model to estimate the level of recombinant protein overexpression for the first time in the literature using a machine learning approach based on the sequence, expression vector, and expression host. The expression host was confined to Escherichia coli which is the most popular bacterial host to overexpress recombinant proteins. To provide a handle to the problem, the overexpression level was categorized as low, medium and high. A set of features which were likely to affect the overexpression level was generated based on the known facts (e.g. gene length) and knowledge gathered from related literature. Then, a representative sub-set of features generated in the previous objective was determined using feature selection techniques. Finally a predictive model was developed using random forest classifier which was able to adequately classify the multi-class imbalanced small dataset constructed. The result showed that the predictive model provided a promising accuracy of 80% on average, in estimating the overexpression level of a recombinant protein. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Antibodies to a recombinant glutamate-rich Plasmodium falciparum protein

    DEFF Research Database (Denmark)

    Hogh, B; Petersen, E; Dziegiel, Morten Hanefeld

    1992-01-01

    A Plasmodium falciparum antigen gene coding for a 220-kD glutamate-rich protein (GLURP) has been cloned, and the 783 C-terminal amino acids of this protein (GLURP489-1271) have been expressed as a beta-galactosidase fusion protein in Escherichia coli. The encoded 783 amino acid residues contain two...

  17. Antibodies to a recombinant glutamate-rich Plasmodium falciparum protein

    DEFF Research Database (Denmark)

    Hogh, B; Petersen, E; Dziegiel, Morten Hanefeld

    1992-01-01

    A Plasmodium falciparum antigen gene coding for a 220-kD glutamate-rich protein (GLURP) has been cloned, and the 783 C-terminal amino acids of this protein (GLURP489-1271) have been expressed as a beta-galactosidase fusion protein in Escherichia coli. The encoded 783 amino acid residues contain t...

  18. A Rapid Method for Determining the Concentration of Recombinant Protein Secreted from Pichia pastoris

    Science.gov (United States)

    Sun, L. W.; Zhao, Y.; Niu, L. P.; Jiang, R.; Song, Y.; Feng, H.; feng, K.; Qi, C.

    2011-02-01

    Pichia secretive expression system is one of powerful eukaryotic expression systems in genetic engineering, which is especially suitable for industrial utilization. Because of the low concentration of the target protein in initial experiment, the methods and conditions for expression of the target protein should be optimized according to the protein yield repetitively. It is necessary to set up a rapid, simple and convenient analysis method for protein expression levels instead of the generally used method such as ultrafiltration, purification, dialysis, lyophilization and so on. In this paper, acetone precipitation method was chosen to concentrate the recombinant protein firstly after comparing with four different protein precipitation methods systematically, and then the protein was analyzed by SDS-Polyacrylamide Gel Electrophoresis. The recombinant protein was determined with the feature of protein band by the Automated Image Capture and 1-D Analysis Software directly. With this method, the optimized expression conditions of basic fibroblast growth factor secreted from pichia were obtained, which is as the same as using traditional methods. Hence, a convenient tool to determine the optimized conditions for the expression of recombinant proteins in Pichia was established.

  19. Diagnostic potential of recombinant scFv antibodies generated against hemagglutinin protein of influenza A virus

    Directory of Open Access Journals (Sweden)

    Roopali eRajput

    2015-09-01

    Full Text Available Human influenza A viruses have been the cause of enormous socio-economic losses worldwide. In order to combat such a notorious pathogen, hemagglutinin protein (HA has been a preferred target for generation of neutralizing-antibodies, as potent therapeutic/ diagnostic agents. In the present study, recombinant anti-HA single chain variable fragment (scFv antibodies were constructed using the phage display technology to aid in diagnosis and treatment of human influenza A virus infections. Spleen cells of mice hyper-immunized with A/New Caledonia/20/99 (H1N1 virus were used as the source for recombinant antibody (rAb production. The antigen-binding phages were quantified after 6 rounds of bio-panning against A/New Caledonia/20/99 (H1N1, A/California/07/2009 (H1N1-like, or A/Udorn/307/72(H3N2 viruses. The phage yield was maximum for the A/New Caledonia/20/99 (H1N1, however, considerable cross-reactivity was observed for the other virus strains as well. The HA-specific polyclonal rAb preparation was subjected to selection of single clones for identification of high reactive relatively conserved epitopes. The high affinity rAbs were tested against certain known conserved HA epitopes by peptide ELISA. Three recombinant mAbs showed reactivity with both the H1N1 strains and one (C5 showed binding with all the three viral strains. The C5 antibody was thus used for development of an ELISA test for diagnosis of influenza virus infection. Based on the sample size in the current analysis, the ELISA test demonstrated 83.9% sensitivity and 100% specificity. Thus, the ELISA, developed in our study, may prove as a cheaper alternative to the presently used real time RT-PCR test for detection of human influenza A viruses in clinical specimens, which will be beneficial, especially in the developing countries. Since, the two antibodies identified in this study are reactive to conserved HA epitopes; these may prove as potential therapeutic agents as well.

  20. Watching single protein molecules in action

    DEFF Research Database (Denmark)

    Heiðarsson, Pétur Orri

    . This knowledge-gap is partly due to our inability to unveil the details of folding mechanisms that can be buried in the ensemble-averaged output of traditional bulk methods. Single-molecule techniques have provided a perspective beyond the ensemble average and enable studying the folding trajectories of protein...... molecules in unprecedented detail. These methods can, in principle, detect rare folding or misfolding events, and ultimately lead to a reconstruction of the free energy landscape. In this thesis, the folding mechanism of both single- and double-domain proteins is unraveled using single-molecule optical......, with transition states located almost halfway between the native and unfolded states. When pulled from the N- and C-termini, both experiments and simulations suggested that the molecule populates a transition state that resembles that observed during chemical denaturation, with respect to structure and position...

  1. Recombinant Plasmodium falciparum glutamate rich protein; purification and use in enzyme-linked immunosorbent assay

    DEFF Research Database (Denmark)

    Dziegiel, Morten Hanefeld; Borre, M B; Jepsen, S

    1991-01-01

    A method for purification of a recombinant Plasmodium falciparum protein produced in E. coli and its use in an enzyme-linked immunosorbent assay (ELISA) is described. The cloned gene fragment encodes GLURP,489-1271 the carboxy-terminal 783 amino acid residue portion of a 1271 amino acid residue P....... falciparum glutamate rich protein (GLURP), with a molecular weight of 220 kilodalton. The protein is associated with all parasite stages in the human host. Examination of sera from 105 adult Liberians living in a malaria endemic area revealed anti-GLURP IgG antibodies in 98% of the sera. The recombinant...... New Guinea (MAD20) and Honduras (HB3) completely absorbed specific antibodies, indicating the presence of conserved epitopes produced by all isolates of P. falciparum. Recombinant GLURP489-1271 ELISA is sensitive and rapid, and therefore well-suited for sero-epidemiological studies, and for control...

  2. Polymyxin B as inhibitor of LPS contamination of Schistosoma mansoni recombinant proteins in human cytokine analysis

    Directory of Open Access Journals (Sweden)

    Pacífico Lucila G

    2007-01-01

    Full Text Available Abstract Background Recombinant proteins expressed in Escherichia coli vectors are generally contaminated with endotoxin. In this study, we evaluated the ability of Polymyxin B to neutralize the effect of LPS present as contaminant on Schistosoma mansoni recombinant proteins produced in E. coli in inducing TNF-α and IL-10. Peripheral blood mononuclear cells from individuals chronically infected with S. mansoni were stimulated in vitro with recombinant Sm22.6, Sm14 and P24 antigens (10 μg/mL in the presence of Polymyxin B (10 μg/mL. Results The levels of cytokines were measured using ELISA. There was greater than 90 % reduction (p S. mansoni recombinant proteins in the presence of Polymyxin B, a reduction in the levels of TNF-α and IL-10 was also observed. However, the percentage of reduction was lower when compared to the cultures stimulated with LPS, probably because these proteins are able to induce the production of these cytokines by themselves. Conclusion This study showed that Polymyxin B was able to neutralize the effect of endotoxin, as contaminant in S. mansoni recombinant antigens produced in E. coli, in inducing TNF-α and IL-10 production.

  3. A Library of Plasmodium vivax Recombinant Merozoite Proteins Reveals New Vaccine Candidates and Protein-Protein Interactions.

    Directory of Open Access Journals (Sweden)

    Jessica B Hostetler

    2015-12-01

    are natively folded and functional. This screen also identified two novel protein-protein interactions, between P12 and PVX_110945, and between MSP3.10 and MSP7.1, the latter of which was confirmed by surface plasmon resonance.We produced a new library of recombinant full-length P. vivax ectodomains, established that the majority of them contain tertiary structure, and used them to identify predicted and novel protein-protein interactions. As well as identifying new interactions for further biological studies, this library will be useful in identifying P. vivax proteins with vaccine potential, and studying P. vivax malaria pathogenesis and immunity.ClinicalTrials.gov NCT00663546.

  4. A Library of Plasmodium vivax Recombinant Merozoite Proteins Reveals New Vaccine Candidates and Protein-Protein Interactions.

    Science.gov (United States)

    Hostetler, Jessica B; Sharma, Sumana; Bartholdson, S Josefin; Wright, Gavin J; Fairhurst, Rick M; Rayner, Julian C

    2015-12-01

    folded and functional. This screen also identified two novel protein-protein interactions, between P12 and PVX_110945, and between MSP3.10 and MSP7.1, the latter of which was confirmed by surface plasmon resonance. We produced a new library of recombinant full-length P. vivax ectodomains, established that the majority of them contain tertiary structure, and used them to identify predicted and novel protein-protein interactions. As well as identifying new interactions for further biological studies, this library will be useful in identifying P. vivax proteins with vaccine potential, and studying P. vivax malaria pathogenesis and immunity. ClinicalTrials.gov NCT00663546.

  5. Practical protocols for production of very high yields of recombinant proteins using Escherichia coli

    OpenAIRE

    Sivashanmugam, Arun; Murray, Victoria; Cui, Chunxian; Zhang, Yonghong; Wang, Jianjun; Li, Qianqian

    2009-01-01

    The gram-negative bacterium Escherichia coli offers a mean for rapid, high yield, and economical production of recombinant proteins. However, high-level production of functional eukaryotic proteins in E. coli may not be a routine matter, sometimes it is quite challenging. Techniques to optimize heterologous protein overproduction in E. coli have been explored for host strain selection, plasmid copy numbers, promoter selection, mRNA stability, and codon usage, significantly enhancing the yield...

  6. Enhanced production of recombinant proteins with Corynebacterium glutamicum by deletion of insertion sequences (IS elements).

    Science.gov (United States)

    Choi, Jae Woong; Yim, Sung Sun; Kim, Min Jeong; Jeong, Ki Jun

    2015-12-29

    In most bacteria, various jumping genetic elements including insertion sequences elements (IS elements) cause a variety of genetic rearrangements resulting in harmful effects such as genome and recombinant plasmid instability. The genetic stability of a plasmid in a host is critical for high-level production of recombinant proteins, and in this regard, the development of an IS element-free strain could be a useful strategy for the enhanced production of recombinant proteins. Corynebacterium glutamicum, which is a workhorse in the industrial-scale production of various biomolecules including recombinant proteins, also has several IS elements, and it is necessary to identify the critical IS elements and to develop IS element deleted strain. From the cultivation of C. glutamicum harboring a plasmid for green fluorescent protein (GFP) gene expression, non-fluorescent clones were isolated by FACS (fluorescent activated cell sorting). All the isolated clones had insertions of IS elements in the GFP coding region, and two major IS elements (ISCg1 and ISCg2 families) were identified. By co-cultivating cells harboring either the isolated IS element-inserted plasmid or intact plasmid, it was clearly confirmed that cells harboring the IS element-inserted plasmids became dominant during the cultivation due to their growth advantage over cells containing intact plasmids, which can cause a significant reduction in recombinant protein production during cultivation. To minimize the harmful effects of IS elements on the expression of heterologous genes in C. glutamicum, two IS element free C. glutamicum strains were developed in which each major IS element was deleted, and enhanced productivity in the engineered C. glutamicum strain was successfully demonstrated with three models: GFP, poly(3-hydroxybutyrate) [P(3HB)] and γ-aminobutyrate (GABA). Our findings clearly indicate that the hopping of IS elements could be detrimental to the production of recombinant proteins in C

  7. Evaluation of the recombinant protein TpF1 of Treponema pallidum for serodiagnosis of syphilis.

    Science.gov (United States)

    Jiang, Chuanhao; Zhao, Feijun; Xiao, Jinhong; Zeng, Tiebing; Yu, Jian; Ma, Xiaohua; Wu, Haiying; Wu, Yimou

    2013-10-01

    Syphilis is a chronic infection caused by Treponema pallidum subsp. pallidum, and diagnosis with sensitive and specific methods is a challenging process that is important for its prevention and treatment. In the present study, we established a recombinant protein TpF1-based indirect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) and a Western blot assay for human and rabbit sera. The 20-kDa recombinant protein TpF1 was detected by Western blotting performed with sera from rabbits immunized with recombinant TpF1 and infected with the T. pallidum Nichols strain and T. pallidum clinical isolates but was not detected by Western blotting with sera from uninfected rabbits. The sensitivity of the recombinant protein was determined by screening sera from individuals with primary, secondary, latent, and congenital syphilis (n = 82). The specificity of the recombinant protein was determined by screening sera from uninfected controls (n = 30) and individuals with potentially cross-reactive infections, including Lyme disease (n = 30) and leptospirosis (n = 5). The sensitivities of TpF1-based ELISAs were 93.3%, 100%, 100%, and 100% for primary, secondary, latent, and congenital syphilis, respectively, and the specificities were all 100% for sera from uninfected controls and individuals with potentially cross-reactive infections. In Western blot assays, the sensitivities and specificities of TpF1 for human sera were all 100%. The reactivities of TpF1 with syphilitic sera were proportional to the titers of the T. pallidum particle agglutination (TPPA) assay. These data indicate that the recombinant protein TpF1 is a highly immunogenic protein in human and rabbit infections and a promising marker for the screening of syphilis.

  8. Efficient secretory expression of recombinant proteins in Escherichia coli with a novel actinomycete signal peptide.

    Science.gov (United States)

    Cui, Yanbing; Meng, Yiwei; Zhang, Juan; Cheng, Bin; Yin, Huijia; Gao, Chao; Xu, Ping; Yang, Chunyu

    2017-01-01

    In well-established heterologous hosts, such as Escherichia coli, recombinant proteins are usually intracellular and frequently found as inclusion bodies-especially proteins possessing high rare codon content. In this study, successful secretory expression of three hydrolases, in a constructed inducible or constitutive system, was achieved by fusion with a novel signal peptide (Kp-SP) from an actinomycete. The signal peptide efficiently enabled extracellular protein secretion and also contributed to the active expression of the intracellular recombinant proteins. The thermophilic α-amylase gene of Bacillus licheniformis was fused with Kp-SP. Both recombinants, carrying inducible and constitutive plasmids, showed remarkable increases in extracellular and intracellular amylolytic activity. Amylase activity was observed to be > 10-fold in recombinant cultures with the constitutive plasmid, pBSPPc, compared to that in recombinants lacking Kp-SP. Further, the signal peptide enabled efficient secretion of a thermophilic cellulase into the culture medium, as demonstrated by larger halo zones and increased enzymatic activities detected in both constructs from different plasmids. For heterologous proteins with a high proportion of rare codons, it is difficult to obtain high expression in E. coli owing to the codon bias. Here, the fusion of an archaeal homologue of the amylase encoding gene, FSA, with Kp-SP resulted in > 5-fold higher extracellular activity. The successful extracellular expression of the amylase indicated that the signal peptide also contributed significantly to its active expression and signified the potential value of this novel and versatile signal peptide in recombinant protein production. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Evaluation of the Recombinant Protein TpF1 of Treponema pallidum for Serodiagnosis of Syphilis

    Science.gov (United States)

    Jiang, Chuanhao; Zhao, Feijun; Xiao, Jinhong; Zeng, Tiebing; Yu, Jian; Ma, Xiaohua; Wu, Haiying

    2013-01-01

    Syphilis is a chronic infection caused by Treponema pallidum subsp. pallidum, and diagnosis with sensitive and specific methods is a challenging process that is important for its prevention and treatment. In the present study, we established a recombinant protein TpF1-based indirect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) and a Western blot assay for human and rabbit sera. The 20-kDa recombinant protein TpF1 was detected by Western blotting performed with sera from rabbits immunized with recombinant TpF1 and infected with the T. pallidum Nichols strain and T. pallidum clinical isolates but was not detected by Western blotting with sera from uninfected rabbits. The sensitivity of the recombinant protein was determined by screening sera from individuals with primary, secondary, latent, and congenital syphilis (n = 82). The specificity of the recombinant protein was determined by screening sera from uninfected controls (n = 30) and individuals with potentially cross-reactive infections, including Lyme disease (n = 30) and leptospirosis (n = 5). The sensitivities of TpF1-based ELISAs were 93.3%, 100%, 100%, and 100% for primary, secondary, latent, and congenital syphilis, respectively, and the specificities were all 100% for sera from uninfected controls and individuals with potentially cross-reactive infections. In Western blot assays, the sensitivities and specificities of TpF1 for human sera were all 100%. The reactivities of TpF1 with syphilitic sera were proportional to the titers of the T. pallidum particle agglutination (TPPA) assay. These data indicate that the recombinant protein TpF1 is a highly immunogenic protein in human and rabbit infections and a promising marker for the screening of syphilis. PMID:23945159

  10. Human Rad51 filaments on double- and single-stranded DNA: correlating regular and irregular forms with recombination function.

    NARCIS (Netherlands)

    D. Ristic (Dejan); M. Modesti (Mauro); T. van der Heijden (Thijn); J. Noort (John); C. Dekker (Cees); R. Kanaar (Roland); C. Wyman (Claire)

    2005-01-01

    textabstractRecombinase proteins assembled into helical filaments on DNA are believed to be the catalytic core of homologous recombination. The assembly, disassembly and dynamic rearrangements of this structure must drive the DNA strand exchange reactions of homologous recombination. The sensitivity

  11. Analysis of Recombinant Proteins in Transgenic Rice Seeds: Identity, Localization, Tolerance to Digestion, and Plant Stress Response.

    Science.gov (United States)

    Wakasa, Yuhya; Takaiwa, Fumio

    2016-01-01

    Rice seeds are an ideal production platform for high-value recombinant proteins in terms of economy, scalability, safety, and stability. Strategies for the expression of large amounts of recombinant proteins in rice seeds have been established in the past decade and transgenic rice seeds that accumulate recombinant products such as bioactive peptides and proteins, which promote the health and quality of life of humans, have been generated in many laboratories worldwide. One of the most important advantages is the potential for direct oral delivery of transgenic rice seeds without the need for recombinant protein purification (downstream processing), which has been attributed to the high expression levels of recombinant products. Transgenic rice will be beneficial as a delivery system for pharmaceuticals and nutraceuticals in the future. This chapter introduces the strategy for producing recombinant protein in the edible part (endosperm) of the rice grain and describes methods for the analysis of transgenic rice seeds in detail.

  12. Production of recombinant proteins in Escherichia coli tagged with the fusion protein CusF3H.

    Science.gov (United States)

    Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Zarate, Xristo

    2017-04-01

    Recombinant protein expression in the bacterium Escherichia coli still is the number one choice for large-scale protein production. Nevertheless, many complications can arise using this microorganism, such as low yields, the formation of inclusion bodies, and the requirement for difficult purification steps. Most of these problems can be solved with the use of fusion proteins. Here, the use of the metal-binding protein CusF3H+ is described as a new fusion protein for recombinant protein expression and purification in E. coli. We have previously shown that CusF produces large amounts of soluble protein, with low levels of formation of inclusion bodies, and that proteins can be purified using IMAC resins charged with Cu(II) ions. CusF3H+ is an enhanced variant of CusF, formed by the addition of three histidine residues at the N-terminus. These residues then can bind Ni(II) ions allowing improved purity after affinity chromatography. Expression and purification of Green Fluorescent Protein tagged with CusF3H+ showed that the mutation did not alter the capacity of the fusion protein to increase protein expression, and purity improved considerably after affinity chromatography with immobilized nickel ions; high yields are obtained after tag-removal since CusF3H+ is a small protein of just 10 kDa. Furthermore, the results of experiments involving expression of tagged proteins having medium to large molecular weights indicate that the presence of the CusF3H+ tag improves protein solubility, as compared to a His-tag. We therefore endorse CusF3H+ as a useful alternative fusion protein/affinity tag for production of recombinant proteins in E. coli. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. High-resolution structure of the recombinant sweet-tasting protein thaumatin I

    International Nuclear Information System (INIS)

    Masuda, Tetsuya; Ohta, Keisuke; Mikami, Bunzo; Kitabatake, Naofumi

    2011-01-01

    The structure of a recombinant form of the sweet-tasting protein thaumatin I was determined at 1.1 Å resolution and refined to an R work of 9.1% and an R free of 11.7%. Comparisons with plant thaumatin revealed the electron density of recombinant thaumatin I to be significantly improved, especially around Asn46 and Ser63. Thaumatin, an intensely sweet-tasting plant protein, elicits a sweet taste at a concentration of 50 nM. The crystal structure of a recombinant form of thaumatin I produced in the yeast Pichia pastoris has been determined to a resolution of 1.1 Å. The model was refined with anisotropic B parameters and riding H atoms. A comparison of the diffraction data and refinement statistics for recombinant thaumatin I with those for plant thaumatin I revealed no significant differences in the diffraction data. The R values for recombinant thaumatin I and plant thaumatin I (F o > 4σ) were 9.11% and 9.91%, respectively, indicating the final model to be of good quality. Notably, the electron-density maps around Asn46 and Ser63, which differ between thaumatin variants, were significantly improved. Furthermore, a number of H atoms became visible in an OMIT map and could be assigned. The high-quality structure of recombinant thaumatin with H atoms should provide details about sweetness determinants in thaumatin and provide valuable insights into the mechanism of its interaction with taste receptors

  14. Cheese whey-induced high-cell-density production of recombinant proteins in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Neubauer Peter

    2003-04-01

    Full Text Available Abstract Background Use of lactose-rich concentrates from dairy processes for the induction of recombinant gene's expression has not received much attention although they are interesting low cost substrates for production of recombinant enzymes. Applicability of dairy waste for induction of recombinant genes in Escherichia coli was studied. Clones expressing Lactobacillus phage muramidase and Lactobacillus alcohol dehydrogenase were used for the experiments. Results Shake flask cultivations in mineral salt medium showed that cheese whey or deproteinised whey induced gene expression as efficiently as IPTG (isopropyl-β-D-thiogalactopyranoside or pure lactose. Addition of yeast extract or proteolytically degraded whey proteins did not improve the recombinant protein yield. In contrast, addition of yeast extract to the well-balanced mineral salt medium decreased the product yield. Feeding with glycerol provided sufficient amount of easily assimilable carbon source during the induction period without preventing lactose intake and induction by lactose. High-cell-density fed-batch cultivations showed that product yields comparable to IPTG-induction can be achieved by feeding bacteria with a mixture of glycerol and concentrated whey permeate during the induction. Conclusion Whey and concentrated whey permeate can be applied as an alternative inducer in recombinant high-cell-density fed-batch fermentations. The yield of the recombinant product was comparable to fermentations induced by IPTG. In low-cell-density shake flask experiments the yield was higher with whey or whey permeate than with IPTG.

  15. Recombinant Sheep Pox Virus Proteins Elicit Neutralizing Antibodies

    Directory of Open Access Journals (Sweden)

    Olga V. Chervyakova

    2016-06-01

    Full Text Available The aim of this work was to evaluate the immunogenicity and neutralizing activity of sheep pox virus (SPPV; genus Capripoxvirus, family Poxviridae structural proteins as candidate subunit vaccines to control sheep pox disease. SPPV structural proteins were identified by sequence homology with proteins of vaccinia virus (VACV strain Copenhagen. Four SPPV proteins (SPPV-ORF 060, SPPV-ORF 095, SPPV-ORF 117, and SPPV-ORF 122, orthologs of immunodominant L1, A4, A27, and A33 VACV proteins, respectively, were produced in Escherichia coli. Western blot analysis revealed the antigenic and immunogenic properties of SPPV-060, SPPV-095, SPPV-117 and SPPV-122 proteins when injected with adjuvant into experimental rabbits. Virus-neutralizing activity against SPPV in lamb kidney cell culture was detected for polyclonal antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins. To our knowledge, this is the first report demonstrating the virus-neutralizing activities of antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins.

  16. Recovering corn germ enriched in recombinant protein by wet-fractionation.

    Science.gov (United States)

    Paraman, Ilankovan; Fox, Steven R; Aspelund, Matthew T; Glatz, Charles E; Johnson, Lawrence A

    2010-01-01

    Corn wet-fractionation processes (quick-germ fractionation and traditional wet milling) were evaluated as means of recovering fractions rich in recombinant collagen-related proteins that were targeted for expression in the germ (embryo) of transgenic corn. Transgenic corn lines accumulating a recombinant full-length human collagen type-I-alpha-1 (full-length rCIalpha1) or a 44-kDa rCIalpha1 fragment targeted for seed expression with an embryo-specific promoter were used. Factors to consider in efficient recovery processes are the distribution of the peptides among botanical parts and process recovery efficiency. Both recombinant proteins were distributed 62-64% in germ comprising about 8.6% of the dry grain mass; 34-38% in the endosperm comprising 84% of the dry grain mass; 1.7% in the pericarp comprising about 5% of the dry mass; and 1% in the tip-cap comprising 1.5-2% of the dry mass. The quick-germ method employed a short steeping period either in water or SO(2)-lactic acid solution followed by wet-milling degermination to recover a germ-rich fraction. Of the total recombinant protein expressed in germ, the quick-germ process recovered 40-43% of the total recombinant protein within 6-8% of the corn mass. The traditional corn wet-milling process produced higher purity germ but with lower recovery (24-26%) of the recombinant protein. The two quick-germ methods, using water alone or SO(2)-lactic acid steeping, did not substantially differ in rCIalpha1 recovery, and the quick-germ processes recovered germ with less leaching and proteolytic losses of the recombinant proteins than did traditional wet milling. Thus, grain fractionation enriched the recombinant proteins 6-fold higher than that of unfractionated kernels. Such enrichment may improve downstream processing efficiency and enable utilizing the protein-lean co-products to produce biofuels and biorenewable chemicals by fermenting the remaining starch-rich fractions.

  17. High-yield secretion of recombinant proteins from the microalga Chlamydomonas reinhardtii.

    Science.gov (United States)

    Ramos-Martinez, Erick Miguel; Fimognari, Lorenzo; Sakuragi, Yumiko

    2017-09-01

    Microalga-based biomanufacturing of recombinant proteins is attracting growing attention due to its advantages in safety, metabolic diversity, scalability and sustainability. Secretion of recombinant proteins can accelerate the use of microalgal platforms by allowing post-translational modifications and easy recovery of products from the culture media. However, currently, the yields of secreted recombinant proteins are low, which hampers the commercial application of this strategy. This study aimed at expanding the genetic tools for enhancing secretion of recombinant proteins in Chlamydomonas reinhardtii, a widely used green microalga as a model organism and a potential industrial biotechnology platform. We demonstrated that the putative signal sequence from C. reinhardtii gametolysin can assist the secretion of the yellow fluorescent protein Venus into the culture media. To increase the secretion yields, Venus was C-terminally fused with synthetic glycomodules comprised of tandem serine (Ser) and proline (Pro) repeats of 10 and 20 units [hereafter (SP) n , wherein n = 10 or 20]. The yields of the (SP) n -fused Venus were higher than Venus without the glycomodule by up to 12-fold, with the maximum yield of 15 mg/L. Moreover, the presence of the glycomodules conferred an enhanced proteolytic protein stability. The Venus-(SP) n proteins were shown to be glycosylated, and a treatment of the cells with brefeldin A led to a suggestion that glycosylation of the (SP) n glycomodules starts in the endoplasmic reticulum (ER). Taken together, the results demonstrate the utility of the gametolysin signal sequence and (SP) n glycomodule to promote a more efficient biomanufacturing of microalgae-based recombinant proteins. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  18. [Expression and characterization of recombinant Sj23HD-HSA fusion protein in Saccharomyces cerevisiae].

    Science.gov (United States)

    Zhang, Wei; Yu, Chuan-Xin; Yang, Jian-Linag; Feng, Wei; Yin, Xu-Ren; Song, Li-Jun; Wang, Jie; Qian, Chun-Yan; Ke, Xue-Dan

    2011-12-01

    To prepare the fusion protein of large hydrophilic domain of 23 kDa membrane protein of Schistosoma japonicum and the mature peptide of human serum albumin (Sj23HD-HSA) and investigate its immunoreactivity. A fusion protein gene encoding Sj23HD-HSA fusion protein was prepared by overlapping PCR, which was confirmed by TA cloning and DNA sequencing. The fusion gene of Sj23HD-HSA was directionally subcloned into yeast expression plasmid pWX530 to construct a recombinant plasmid Sj23HD-HSA/pWX530. The transformants of Saccharomyces cerevisiae containing the recombinant plasmid Sj23HD-HSA/pWX530 were screened on leu deficient SD medium after yeast competent cells were transformed with recombinant plasmid. The excretive Sj23HD-HSA protein was expressed by culturing the yeast transformants at 30 degrees C for 1 week, and the protein component of culture supernatant was analyzed by SDS-PAGE. Sj23HD-HSA fusion protein was purified through Ion Exchange Chromatography. The immunoreactivity of recombinant Sj23HD-HSA fusion protein was determined by Western blotting with sera of schistosomiasis, clonorchiasis and healthy. The gene encoding the Sj23HD-HSA fusion protein was constructed successfully, which was confirmed by DNA sequencing. The yeast transformants containing plasmid Sj23HD-HSA/pWX530 could express the excretive Sj23HD-HSA fusion protein without inducing. The results of Western blotting indicated Sj23HD-HSA could be recognized by the sera of schistosomiasis, but could not be recognized by the sera of clonorchiasis and healthy respectively. Sj23HD-HSA fusion protein with good immune reactivity is prepared successfully, which will be a potential antigen for schistosomiasis immunodiagnosis.

  19. Use of Cre/loxP recombination to swap cell binding motifs on the adenoviral capsid protein IX

    International Nuclear Information System (INIS)

    Poulin, Kathy L.; Tong, Grace; Vorobyova, Olga; Pool, Madeline; Kothary, Rashmi; Parks, Robin J.

    2011-01-01

    We used Cre/loxP recombination to swap targeting ligands present on the adenoviral capsid protein IX (pIX). A loxP-flanked sequence encoding poly-lysine (pK-binds heparan sulfate proteoglycans) was engineered onto the 3'-terminus of pIX, and the resulting fusion protein allowed for routine virus propagation. Growth of this virus on Cre-expressing cells removed the pK coding sequence, generating virus that could only infect through alternative ligands, such as a tyrosine kinase receptor A (TrkA)-binding motif engineered into the capsid fibre protein for enhanced infection of neuronal cells. We used a similar approach to swap the pK motif on pIX for a sequence encoding a single-domain antibody directed towards CD66c for targeted infection of cancer cells; Cre-mediated removal of the pK-coding sequence simultaneously placed the single-domain antibody coding sequence in frame with pIX. Thus, we have developed a simple method to propagate virus lacking native viral tropism but containing cell-specific binding ligands. - Highlights: → We describe a method to grow virus lacking native tropism but containing novel cell-binding ligands. → Cre/loxP recombination was used to modify the adenovirus genome. → A targeting ligand present on capsid protein IX was removed or replaced using recombination. → Cre-loxP was also used to 'swap' the identity of the targeting ligand present on pIX.

  20. Expression and affinity purification of recombinant proteins from plants

    Science.gov (United States)

    Desai, Urvee A.; Sur, Gargi; Daunert, Sylvia; Babbitt, Ruth; Li, Qingshun

    2002-01-01

    With recent advances in plant biotechnology, transgenic plants have been targeted as an inexpensive means for the mass production of proteins for biopharmaceutical and industrial uses. However, the current plant purification techniques lack a generally applicable, economic, large-scale strategy. In this study, we demonstrate the purification of a model protein, beta-glucuronidase (GUS), by employing the protein calmodulin (CaM) as an affinity tag. In the proposed system, CaM is fused to GUS. In the presence of calcium, the calmodulin fusion protein binds specifically to a phenothiazine-modified surface of an affinity column. When calcium is removed with a complexing agent, e.g., EDTA, calmodulin undergoes a conformational change allowing the dissociation of the calmodulin-phenothiazine complex and, therefore, permitting the elution of the GUS-CaM fusion protein. The advantages of this approach are the fast, efficient, and economical isolation of the target protein under mild elution conditions, thus preserving the activity of the target protein. Two types of transformation methods were used in this study, namely, the Agrobacterium-mediated system and the viral-vector-mediated transformation system. Copyright 2002 Elsevier Science (USA).

  1. Overview of systems and techniques for surface display of recombinant proteins in yeast S. cerevisiae

    Directory of Open Access Journals (Sweden)

    Renata Teparic

    2015-12-01

    Full Text Available In the past decade much effort has been devoted to the development of new expression systems and novel techniques for the surface display of heterologous proteins in yeast in order to improve their applications in biotechnology, food technology, pharmacology and medicine. Heterologous protein-encoding genes are generally fused with genes coding for yeast cell wall proteins or their fragments required for anchoring. The variety of reactions by which a protein can be displayed at the cell surface enables finding the appropriate one for each individual protein. However, it is still challenging how to improve the efficiency of display of protein complexes and increase the quantity of protein displayed on the yeast surface. Recently, synthetic protein chimeras that self-assemble into the scaffolds on the yeast surface displaying different proteins have been constructed. This review focuses on systems and techniques for display of recombinant proteins on the yeast cell surfaces and applications afforded by this technology.

  2. Recombinant fusion proteins TAT-Mu, Mu and Mu-Mu mediate efficient non-viral gene delivery.

    Science.gov (United States)

    Rajagopalan, Rukkumani; Xavier, Jennifer; Rangaraj, Nandini; Rao, Nalam Madhusudhana; Gopal, Vijaya

    2007-04-01

    The inherent ability of certain peptides or proteins of viral, prokaryotic and eukaryotic origin to bind DNA was used to generate novel peptide-based DNA delivery protocols. We have developed a recombinant approach to make fusion proteins with motifs for DNA-binding ability, Mu and membrane transduction domains, TAT, and tested them for their DNA-binding, uptake and transfection efficiencies. In one of the constructs, the recombinant plasmid was designed to encode the Mu moiety of sequence MRRAHHRRRRASHRRMRGG in-frame with TAT of sequence YGRKKRRQRRR to generate TAT-Mu, while the other two constructs, Mu and Mu-Mu, harbor a single copy or two copies of the Mu moiety. Recombinant his-tag fusion proteins TAT-Mu, Mu and Mu-Mu were purified by overexpression of plasmid constructs using cobalt-based affinity resins. The peptides were characterized for their size and interaction with DNA, complexed with plasmid pCMVbeta-gal, and shown to transfect MCF-7, COS and CHOK-1 cells efficiently. Recombinant fusion proteins TAT-Mu, Mu and Mu-Mu were cloned and overexpressed in BL21(DE3)pLysS with greater than 95% purity. The molecular weight of TAT-Mu was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to be 11.34 kDa while those of Mu and Mu-Mu were 7.78 and 9.83 kDa, respectively. Live uptake analysis of TAT-Mu, Mu and Mu-Mu as DP (DNA+peptide) or DPL (DNA+peptide+lipid) complexes into MCF-7 cells, followed by immunostaining and laser scanning confocal microscopy, demonstrated that the complexes are internalized very efficiently and localized in the nucleus. DNA:peptide complexes (DP) transfect MCF-7, COS and CHOK-1 cells. The addition of cationic liposomes enhances the uptake of the ternary complexes (DPL) further and also brings about 3-7-fold enhancement in reporter gene expression compared to DP alone. Recombinant proteins that are heterologous fusions, having DNA-binding domains and nuclear localization epitopes

  3. Detoxifying Escherichia coli for endotoxin-free production of recombinant proteins.

    Science.gov (United States)

    Mamat, Uwe; Wilke, Kathleen; Bramhill, David; Schromm, Andra Beate; Lindner, Buko; Kohl, Thomas Andreas; Corchero, José Luis; Villaverde, Antonio; Schaffer, Lana; Head, Steven Robert; Souvignier, Chad; Meredith, Timothy Charles; Woodard, Ronald Wesley

    2015-04-16

    Lipopolysaccharide (LPS), also referred to as endotoxin, is the major constituent of the outer leaflet of the outer membrane of virtually all Gram-negative bacteria. The lipid A moiety, which anchors the LPS molecule to the outer membrane, acts as a potent agonist for Toll-like receptor 4/myeloid differentiation factor 2-mediated pro-inflammatory activity in mammals and, thus, represents the endotoxic principle of LPS. Recombinant proteins, commonly manufactured in Escherichia coli, are generally contaminated with endotoxin. Removal of bacterial endotoxin from recombinant therapeutic proteins is a challenging and expensive process that has been necessary to ensure the safety of the final product. As an alternative strategy for common endotoxin removal methods, we have developed a series of E. coli strains that are able to grow and express recombinant proteins with the endotoxin precursor lipid IVA as the only LPS-related molecule in their outer membranes. Lipid IVA does not trigger an endotoxic response in humans typical of bacterial LPS chemotypes. Hence the engineered cells themselves, and the purified proteins expressed within these cells display extremely low endotoxin levels. This paper describes the preparation and characterization of endotoxin-free E. coli strains, and demonstrates the direct production of recombinant proteins with negligible endotoxin contamination.

  4. Construction and expression of recombinant fusion protein of thioredoxin-ApoO.

    Science.gov (United States)

    Wu, Chenlu; Zhao, Shuiping; Yu, Bilian; Xiong, Dan

    2011-02-01

    To construct human apolipoprotein O (apolipoprotein O, ApoO) expression vector and obtain recombinant fusion protein thioredoxin (Trx)-ApoO by pET prokaryotic expression system. The ApoO gene fragment from the human liver cDNA library was amplified by PCR. The resulting product was cloned into pET-32a(+) vector and sequenced. The confirmed cDNA was cloned into plasmid E.coli DH10B and then transformed into E.coli BL 21 (DE3) where it was induced to express protein by isopropyl β-D-1-thiogalactopyranoside (IPTG). The fusion protein was purified by Ni-NTA resin. The ApoO gene was cloned by PCR and a 519 bp DNA fragment was shown on the agarose electrophoresis. The cloned gene was sequenced and demonstrated to have the same sequence as that of human ApoO gene in GenBank which justified a successful construction of recombinant plasmid. ApoO cDNA gene fragment was induced by IPTG, and a 34 kD recombinant fusion protein Trx-ApoO was tested on sodium dodecyl sulfate polyacrylamide (SDS-PAGE). Human ApoO gene is successfully cloned and its recombinant fusion protein Trx-ApoO is expressed.

  5. TA-GC cloning: A new simple and versatile technique for the directional cloning of PCR products for recombinant protein expression.

    Directory of Open Access Journals (Sweden)

    Athanasios Niarchos

    Full Text Available During the last few decades, the recombinant protein expression finds more and more applications. The cloning of protein-coding genes into expression vectors is required to be directional for proper expression, and versatile in order to facilitate gene insertion in multiple different vectors for expression tests. In this study, the TA-GC cloning method is proposed, as a new, simple and efficient method for the directional cloning of protein-coding genes in expression vectors. The presented method features several advantages over existing methods, which tend to be relatively more labour intensive, inflexible or expensive. The proposed method relies on the complementarity between single A- and G-overhangs of the protein-coding gene, obtained after a short incubation with T4 DNA polymerase, and T and C overhangs of the novel vector pET-BccI, created after digestion with the restriction endonuclease BccI. The novel protein-expression vector pET-BccI also facilitates the screening of transformed colonies for recombinant transformants. Evaluation experiments of the proposed TA-GC cloning method showed that 81% of the transformed colonies contained recombinant pET-BccI plasmids, and 98% of the recombinant colonies expressed the desired protein. This demonstrates that TA-GC cloning could be a valuable method for cloning protein-coding genes in expression vectors.

  6. Contraceptive efficacy of recombinant fusion protein comprising zona pellucida glycoprotein-3 fragment and gonadotropin releasing hormone.

    Science.gov (United States)

    Arukha, Ananta Prasad; Minhas, Vidisha; Shrestha, Abhinav; Gupta, Satish Kumar

    2016-04-01

    Contraceptive vaccines have been used for the management of wildlife population. In the present study, we have examined the contraceptive potential of Escherichia coli-expressed recombinant fusion protein comprising of 'promiscuous' T cell epitope of tetanus toxoid [TT; amino acid (aa) residues 830-844] followed by dilysine linker (KK), dog ZP3 fragment (aa residues 307-346), triglycine spacer (GGG), T cell epitope of bovine RNase (bRNase; aa residues 94-104), GnRH, T cell epitope of circumsporozoite protein of Plasmodium falciparum (CSP; aa residues 362-383), and GnRH. SDS-PAGE analysis of the purified refolded protein revealed a dominant ∼12 kDa band, which in Western blot reacted with mouse polyclonal antibodies against dog ZP3 fragment and mouse monoclonal antibodies against GnRH. Immunization of female FvB/J mice following two booster schedule with the above recombinant protein supplemented with alum led to high antibody titres against the immunogen as well as ZP3 and GnRH as determined by ELISA. The immune sera reacted with zona pellucida of mouse oocyte and also inhibited in-vitro fertilization. The qRT-PCR studies showed decrease in the ovarian GnRH receptor in mice immunized with the recombinant fusion protein. Mating studies revealed high contraceptive efficacy of the recombinant protein as in two independent experiments, 90% of the immunized female mice failed to conceive. Following one booster immunization schedule, 50% of the immunized female mice failed to conceive. However, in adjuvanted controls, all the female mice became pregnant. To conclude, the recombinant protein described herein has a good potential to be developed as candidate contraceptive vaccine. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  7. The effect of alcohol on recombinant proteins derived from mammalian adenylyl cyclase

    Directory of Open Access Journals (Sweden)

    Emily Qualls-Creekmore

    2017-07-01

    Full Text Available The cyclic AMP (cAMP signaling pathway is implicated in the development of alcohol use disorder. Previous studies have demonstrated that ethanol enhances the activity of adenylyl cyclase (AC in an isoform specific manner; AC7 is most enhanced by ethanol, and regions responsible for enhancement by ethanol are located in the cytoplasmic domains of the AC7 protein. We hypothesize that ethanol modulates AC activity by directly interacting with the protein and that ethanol effects on AC can be studied using recombinant AC in vitro. AC recombinant proteins containing only the C1a or C2 domains of AC7 and AC9 individually were expressed in bacteria, and purified. The purified recombinant AC proteins retained enzymatic activity and isoform specific alcohol responsiveness. The combination of the C1a or C2 domains of AC7 maintained the same alcohol cutoff point as full-length AC7. We also find that the recombinant AC7 responds to alcohol differently in the presence of different combinations of activators including MnCl2, forskolin, and Gsα. Through a series of concentration-response experiments and curve fitting, the values for maximum activities, Hill coefficients, and EC50 were determined in the absence and presence of butanol as a surrogate of ethanol. The results suggest that alcohol modulates AC activity by directly interacting with the AC protein and that the alcohol interaction with the AC protein occurs at multiple sites with positive cooperativity. This study indicates that the recombinant AC proteins expressed in bacteria can provide a useful model system to investigate the mechanism of alcohol action on their activity.

  8. The Mycoplasma hyopneumoniae recombinant heat shock protein P42 induces an immune response in pigs under field conditions.

    Science.gov (United States)

    Jorge, Sérgio; de Oliveira, Natasha Rodrigues; Marchioro, Silvana Beutinger; Fisch, Andressa; Gomes, Charles Klazer; Hartleben, Cláudia Pinho; Conceição, Fabricio Rochedo; Dellagostin, Odir Antonio

    2014-09-01

    Enzootic pneumonia (EP), resulting from Mycoplasma hyopneumoniae infection is one of the most prevalent diseases in pigs and is a major cause of economic losses to the swine industry worldwide. EP is often controlled by vaccination with inactivated, adjuvanted whole-cell bacterin. However, these bacterins provide only partial protection and do not prevent M. hyopneumoniae colonization. Attempts to develop vaccines that are more efficient have made use of the recombinant DNA technology. The objective of this study was to assess the potential of recombinant M. hyopneumoniae heat shock protein P42 in vaccine preparations against EP, using piglets housed under field conditions in a M. hyopneumoniae-positive farm. The cellular and humoral immune responses were elicited after a single intramuscular inoculation of rP42 in an oil-based adjuvant, or in conjunction with whole-cell vaccine preparation. The production of INF-γ and IL-10 cytokines was quantified in the supernatant of the cultured mononuclear cells. The rP42 emulsified in oil-based adjuvant was able to trigger a strong humoral immune response. Further, it induced a cellular immune response, accompanied by the production of antibodies that reacted with the native M. hyopneumoniae protein. The rP42 mediated induction of cellular and humoral immune response in the host suggests that rP42 emulsified in an oil-based adjuvant holds promise as an effective recombinant subunit vaccine against EP. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Expression and diagnostic use of recombinant M protein of the porcine reproductive and respiratory syndrome virus

    Directory of Open Access Journals (Sweden)

    Jitka Frölichová

    2017-01-01

    Full Text Available Matrix M protein combined with nucleocapsid N protein could be a promising combination of virus antigens for diagnosing the porcine reproductive and respiratory syndrome. The goal of this work was to express the recombinant M protein of the porcine reproductive and respiratory syndrome virus in Escherichia coli cells and compare its serological reactivity with the N protein of the virus. The gene coding for the M protein was cloned into the pDest17 vector. The resulting protein was purified by metalochelating affinity chromatography. Recombinant M protein was applied as an antigen in immunoblot test and compared on a panel of porcine sera with N protein based IDEXX test. Of 120 examined samples, the majority (78.3% gave identical results using both compared tests. From the group of discrepant results, IDEXX test identified considerably more positive sera (17.5% than M protein based test (4.2%. The main contribution of the work is finding that although IDEXX test proved to be more sensitive than M protein based test, 4.2% of sera would escape detection by serological test based on N protein. Further development and purification of the M protein for the use in Enzyme Linked Immunosorbent Assay format test could increase the performance of serological testing.

  10. Acting on Folding Effectors to Improve Recombinant Protein Yields and Functional Quality.

    Science.gov (United States)

    de Marco, Ario

    2017-01-01

    Molecular and chemical chaperones /foldases can strongly contribute to improve the amounts and the structural quality of recombinant proteins. Several methodologies have been proposed to optimize their beneficial effects. This chapter presents a condensed summary of the biotechnological opportunities offered by this approach followed by a protocol describing the method we use for expressing disulfide bond-dependent recombinant antibodies in the cytoplasm of bacteria engineered to overexpress sulfhydryl oxidase and DsbC isomerase. The system is based on the possibility to trigger the foldase expression independently and before the induction of the target protein. As a consequence, the recombinant antibody synthesis starts only after enough foldases have accumulated to promote correct folding of the antibody.

  11. Effect of Plasmid Design and Type of Integration Event on Recombinant Protein Expression in Pichia pastoris.

    Science.gov (United States)

    Vogl, Thomas; Gebbie, Leigh; Palfreyman, Robin W; Speight, Robert

    2018-03-15

    Pichia pastoris (syn. Komagataella phaffii ) is one of the most common eukaryotic expression systems for heterologous protein production. Expression cassettes are typically integrated in the genome to obtain stable expression strains. In contrast to Saccharomyces cerevisiae , where short overhangs are sufficient to target highly specific integration, long overhangs are more efficient in P. pastoris and ectopic integration of foreign DNA can occur. Here, we aimed to elucidate the influence of ectopic integration by high-throughput screening of >700 transformants and whole-genome sequencing of 27 transformants. Different vector designs and linearization approaches were used to mimic the most common integration events targeted in P. pastoris Fluorescence of an enhanced green fluorescent protein (eGFP) reporter protein was highly uniform among transformants when the expression cassettes were correctly integrated in the targeted locus. Surprisingly, most nonspecifically integrated transformants showed highly uniform expression that was comparable to specific integration, suggesting that nonspecific integration does not necessarily influence expression. However, a few clones (integrated cassettes showed a greater variation spanning a 25-fold range, surpassing specifically integrated reference strains up to 6-fold. High-expression strains showed a correlation between increased gene copy numbers and high reporter protein fluorescence levels. Our results suggest that for comparing expression levels between strains, the integration locus can be neglected as long as a sufficient numbers of transformed strains are compared. For expression optimization of highly expressible proteins, increasing copy number appears to be the dominant positive influence rather than the integration locus, genomic rearrangements, deletions, or single-nucleotide polymorphisms (SNPs). IMPORTANCE Yeasts are commonly used as biotechnological production hosts for proteins and metabolites. In the yeast

  12. Automated production of recombinant human proteins as resource for proteome research

    Directory of Open Access Journals (Sweden)

    Poustka Annemarie

    2008-01-01

    Full Text Available Abstract Background An arbitrary set of 96 human proteins was selected and tested to set-up a fully automated protein production strategy, covering all steps from DNA preparation to protein purification and analysis. The target proteins are encoded by functionally uncharacterized open reading frames (ORF identified by the German cDNA consortium. Fusion proteins were produced in E. coli with four different fusion tags and tested in five different purification strategies depending on the respective fusion tag. The automated strategy relies on standard liquid handling and clone picking equipment. Results A robust automated strategy for the production of recombinant human proteins in E. coli was established based on a set of four different protein expression vectors resulting in NusA/His, MBP/His, GST and His-tagged proteins. The yield of soluble fusion protein was correlated with the induction temperature and the respective fusion tag. NusA/His and MBP/His fusion proteins are best expressed at low temperature (25°C, whereas the yield of soluble GST fusion proteins was higher when protein expression was induced at elevated temperature. In contrast, the induction of soluble His-tagged fusion proteins was independent of the temperature. Amylose was not found useful for affinity-purification of MBP/His fusion proteins in a high-throughput setting, and metal chelating chromatography is recommended instead. Conclusion Soluble fusion proteins can be produced in E. coli in sufficient qualities and μg/ml culture quantities for downstream applications like microarray-based assays, and studies on protein-protein interactions employing a fully automated protein expression and purification strategy. Future applications might include the optimization of experimental conditions for the large-scale production of soluble recombinant proteins from libraries of open reading frames.

  13. A novel, lactase-based selection and strain improvement strategy for recombinant protein expression in Kluyveromyces lactis

    Directory of Open Access Journals (Sweden)

    Krijger Jorrit-Jan

    2012-08-01

    Full Text Available Abstract Background The Crabtree-negative yeast species Kluyveromyces lactis has been established as an attractive microbial expression system for recombinant proteins at industrial scale. Its LAC genes allow for utilization of the inexpensive sugar lactose as a sole source of carbon and energy. Lactose efficiently induces the LAC4 promoter, which can be used to drive regulated expression of heterologous genes. So far, strain manipulation of K. lactis by homologous recombination was hampered by the high rate of non-homologous end-joining. Results Selection for growth on lactose was applied to target the insertion of heterologous genes downstream of the LAC4 promoter into the K. lactis genome and found to yield high numbers of positive transformants. Concurrent reconstitution of the β-galactosidase gene indicated the desired integration event of the expression cassette, and β-galactosidase activity measurements were used to monitor gene expression for strain improvement and fermentation optimization. The system was particularly improved by usage of a cell lysis resistant strain, VAK367-D4, which allowed for protein accumulation in long-term fermentation. Further optimization was achieved by increased gene dosage of KlGAL4 encoding the activator of lactose and galactose metabolic genes that led to elevated transcription rates. Pilot experiments were performed with strains expressing a single-chain antibody fragment (scFvox and a viral envelope protein (BVDV-E2, respectively. scFvox was shown to be secreted into the culture medium in an active, epitope-binding form indicating correct processing and protein folding; the E2 protein could be expressed intracellularly. Further data on the influence of protein toxicity on batch fermentation and potential post-transcriptional bottlenecks in protein accumulation were obtained. Conclusions A novel Kluyveromyces lactis host-vector system was developed that places heterologous genes under the control of

  14. [Expression and activity determination of recombinant capsid protein VP2 gene of enterovirus type 71].

    Science.gov (United States)

    Huang, Xueyong; Liu, Guohua; Hu, Xiaoning; Du, Yanhua; Li, Xingle; Xu, Yuling; Chen, Haomin; Xu, Bianli

    2014-04-01

    To clone and express the recombinant capsid protein VP2 of enterovirus type 71 (EV71) and to identify the immune activity of expressed protein in order to build a basis for the investigation work of vaccine and diagnostic antigen. VP2 gene of EV71 was amplified by PCR, and then was cut by restriction enzyme and inserted into expression vector pMAL-c2X. The positive recombinants were transferred into E.coli TB1, the genetically engineered bacteria including pMAL-c2X-VP2 plasmids were induced by isopropyl thiogalactoside ( IPTG) , and the expression products were analyzed by SDS-PAGE and western blotting method. EV71 IgM antibody detection method by ELISA was set up, and the sensitivity and specificity of this method was assessed; 60 neutralizing antibody positive serum samples from hand foot and mouth disease (HFMD) patients were determined, of which 52 samples were positive and 8 samples were negative; a total of 88 acute phase serum samples of HFMD patients diagnosed in clinical were also detected. VP2 gene of 762 bp was obtained by PCR, the gene segment inserted into the recombinant vector was identified using restriction enzyme digestion. The recombinant vector could express a specific about 71 500 fusion protein in E.coli by SDS-PAGE. The purified recombinant protein of EV71-VP2 can react with the serum of HFMD patients to produce a specific band by western blotting. The sensitivity and specificity of ELISA was 87% and 83%, respectively. Of the 88 acute phase serum samples from children with HFMD, 48 samples (55%) were positive by the ELISA assay. VP2 gene of EV71 has been cloned and a prokaryotic high expression system for VP2 gene was successfully constructed in the present study. The recombination EV71-VP2 has well antigenicity, which could be useful for developing diagnose reagent or vaccine of EV71.

  15. PCR-based gene synthesis to produce recombinant proteins for crystallization

    Directory of Open Access Journals (Sweden)

    Byrne-Steele Miranda L

    2008-04-01

    Full Text Available Abstract Background Gene synthesis technologies are an important tool for structural biology projects, allowing increased protein expression through codon optimization and facilitating sequence alterations. Existing methods, however, can be complex and not always reproducible, prompting researchers to use commercial suppliers rather than synthesize genes themselves. Results A PCR-based gene synthesis method, referred to as SeqTBIO, is described to efficiently assemble the coding regions of two novel hyperthermophilic proteins, PAZ (Piwi/Argonaute/Zwille domain, a siRNA-binding domain of an Argonaute protein homologue and a deletion mutant of a family A DNA polymerase (PolA. The gene synthesis procedure is based on sequential assembly such that homogeneous DNA products can be obtained after each synthesis step without extensive manipulation or purification requirements. Coupling the gene synthesis procedure to in vivo homologous recombination techniques allows efficient subcloning and site-directed mutagenesis for error correction. The recombinant proteins of PAZ and PolA were subsequently overexpressed in E. coli and used for protein crystallization. Crystals of both proteins were obtained and they were suitable for X-ray analysis. Conclusion We demonstrate, by using PAZ and PolA as examples, the feasibility of integrating the gene synthesis, error correction and subcloning techniques into a non-automated gene to crystal pipeline such that genes can be designed, synthesized and implemented for recombinant expression and protein crystallization.

  16. A dual protease approach for expression and affinity purification of recombinant proteins.

    Science.gov (United States)

    Raran-Kurussi, Sreejith; Waugh, David S

    2016-07-01

    We describe a new method for affinity purification of recombinant proteins using a dual protease protocol. Escherichia coli maltose binding protein (MBP) is employed as an N-terminal tag to increase the yield and solubility of its fusion partners. The MBP moiety is then removed by rhinovirus 3C protease, prior to purification, to yield an N-terminally His6-tagged protein. Proteins that are only temporarily rendered soluble by fusing them to MBP are readily identified at this stage because they will precipitate after the MBP tag is removed by 3C protease. The remaining soluble His6-tagged protein, if any, is subsequently purified by immobilized metal affinity chromatography (IMAC). Finally, the N-terminal His6 tag is removed by His6-tagged tobacco etch virus (TEV) protease to yield the native recombinant protein, and the His6-tagged contaminants are removed by adsorption during a second round of IMAC, leaving only the untagged recombinant protein in the column effluent. The generic strategy described here saves time and effort by removing insoluble aggregates at an early stage in the process while also reducing the tendency of MBP to "stick" to its fusion partners during affinity purification. Published by Elsevier Inc.

  17. Roles of silkworm endoplasmic reticulum chaperones in the secretion of recombinant proteins expressed by baculovirus system.

    Science.gov (United States)

    Imai, Saki; Kusakabe, Takahiro; Xu, Jian; Li, Zhiqing; Shirai, Shintaro; Mon, Hiroaki; Morokuma, Daisuke; Lee, Jae Man

    2015-11-01

    Baculovirus expression vector system (BEVS) is widely used for production of recombinant eukaryotic proteins in insect larvae or cultured cells. BEVS has advantages over bacterial expression system in producing post-translationally modified secreted proteins. However, for some unknown reason, it is very difficult for insects to secrete sufficiently for certain proteins of interest. To understand the reasons why insect cells fail to secrete some kinds of recombinant proteins, we here employed three mammalian proteins as targets, EPO, HGF, and Wnt3A, with different secretion levels in BEVS and investigated their mRNA transcriptions from the viral genome, subcellular localizations, and interactions with silkworm ER chaperones. Moreover, we observed that no significantly influence on the secretion amounts of all three proteins when depleting or overexpressing most endogenous ER chaperone genes in cultured silkworm cells. However, among all detected ER chaperones, the depletion of BiP severely decreased the recombinant protein secretion in BEVS, indicating the possible central role of Bip in silkworm secretion pathway.

  18. Genome-scale metabolic model of Pichia pastoris with native and humanized glycosylation of recombinant proteins.

    Science.gov (United States)

    Irani, Zahra Azimzadeh; Kerkhoven, Eduard J; Shojaosadati, Seyed Abbas; Nielsen, Jens

    2016-05-01

    Pichia pastoris is used for commercial production of human therapeutic proteins, and genome-scale models of P. pastoris metabolism have been generated in the past to study the metabolism and associated protein production by this yeast. A major challenge with clinical usage of recombinant proteins produced by P. pastoris is the difference in N-glycosylation of proteins produced by humans and this yeast. However, through metabolic engineering, a P. pastoris strain capable of producing humanized N-glycosylated proteins was constructed. The current genome-scale models of P. pastoris do not address native nor humanized N-glycosylation, and we therefore developed ihGlycopastoris, an extension to the iLC915 model with both native and humanized N-glycosylation for recombinant protein production, but also an estimation of N-glycosylation of P. pastoris native proteins. This new model gives a better prediction of protein yield, demonstrates the effect of the different types of N-glycosylation of protein yield, and can be used to predict potential targets for strain improvement. The model represents a step towards a more complete description of protein production in P. pastoris, which is required for using these models to understand and optimize protein production processes. © 2015 Wiley Periodicals, Inc.

  19. Recombinant protein-based assays for detection of antibodies to severe acute respiratory syndrome coronavirus spike and nucleocapsid proteins.

    Science.gov (United States)

    Haynes, Lia M; Miao, Congrong; Harcourt, Jennifer L; Montgomery, Joel M; Le, Mai Quynh; Dryga, Sergey A; Kamrud, Kurt I; Rivers, Bryan; Babcock, Gregory J; Oliver, Jennifer Betts; Comer, James A; Reynolds, Mary; Uyeki, Timothy M; Bausch, Daniel; Ksiazek, Thomas; Thomas, William; Alterson, Harold; Smith, Jonathan; Ambrosino, Donna M; Anderson, Larry J

    2007-03-01

    Recombinant severe acute respiratory syndrome (SARS) nucleocapsid and spike protein-based immunoglobulin G immunoassays were developed and evaluated. Our assays demonstrated high sensitivity and specificity to the SARS coronavirus in sera collected from patients as late as 2 years postonset of symptoms. These assays will be useful not only for routine SARS coronavirus diagnostics but also for epidemiological and antibody kinetic studies.

  20. Recombinant activated protein C attenuates coagulopathy and inflammation when administered early in murine pneumococcal pneumonia

    NARCIS (Netherlands)

    Schouten, Marcel; van 't Veer, Cornelis; Roelofs, Joris J. T. H.; Gerlitz, Bruce; Grinnell, Brian W.; Levi, Marcel; van der Poll, Tom

    2011-01-01

    Recombinant human activated protein C (APC), which has both anticoagulant and anti-inflammatory properties, improves survival of patients with severe sepsis. This beneficial effect is especially apparent in patients with pneumococcal pneumonia. Earlier treatment with APC in sepsis has been

  1. Production of recombinant proteins GST L1, E6 and E7 tag HPV 16 ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-02-04

    goat anti-human IgG diluted 1:1000 in casein buffer) was added .... The purified recombinant proteins were separated in silver-stained sodium dodecyl sulphate polyacrylamide gels. Production of antigens in plants is known to ...

  2. Human recombinant protein C for severe sepsis and septic shock in adult and paediatric patients

    DEFF Research Database (Denmark)

    Martí-Carvajal, Arturo J; Solà, Ivan; Gluud, Christian

    2012-01-01

    Sepsis is a common and frequently fatal condition. Human recombinant activated protein C (APC) has been introduced to reduce the high risk of death associated with severe sepsis or septic shock. This systematic review is an update of a Cochrane review originally published in 2007....

  3. Production of recombinant proteins GST L1, E6 and E7 tag HPV 16 ...

    African Journals Online (AJOL)

    In the present work recombinant proteins were produced for used in LUMINEX in order to undergo serological study of Tunisian female population. HPV types 16 L1, E6 and E7 sequences fused to their 3'-end to a sequence encoding the terminal undecapeptide of the SV40 large T-antigen (tag) were isolated from plasmids ...

  4. Preparation and use of recombinant protein G-gold complexes as markers in double labelling immunocytochemistry

    DEFF Research Database (Denmark)

    Balslev, Y; Hansen, Gert Helge

    1989-01-01

    Recombinant protein G (RPG) was conjugated to colloidal gold particles and used for immunocytochemistry. In this report, the preparation of RPG-gold conjugates (RPGG) and the application of these conjugates in spot blot tests and in double immunolabelling are described. The immunolabelling...

  5. Purification and characterization of recombinant protein kinase CK2 from Zea mays expressed in Escherichia coli

    DEFF Research Database (Denmark)

    Riera, Marta; Pages, Montserrat; Issinger, Olaf Georg

    2003-01-01

    Recombinant protein kinase subunits rmCK2alpha-1 and rmCK2beta-1 from Zea mays were expressed separately in Escherichia coli and assembled to a fully active tetrameric holoenzyme complex in vitro. The obtained maize holoenzyme was purified to homogeneity, biochemically characterized, and compared...

  6. Polyclonal Antibodies to a Recombinant Coat Protein of Potato Virus A

    Czech Academy of Sciences Publication Activity Database

    Čeřovská, Noemi; Moravec, Tomáš; Velemínský, Jiří

    2002-01-01

    Roč. 46, - (2002), s. 147-151 ISSN 0001-723X R&D Projects: GA ČR GA310/00/0381 Institutional research plan: CEZ:AV0Z5038910 Keywords : Potato virus A * recombinant coat protein * Escherichia coli Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.660, year: 2002

  7. Analysis of the lipidated recombinant outer surface protein A from Borrelia burgdorferi by mass spectrometry

    NARCIS (Netherlands)

    Bouchon, B.; Klein, Michele; Bischoff, Rainer; Van Dorsselaer, A.; Roitsch, C.

    1997-01-01

    The outer surface protein A, OspA, from the spirochete Borrelia burgdorferi is a lipoprotein of 25 kDa. The recombinant OspA (rOspA) expressed in Escherichia coli has been purified and analyzed by electrospray mass spectrometry (ESMS). A heterogenous spectrum gave a measured mass of 28,462 +/- 9 Da

  8. Production of Polyclonal Antibodies to a Recombinant Coat Protein of Potato mop-top virus

    Czech Academy of Sciences Publication Activity Database

    Čeřovská, Noemi; Moravec, Tomáš; Rosecká, Pavla; Dědič, P.; Filigarová, Marie

    2003-01-01

    Roč. 151, č. 4 (2003), s. 195-200 ISSN 0931-1785 R&D Projects: GA ČR GA522/01/1121 Institutional research plan: CEZ:AV0Z5038910 Keywords : potato mop-top virus * recombinant coat protein * Escherichia Coli Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.557, year: 2003

  9. Diagnosis of paracoccidioidomycosis by a dot blot assay using a recombinant Paracoccidioides brasiliensis p27 protein.

    Science.gov (United States)

    Correa, M M; Bedoya, A M; Guerrero, M P; Méndez, J; Restrepo, A; McEwen, J G

    2007-01-01

    A variety of immunological methods have proven useful for Paracoccidioidomycosis (PCM) diagnosis; however, they are often time consuming and many lack sensitivity and specificity, partially attributed to the use of crude antigens, which give cross reactivity. Until now, attempts to clone and express Paracoccidioides brasiliensis immunodominant antigens have presented difficulties of process and problems of cost. In an attempt to obtain a more rapid, sensitive, and specific test for PCM diagnosis, we subcloned the P. brasiliensis p27 gene and used the recombinant protein as the antigen in dot blot assays to evaluate its usefulness in paracoccidioidomicosis diagnosis. The development of an optimised procedure for p27 recombinant protein purification and production led to an easier and less expensive process than the one previously used in our laboratory and allowed the availability of enough purified protein for its evaluation as the antigen in the dot blot assays. In these assays, antibodies present in ten serum samples from seven patients with PCM recognised the recombinant protein showing a sensitivity of 100% with a specificity of 98%. These results confirm the value of the 27-kDa recombinant antigen in the serodiagnosis of paracoccidioidomycosis and that the dot blot format is an alternative to the immunoenzymatic assay procedure.

  10. A novel multi-epitope recombined protein for diagnosis of human brucellosis.

    Science.gov (United States)

    Yin, Dehui; Li, Li; Song, Xiuling; Li, Han; Wang, Juan; Ju, Wen; Qu, Xiaofeng; Song, Dandan; Liu, Yushen; Meng, Xiangjun; Cao, Hongqian; Song, Weiyi; Meng, Rizeng; Liu, Jinhua; Li, Juan; Xu, Kun

    2016-05-21

    In epidemic regions of the world, brucellosis is a reemerging zoonosis with minimal mortality but is a serious public hygiene problem. Currently, there are various methods for brucellosis diagnosis, however few of them are available to be used to diagnose, especially for serious cross-reaction with other bacteria. To overcome this disadvantage, we explored a novel multi-epitope recombinant protein as human brucellosis diagnostic antigen. We established an indirect enzyme-linked immunosorbent assay (ELISA) based on this recombinant protein. 248 sera obtained from three different groups including patients with brucellosis (146 samples), non-brucellosis patients (82 samples), and healthy individuals (20 samples) were tested by indirect ELISA. To evaluate the assay, a receiver-operating characteristic (ROC) analysis and immunoblotting were carried out using these characterized serum samples. For this test, the area under the ROC curve was 0.9409 (95 % confidence interval, 0.9108 to 0.9709), and a sensitivity of 88.89 % and a specificity of 85.54 % was given with a cutoff value of 0.3865 from this ROC analysis. The Western blot results indicate that it is feasible to differentiate human brucellosis and non-brucellosis with the newly established method based on this recombinant protein. Our results obtained high diagnostic accuracy of the ELISA assay which encourage the use of this novel recombinant protein as diagnostic antigen to implement serological diagnosis of brucellosis.

  11. Recent advances in recombinant protein-based malaria vaccines.

    Science.gov (United States)

    Draper, Simon J; Angov, Evelina; Horii, Toshihiro; Miller, Louis H; Srinivasan, Prakash; Theisen, Michael; Biswas, Sumi

    2015-12-22

    Plasmodium parasites are the causative agent of human malaria, and the development of a highly effective vaccine against infection, disease and transmission remains a key priority. It is widely established that multiple stages of the parasite's complex lifecycle within the human host and mosquito vector are susceptible to vaccine-induced antibodies. The mainstay approach to antibody induction by subunit vaccination has been the delivery of protein antigen formulated in adjuvant. Extensive efforts have been made in this endeavor with respect to malaria vaccine development, especially with regard to target antigen discovery, protein expression platforms, adjuvant testing, and development of soluble and virus-like particle (VLP) delivery platforms. The breadth of approaches to protein-based vaccines is continuing to expand as innovative new concepts in next-generation subunit design are explored, with the prospects for the development of a highly effective multi-component/multi-stage/multi-antigen formulation seeming ever more likely. This review will focus on recent progress in protein vaccine design, development and/or clinical testing for a number of leading malaria antigens from the sporozoite-, merozoite- and sexual-stages of the parasite's lifecycle-including PfCelTOS, PfMSP1, PfAMA1, PfRH5, PfSERA5, PfGLURP, PfMSP3, Pfs48/45 and Pfs25. Future prospects and challenges for the development, production, human delivery and assessment of protein-based malaria vaccines are discussed. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  12. [Rapid selection of recombinant orf virus expression vectors using green fluorescent protein].

    Science.gov (United States)

    Zhang, Jiachun; Guo, Xianfeng; Zhang, Min; Wu, Feifan; Peng, Yongzheng

    2016-01-01

    To construct a universal, highly attenuated orf virus expression vector for exogenous genes using green fluorescent protein (GFP) as the reporter gene. The flanking regions of the ORFV132 of orf virus DNA were amplified by PCR to construct the shuttle plasmid pSPV-132LF-EGFP-132RF. The shuttle plasmid was transfected into OFTu cells and GFP was incorporated into orf virus IA82Delta 121 by homologous recombination. The recombinant IA82Delta121-V was selected by green fluorescent signal. The deletion gene was identified by PCR and sequencing. The effects of ORFV132 knockout were evaluated by virus titration and by observing the proliferation of the infected vascular endothelial cells in vitro. The recombinant orf virus IA82Delta121-V was obtained successfully and quickly, and the deletion of ORFV132 did not affect the replication of the virus in vitro but reduced its virulence. Green fluorescent protein is a selectable marker for rapid, convenient and stable selection of the recombinant viruses. Highly attenuated recombinant orf virus IA82Delta121-V can serve as a new expression vector for exogenous genes.

  13. High-throughput recombinant protein expression in Escherichia coli: current status and future perspectives

    OpenAIRE

    Jia, Baolei; Jeon, Che Ok

    2016-01-01

    The ease of genetic manipulation, low cost, rapid growth and number of previous studies have made Escherichia coli one of the most widely used microorganism species for producing recombinant proteins. In this post-genomic era, challenges remain to rapidly express and purify large numbers of proteins for academic and commercial purposes in a high-throughput manner. In this review, we describe several state-of-the-art approaches that are suitable for the cloning, expression and purification, co...

  14. Single-domain antibody-based ligands for immunoaffinity separation of recombinant human lactoferrin from the goat lactoferrin of transgenic goat milk.

    Science.gov (United States)

    Tillib, S V; Privezentseva, M E; Ivanova, T I; Vasilev, L F; Efimov, G A; Gursky, Y G; Georgiev, G P; Goldman, I L; Sadchikova, E R

    2014-02-15

    Single-domain antibody generation technology was applied to make new Sepharose-bound ligands for affinity separation of closely related proteins, such as human and goat lactoferrin. We generated recombinant antibodies that can selectively bind/recognize only lactoferrins having amino acid sequences identical to that of human natural lactoferrin (anti-hLF Ab). Selected and purified histidine-tagged single-domain antibodies were used as ligands, and different lactoferrins were used as analytes in the kinetics analysis of lactoferrin binding to captured anti-hLF Abs using the Bio-Rad ProteOn XPR36 protein interaction array system. The data obtained were consistent with a 1:1 binding model with very high affinity, practically equal in the case of hLF and rec-hLF (calculated KD varied from 0.43nM to 3.7nM). Interaction of captured fsdAbs with goat LF was significantly weaker and not detectable under the same analysis conditions. We demonstrated the high efficiency of the recombinant human lactoferrin purification from goat lactoferrin and other proteins using the obtained single domain antibody-based affinity ligands. We believe this approach can be used for the generation of single-domain antibody-based affinity media for the efficient separation/purification of a wide spectrum of other highly homologous proteins. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. Recent advances in recombinant protein-based malaria vaccines

    DEFF Research Database (Denmark)

    Draper, Simon J; Angov, Evelina; Horii, Toshihiro

    2015-01-01

    Plasmodium parasites are the causative agent of human malaria, and the development of a highly effective vaccine against infection, disease and transmission remains a key priority. It is widely established that multiple stages of the parasite's complex lifecycle within the human host and mosquito...... vector are susceptible to vaccine-induced antibodies. The mainstay approach to antibody induction by subunit vaccination has been the delivery of protein antigen formulated in adjuvant. Extensive efforts have been made in this endeavor with respect to malaria vaccine development, especially with regard......, with the prospects for the development of a highly effective multi-component/multi-stage/multi-antigen formulation seeming ever more likely. This review will focus on recent progress in protein vaccine design, development and/or clinical testing for a number of leading malaria antigens from the sporozoite...

  16. Viral meningitis epidemics and a single, recent, recombinant and anthroponotic origin of swine vesicular disease virus

    DEFF Research Database (Denmark)

    Bruhn, Christian Anders Wathne; Nielsen, Sandra Cathrine Abel; Samaniego Castruita, Jose Alfredo

    2015-01-01

    BACKGROUND AND OBJECTIVES: Swine vesicular disease virus (SVDV) is a close relative of the human Enterovirus B serotype, coxsackievirus B5. As the etiological agent of a significant emergent veterinary disease, several studies have attempted to explain its origin. However, several key questions r...... stating that SVDV originated through co-infection, recombination, and a single anthroponotic event, during large viral meningitis epidemics around 1960/1961 involving the ancestral serotypes. The exact geographical origin of SVDV may remain untestable due to historical aspects....

  17. Protection by recombinant viral proteins against a respiratory challenge with virulent avian metapneumovirus.

    Science.gov (United States)

    Chary, Parag; Njenga, M Kariuki; Sharma, Jagdev M

    2005-12-15

    Protection by recombinant avian metapneumovirus (aMPV) N or M proteins against a respiratory challenge with virulent aMPV was examined. N, M or N+M proteins were administered intramuscularly (IM) with incomplete Freund's adjuvant (IFA) or by the oculonasal (ON) route with cholera toxin-B (CTB). Each turkey received 40 or 80 microg of each recombinant protein. Birds were considered protected against challenge if the challenge virus was not detectable in the choanal swabs by RT-PCR. At a dose of 40 microg/bird, N protein given with IFA by the IM route protected eight out of nine birds. M protein at the same dose protected three out of seven birds, while a combination of N+M proteins (40 microg each) protected three out of four birds. At a dose of 80 microg of each of N and M proteins per bird given with IFA by the IM route, 100% protection was achieved. ON immunization with a mixture of N and M proteins induced partial protection when the proteins were given with CTB; no detectable protection was noted without CTB. N and M proteins induced anti-aMPV antibodies, although protection against virulent virus challenge did not appear to be associated with the level or presence of antibodies.

  18. Expression of Recombinant Proteins with Uniform N-Termini

    OpenAIRE

    Király, Orsolya; Guan, Lan; Sahin-Tóth, Miklós

    2011-01-01

    Heterologously expressed proteins in Escherichia coli may undergo unwanted N-terminal processing by methionine and proline aminopeptidases. To overcome this problem, we present a system where the gene of interest is cloned as a fusion to a self-splicing mini-intein. Furthermore, this fusion construct is expressed in an engineered Escherichia coli strain from which the pepP gene coding for aminopeptidase P has been deleted. We describe a protocol using human cationic trypsinogen as an example ...

  19. The potential of transgenic green microalgae; a robust photobioreactor to produce recombinant therapeutic proteins.

    Science.gov (United States)

    Akbari, Fariba; Eskandani, Morteza; Khosroushahi, Ahmad Yari

    2014-11-01

    Microalgae have been used in food, cosmetic, and biofuel industries as a natural source of lipids, vitamins, pigments and antioxidants for a long time. Green microalgae, as potent photobioreactors, can be considered as an economical expression system to produce recombinant therapeutical proteins at large-scale due to low cost of production and scaling-up capitalization owning to the inexpensive medium requirement, fast growth rate, and the ease of manipulation. These microalgae possess all benefit eukaryotic expression systems including the ability of post-translational modifications required for proper folding and stability of active proteins. Among the many items regarded as recombinant protein production, this review compares the different expression systems with green microalgae like Dunaliella by viewing the nuclear/chloroplast transformation challenges/benefits, related selection markers/reporter genes, and crucial factors/strategies affecting the increase of foreign protein expression in microalgae transformants. Some important factors were discussed regarding the increase of protein yielding in microalgae transformants including: transformation-associated genotypic modifications, endogenous regulatory factors, promoters, codon optimization, enhancer elements, and milking of recombinant protein.

  20. Immunization against Rumen Methanogenesis by Vaccination with a New Recombinant Protein.

    Directory of Open Access Journals (Sweden)

    Litai Zhang

    Full Text Available Vaccination through recombinant proteins against rumen methanogenesis provides a mitigation approach to reduce enteric methane (CH4 emissions in ruminants. The objective of present study was to evaluate the in vivo efficacy of a new vaccine candidate protein (EhaF on methanogenesis and microbial population in the rumen of goats. We amplified the gene mru 1407 encoding protein EhaF using fresh rumen fluid samples of mature goats and successfully expressed recombinant protein (EhaF in Escherichia coli Rosetta. This product was evaluated using 12 mature goats with half for control and other half injected with 400ug/goat the purified recombinant protein in day 1 and two subsequent booster immunizations in day 35 and 49. All measurements were undertaken from 63 to 68 days after the initial vaccination, with CH4 emissions determined using respiration calorimeter chambers. The results showed that the vaccination caused intensive immune responses in serum and saliva, although it had no significant effect on total enteric CH4 emissions and methanogen population in the rumen, when compared with the control goats. However, the vaccination altered the composition of rumen bacteria, especially the abundance of main phylum Firmicutes and genus Prevotella. The results indicate that protein EhaF might not be an effective vaccine to reduce enteric CH4 emissions but our vaccine have potential to influence the rumen ecosystem of goats.

  1. Expression, Delivery and Function of Insecticidal Proteins Expressed by Recombinant Baculoviruses

    Science.gov (United States)

    Kroemer, Jeremy A.; Bonning, Bryony C.; Harrison, Robert L.

    2015-01-01

    Since the development of methods for inserting and expressing genes in baculoviruses, a line of research has focused on developing recombinant baculoviruses that express insecticidal peptides and proteins. These recombinant viruses have been engineered with the goal of improving their pesticidal potential by shortening the time required for infection to kill or incapacitate insect pests and reducing the quantity of crop damage as a consequence. A wide variety of neurotoxic peptides, proteins that regulate insect physiology, degradative enzymes, and other potentially insecticidal proteins have been evaluated for their capacity to reduce the survival time of baculovirus-infected lepidopteran host larvae. Researchers have investigated the factors involved in the efficient expression and delivery of baculovirus-encoded insecticidal peptides and proteins, with much effort dedicated to identifying ideal promoters for driving transcription and signal peptides that mediate secretion of the expressed target protein. Other factors, particularly translational efficiency of transcripts derived from recombinant insecticidal genes and post-translational folding and processing of insecticidal proteins, remain relatively unexplored. The discovery of RNA interference as a gene-specific regulation mechanism offers a new approach for improvement of baculovirus biopesticidal efficacy through genetic modification. PMID:25609310

  2. Industrial PE-2 strain of Saccharomyces cerevisiae: from alcoholic fermentation to the production of recombinant proteins.

    Science.gov (United States)

    Soares-Costa, Andrea; Nakayama, Darlan Gonçalves; Andrade, Letícia de Freitas; Catelli, Lucas Ferioli; Bassi, Ana Paula Guarnieri; Ceccato-Antonini, Sandra Regina; Henrique-Silva, Flavio

    2014-01-25

    Saccharomyces cerevisiae is the most important microorganism used in the ethanol fermentation process. The PE-2 strain of this yeast is widely used to produce alcohol in Brazil due to its high fermentation capacity. The aim of the present study was to develop an expression system for recombinant proteins using the industrial PE-2 strain of S. cerevisiae during the alcoholic fermentation process. The protein chosen as a model for this system was CaneCPI-1, a cysteine peptidase inhibitor. A plasmid containing the CaneCPI-1 gene was constructed and yeast cells were transformed with the pYADE4_CaneCPI-1 construct. To evaluate the effect on fermentation ability, the transformed strain was used in the fermentation process with cell recycling. During the nine-hour fermentative cycles the transformed strain did not have its viability and fermentation ability affected. In the last cycle, when the fermentation lasted longer, the protein was expressed probably at the expense of ethanol once the sugars were exhausted. The recombinant protein was expressed in yeast cells, purified and submitted to assays of activity that demonstrated its functionality. Thus, the industrial PE-2 strain of S. cerevisiae can be used as a viable system for protein expression and to produce alcohol simultaneously. The findings of the present study demonstrate the possibility of producing recombinant proteins with biotechnological applications during the ethanol fermentation process. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. [Cloning and prokaryotic expression of malate dehydrogenase gene of Taenia saginata asiatica and immunogenicity analysis of the recombinant protein].

    Science.gov (United States)

    Huang, Jiang; Hu, Xu-Chu; Wu, Xuan; Xu, Jin; Yu, Xin-Bing; Bao, Huai-En; Lang, Shu-Yuan; Liao, Xing-Jiang

    2008-08-01

    To clone and express the lactate dehydrogenase (LDH) gene of Taenia saginata asiatica and analyze the immunogenicity of the recombinant protein. By screening the full length cDNA plasmid library, the coding region of LDH was amplified with PCR, and cloned into the prokaryotic expression vector pET-30a (+), then expressed in E. coli BL21 with IPTG induction. The recombinant protein was detected by SDS-PAGE and purified by Ni-IDA affinity chromatography, and its immunogenicity was analyzed by Western blotting. PCR, double enzyme digestion and DNA sequencing confirmed that the recombinant expression plasmid was constructed. The expression products were obtained and purified by Ni-IDA affinity chromatography. Western blotting analysis of LDH recombinant protein testified that the recombinant protein could be recognized by sera of the Taenia saginata asiatica infected swine and the patient. The LDH gene of Taenia saginata asiatica has been cloned and expressed, and the purified protein has been confirmed with immunogenicity.

  4. Expression of active secreted forms of human amyloid beta-protein precursor by recombinant baculovirus-infected insect cells.

    OpenAIRE

    Bhasin, R; Van Nostrand, W E; Saitoh, T; Donets, M A; Barnes, E A; Quitschke, W W; Goldgaber, D

    1991-01-01

    Three alternatively spliced forms of the amyloid precursor protein (APP), APP-695, APP-751, and APP-770, were expressed in the baculovirus expression vector system. The recombinant proteins were secreted into the culture medium by infected insect cells, and APP molecules were detected in insect cells and medium 2 days after infection with the recombinant APP-baculoviruses. A partial sequence of the NH2 terminus of the secreted protein revealed identity with the native secreted protein and sho...

  5. SCHEMA recombination of a fungal cellulase uncovers a single mutation that contributes markedly to stability.

    Science.gov (United States)

    Heinzelman, Pete; Snow, Christopher D; Smith, Matthew A; Yu, Xinlin; Kannan, Arvind; Boulware, Kevin; Villalobos, Alan; Govindarajan, Sridhar; Minshull, Jeremy; Arnold, Frances H

    2009-09-25

    A quantitative linear model accurately (R(2) = 0.88) describes the thermostabilities of 54 characterized members of a family of fungal cellobiohydrolase class II (CBH II) cellulase chimeras made by SCHEMA recombination of three fungal enzymes, demonstrating that the contributions of SCHEMA sequence blocks to stability are predominantly additive. Thirty-one of 31 predicted thermostable CBH II chimeras have thermal inactivation temperatures higher than the most thermostable parent CBH II, from Humicola insolens, and the model predicts that hundreds more CBH II chimeras share this superior thermostability. Eight of eight thermostable chimeras assayed hydrolyze the solid cellulosic substrate Avicel at temperatures at least 5 degrees C above the most stable parent, and seven of these showed superior activity in 16-h Avicel hydrolysis assays. The sequence-stability model identified a single block of sequence that adds 8.5 degrees C to chimera thermostability. Mutating individual residues in this block identified the C313S substitution as responsible for the entire thermostabilizing effect. Introducing this mutation into the two recombination parent CBH IIs not featuring it (Hypocrea jecorina and H. insolens) decreased inactivation, increased maximum Avicel hydrolysis temperature, and improved long time hydrolysis performance. This mutation also stabilized and improved Avicel hydrolysis by Phanerochaete chrysosporium CBH II, which is only 55-56% identical to recombination parent CBH IIs. Furthermore, the C313S mutation increased total H. jecorina CBH II activity secreted by the Saccharomyces cerevisiae expression host more than 10-fold. Our results show that SCHEMA structure-guided recombination enables quantitative prediction of cellulase chimera thermostability and efficient identification of stabilizing mutations.

  6. Chloroplast-Based Expression of Recombinant Proteins by Gateway® Cloning Technology.

    Science.gov (United States)

    Gottschamel, Johanna; Lössl, Andreas

    2016-01-01

    Plastid transformation for the expression of recombinant proteins and entire enzymatic pathways has become a promising tool for plant biotechnology in the past decade. Several improvements of the technology have turned plant plastids into robust and dependable expression platforms for multiple high value compounds. In this chapter, we describe our current methodology based on Gateway(®) recombinant cloning, which we have adapted for plastid transformation. We describe the steps required for cloning, biolistic transformation, identification, and regeneration of transplastomic plant lines and Western blot analysis.

  7. Expression and purification of functional, recombinant Trypanosoma cruzi complement regulatory protein.

    Science.gov (United States)

    Beucher, Margaret; Meira, Wendell S F; Zegarra, Vasthy; Galvão, Lúcia M C; Chiari, Egler; Norris, Karen A

    2003-01-01

    The complement regulatory protein (CRP) of Trypanosoma cruzi is a developmentally regulated glycosylphosphatidylinositol (GPI)-anchored membrane protein that protects the parasite from complement-mediated killing, and is an important virulence determinant of the microorganism. CRP binds human complement components C3b and C4b to restrict activation of the complement cascade. Here, we report production of functional, recombinant T. cruzi CRP in mammalian cells and a one-step purification of the recombinant protein. Exchange of the crp DNA sequence encoding the carboxy-terminal GPI signal sequence with the corresponding sequence of decay accelerating factor (DAF) was necessary for recognition, cleavage, and addition of GPI in mammalian cells. CRP production was assessed in two mammalian cell lines with crp-daf gene expression driven by three different transcription control regions: Rous sarcoma virus long terminal repeat, cytomegalovirus (CMV) immediate early gene, and chicken beta-actin promoter/CMV enhancer. We present evidence that CRP produced in transfected Chinese hamster Ovary (CHO) cells was functional and protected the cells from complement-mediated lysis. To facilitate purification of the recombinant protein, a hexahistidyl tag was incorporated at 3(') end of the cDNA upstream of the GPI anchor addition sequence. An additional histidine fusion construct was made that allowed for secretion and recovery of recombinant protein from culture supernatant fluid. Both membrane and secreted forms of the protein were purified in one step by nickel nitrilotriacetic acid. The production and purification of functionally active CRP in a non-infectious expression system will allow for structure and function studies aimed at identifying the active site(s) of this protein.

  8. Tunable recombinant protein expression with E. coli in a mixed-feed environment.

    Science.gov (United States)

    Sagmeister, Patrick; Schimek, Clemens; Meitz, Andrea; Herwig, Christoph; Spadiut, Oliver

    2014-04-01

    Controlling the recombinant protein production rate in Escherichia coli is of utmost importance to ensure product quality and quantity. Up to now, only the genetic construct, introduced into E. coli, and the specific growth rate of the culture were used to influence and stir the productivity. However, bioprocess technological means to control or even tune the productivity of E. coli are scarce. Here, we present a novel method for the process-technological control over the recombinant protein expression rate in E. coli. A mixed-feed fed-batch bioprocess based on the araBAD promoter expression system using both D-glucose and L-arabinose as assimilable C-sources was designed. Using the model product green fluorescent protein, we show that the specific product formation rate can be efficiently tuned even on the cellular level only via the uptake rate of L-arabinose. This novel approach introduces an additional degree of freedom for the design of recombinant bioprocesses with E. coli. We anticipate that the presented method will result in significant quality and robustness improvement as well as cost and process time reduction for recombinant bacterial bioprocesses in the future.

  9. [Construction and expression of recombinant human serum albumin-EPO fusion protein].

    Science.gov (United States)

    Huang, Ying-Chun; Gou, Xing-Hua; Han, Lei; Li, De-Hua; Zhao, Lan-Ying; Wu, Qia-Qing

    2011-05-01

    OBJECTIVE To construct the recombinant plasmid pCI-HLE encoding human serum album-EPO (HSA-EPO) fusion protein and to express it in CHO cell. The cDNA encoding human serum album and EPO were amplified by PCR, and then spliced with the synsitic DNA fragment encoding GS (GGGGS), by overlap PCR extension to form LEPO. After BamH I digestion, the HSA and LEPO was ligated to generate the fusion HSA-EPO gene and was then cloned into the expression vector pCI-neo to generate the recombinant plasmid pCI-HLE. The plasmid pCI-HLE was transfected into CHO cell by liposome protocol. Then, the recombinant cells were screened by G418 and identified by PCR and Western blot. Expression of fusion protein was evaluated by Enzyme Linked Immunosorbent Assay (ELISA). Restrictive enzymes digestion and DNA sequencing revealed that HSA-EPO fusion gene was cloned into expression vector pCI-neo successfully. PCR and Western blot analysis confirmed that the fusion gene was integrated in the genome of CHO cells and expressed successfully. The HSA-EPO production varied from 86 Iu/(mL x 10(6) x 72 h) to 637 IU/(mLx 10(6) x 72 h). The results confirmed that HSA-EPO fusion gene can be expressed in the CHO cells, with EPO immunogenicity, which could serve as foundation for the development of long-lasting recombinant HSA-EPO protein.

  10. Development of a diagnostic method for neosporosis in cattle using recombinant Neospora caninum proteins

    Directory of Open Access Journals (Sweden)

    Dong Jinhua

    2012-05-01

    Full Text Available Abstract Background Neosporosis is an infectious disease primarily of cattle and dogs, caused by intracellular parasite, Neospora caninum. Neosporosis appears to be a major cause of abortion in dairy cattle worldwide and causes to huge economic loss to dairy industry. Results Recombinant surface associated antigen 1 (NcSAG1, NcSAG1 related sequence 2 (NcSRS2 and the dense granule antigen 2 (NcGRA2 of N. caninum were expressed either in silkworm or in Escherichia coli and purified. The purified recombinant proteins bound to the N. caninum-specific antibodies in serum samples from infected cattle as revealed by an enzyme-linked immunosorbent assay (ELISA. By co-immobilizing these recombinant proteins, a novel indirect ELISA was developed for detection of neosporosis. With the use of 32 serum samples, comprising 12 positive serum samples and 20 negative serum samples, the sensitivity and specificity of the assay were found to be 91.7 and 100%, respectively. Seventy-two serum samples from dairy farms were also tested and one was diagnosed with neosporasis with both this method and a commercial assay. Conclusions A diagnostic method employing recombinant proteins of N. caninum was developed. The method showed high sensitivity and specificity. Diagnostic test with field serum samples suggested its applicability to the practical diagnosis of neosporosis.

  11. Development of a diagnostic method for neosporosis in cattle using recombinant Neospora caninum proteins

    Science.gov (United States)

    2012-01-01

    Background Neosporosis is an infectious disease primarily of cattle and dogs, caused by intracellular parasite, Neospora caninum. Neosporosis appears to be a major cause of abortion in dairy cattle worldwide and causes to huge economic loss to dairy industry. Results Recombinant surface associated antigen 1 (NcSAG1), NcSAG1 related sequence 2 (NcSRS2) and the dense granule antigen 2 (NcGRA2) of N. caninum were expressed either in silkworm or in Escherichia coli and purified. The purified recombinant proteins bound to the N. caninum-specific antibodies in serum samples from infected cattle as revealed by an enzyme-linked immunosorbent assay (ELISA). By co-immobilizing these recombinant proteins, a novel indirect ELISA was developed for detection of neosporosis. With the use of 32 serum samples, comprising 12 positive serum samples and 20 negative serum samples, the sensitivity and specificity of the assay were found to be 91.7 and 100%, respectively. Seventy-two serum samples from dairy farms were also tested and one was diagnosed with neosporasis with both this method and a commercial assay. Conclusions A diagnostic method employing recombinant proteins of N. caninum was developed. The method showed high sensitivity and specificity. Diagnostic test with field serum samples suggested its applicability to the practical diagnosis of neosporosis. PMID:22558916

  12. Development of a diagnostic method for neosporosis in cattle using recombinant Neospora caninum proteins.

    Science.gov (United States)

    Dong, Jinhua; Otsuki, Takahiro; Kato, Tatsuya; Park, Enoch Y

    2012-05-04

    Neosporosis is an infectious disease primarily of cattle and dogs, caused by intracellular parasite, Neospora caninum. Neosporosis appears to be a major cause of abortion in dairy cattle worldwide and causes to huge economic loss to dairy industry. Recombinant surface associated antigen 1 (NcSAG1), NcSAG1 related sequence 2 (NcSRS2) and the dense granule antigen 2 (NcGRA2) of N. caninum were expressed either in silkworm or in Escherichia coli and purified. The purified recombinant proteins bound to the N. caninum-specific antibodies in serum samples from infected cattle as revealed by an enzyme-linked immunosorbent assay (ELISA). By co-immobilizing these recombinant proteins, a novel indirect ELISA was developed for detection of neosporosis. With the use of 32 serum samples, comprising 12 positive serum samples and 20 negative serum samples, the sensitivity and specificity of the assay were found to be 91.7 and 100%, respectively. Seventy-two serum samples from dairy farms were also tested and one was diagnosed with neosporasis with both this method and a commercial assay. A diagnostic method employing recombinant proteins of N. caninum was developed. The method showed high sensitivity and specificity. Diagnostic test with field serum samples suggested its applicability to the practical diagnosis of neosporosis.

  13. Choreography of recombination proteins during the DNA damage response

    DEFF Research Database (Denmark)

    Lisby, Michael; Rothstein, Rodney

    2009-01-01

    Genome integrity is frequently challenged by DNA lesions from both endogenous and exogenous sources. A single DNA double-strand break (DSB) is lethal if unrepaired and may lead to loss of heterozygosity, mutations, deletions, genomic rearrangements and chromosome loss if repaired improperly....... Such genetic alterations are the main causes of cancer and other genetic diseases. Consequently, DNA double-strand break repair (DSBR) is an important process in all living organisms. DSBR is also the driving mechanism in most strategies of gene targeting, which has applications in both genetic and clinical...

  14. Crystallization and preliminary crystallographic analysis of recombinant immunoglobulin G-binding protein from Streptococcus suis

    Energy Technology Data Exchange (ETDEWEB)

    Khan, Abdul Hamid; Chu, Fuliang; Feng, Youjun; Zhang, Qinagmin [Center for Molecular Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101 (China); Qi, Jianxun [Institute of Physics, Chinese Academy of Sciences, Beijing 100080 (China); Gao, George Fu, E-mail: gaof@im.ac.cn [Center for Molecular Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101 (China)

    2008-08-01

    Crystallization of recombinant IgG-binding protein expressed in Escherichia coli using the hanging-drop vapour-diffusion method is described. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 38.98, b = 43.94, c = 78.17 Å. Streptococcus suis, an important zoonotic pathogen, expresses immunoglobulin G-binding protein, which is thought to be helpful to the organism in eluding the host defence system. Recombinant IgG-binding protein expressed in Escherichia coli has been crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 38.98, b = 43.94, c = 78.17 Å and one molecule in the asymmetric unit. Diffraction data were collected to 2.60 Å resolution.

  15. Cross-system excision of chaperone-mediated proteolysis in chaperone-assisted recombinant protein production

    Science.gov (United States)

    Martínez-Alonso, Mónica; Villaverde, Antonio

    2010-01-01

    Main Escherichia coli cytosolic chaperones such as DnaK are key components of the control quality network designed to minimize the prevalence of polypeptides with aberrant conformations. This is achieved by both favoring refolding activities but also stimulating proteolytic degradation of folding reluctant species. This last activity is responsible for the decrease of the proteolytic stability of recombinant proteins when co-produced along with DnaK, where an increase in solubility might be associated to a decrease in protein yield. However, when DnaK and its co-chaperone DnaJ are co-produced in cultured insect cells or whole insect larvae (and expectedly, in other heterologous hosts), only positive, folding-related effects of these chaperones are observed, in absence of proteolysis-mediated reduction of recombinant protein yield. PMID:21326941

  16. Secretion of a recombinant protein without a signal peptide by the exocrine glands of transgenic rabbits.

    Directory of Open Access Journals (Sweden)

    Andrea Kerekes

    Full Text Available Transgenic rabbits carrying mammary gland specific gene constructs are extensively used for excreting recombinant proteins into the milk. Here, we report refined phenotyping of previously generated Venus transposon-carrying transgenic rabbits with particular emphasis on the secretion of the reporter protein by exocrine glands, such as mammary, salivary, tear and seminal glands. The Sleeping Beauty (SB transposon transgenic construct contains the Venus fluorophore cDNA, but without a signal peptide for the secretory pathway, driven by the ubiquitous CAGGS (CAG promoter. Despite the absence of a signal peptide, the fluorophore protein was readily detected in milk, tear, saliva and seminal fluids. The expression pattern was verified by Western blot analysis. Mammary gland epithelial cells of SB-CAG-Venus transgenic lactating does also showed Venus-specific expression by tissue histology and fluorescence microscopy. In summary, the SB-CAG-Venus transgenic rabbits secrete the recombinant protein by different glands. This finding has relevance not only for the understanding of the biological function of exocrine glands, but also for the design of constructs for expression of recombinant proteins in dairy animals.

  17. Methods for Using Small Non-Coding RNAs to Improve Recombinant Protein Expression in Mammalian Cells

    Directory of Open Access Journals (Sweden)

    Sarah Inwood

    2018-01-01

    Full Text Available The ability to produce recombinant proteins by utilizing different “cell factories” revolutionized the biotherapeutic and pharmaceutical industry. Chinese hamster ovary (CHO cells are the dominant industrial producer, especially for antibodies. Human embryonic kidney cells (HEK, while not being as widely used as CHO cells, are used where CHO cells are unable to meet the needs for expression, such as growth factors. Therefore, improving recombinant protein expression from mammalian cells is a priority, and continuing effort is being devoted to this topic. Non-coding RNAs are RNA segments that are not translated into a protein and often have a regulatory role. Since their discovery, major progress has been made towards understanding their functions. Non-coding RNA has been investigated extensively in relation to disease, especially cancer, and recently they have also been used as a method for engineering cells to improve their protein expression capability. In this review, we provide information about methods used to identify non-coding RNAs with the potential of improving recombinant protein expression in mammalian cell lines.

  18. Secretion of a recombinant protein without a signal peptide by the exocrine glands of transgenic rabbits.

    Science.gov (United States)

    Kerekes, Andrea; Hoffmann, Orsolya Ivett; Iski, Gergely; Lipták, Nándor; Gócza, Elen; Kues, Wilfried A; Bősze, Zsuzsanna; Hiripi, László

    2017-01-01

    Transgenic rabbits carrying mammary gland specific gene constructs are extensively used for excreting recombinant proteins into the milk. Here, we report refined phenotyping of previously generated Venus transposon-carrying transgenic rabbits with particular emphasis on the secretion of the reporter protein by exocrine glands, such as mammary, salivary, tear and seminal glands. The Sleeping Beauty (SB) transposon transgenic construct contains the Venus fluorophore cDNA, but without a signal peptide for the secretory pathway, driven by the ubiquitous CAGGS (CAG) promoter. Despite the absence of a signal peptide, the fluorophore protein was readily detected in milk, tear, saliva and seminal fluids. The expression pattern was verified by Western blot analysis. Mammary gland epithelial cells of SB-CAG-Venus transgenic lactating does also showed Venus-specific expression by tissue histology and fluorescence microscopy. In summary, the SB-CAG-Venus transgenic rabbits secrete the recombinant protein by different glands. This finding has relevance not only for the understanding of the biological function of exocrine glands, but also for the design of constructs for expression of recombinant proteins in dairy animals.

  19. Purification and characterization of recombinant protein kinase CK2 from Zea mays expressed in Escherichia coli

    DEFF Research Database (Denmark)

    Riera, Marta; Pages, Montserrat; Issinger, Olaf Georg

    2003-01-01

    Recombinant protein kinase subunits rmCK2alpha-1 and rmCK2beta-1 from Zea mays were expressed separately in Escherichia coli and assembled to a fully active tetrameric holoenzyme complex in vitro. The obtained maize holoenzyme was purified to homogeneity, biochemically characterized, and compared...... to CK2 from human. Kinetic measurements of the recombinant maize holoenzyme (rmCK2) revealed k(cat) values for ATP and GTP of 4 and 2s(-1), respectively; whereas the recombinant maize catalytic subunit showed almost equal values for ATP and GTP, i.e., ca. 0.8s(-1). A comparison of the k(cat)/K(m) ratio...

  20. Analysis of two lysozyme genes and antimicrobial functions of their recombinant proteins in Asian seabass.

    Directory of Open Access Journals (Sweden)

    Gui Hong Fu

    Full Text Available Lysozymes are important proteins of the innate immune system for the defense against bacterial infection. We cloned and analyzed chicken-type (c-type and goose-type (g-type lysozymes from Asian seabass (Lates calcarifer. The deduced amino acid sequence of the c-type lysozyme contained 144 residues and possessed typical structure residues, conserved catalytic residues (Glu(50 and Asp(67 and a "GSTDYGIFQINS" motif. The deduced g-type lysozyme contained 187 residues and possessed a goose egg white lysozyme (GEWL domain containing three conserved catalytic residues (Glu(71, Asp(84, Asp(95 essential for catalytic activity. Real time quantitative PCR (qRT-PCR revealed that the two lysozyme genes were constitutively expressed in all the examined tissues. The c-type lysozyme was most abundant in liver, while the g-type lysozyme was predominantly expressed in intestine and weakly expressed in muscle. The c-type and g-type transcripts were up-regulated in the kidney, spleen and liver in response to a challenge with Vibrio harveyi. The up-regulation of the c-type lysozyme was much stronger than that of the g-type lysozyme in kidney and spleen. The recombinant proteins of the c-type and g-type lysozymes showed lytic activities against the bacterial pathogens Vibrio harveyi and Photobacterium damselae in a dosage-dependent manner. We identified single nucleotide polymorphisms (SNPs in the two lysozyme genes. There were significant associations of these polymorphisms with resistance to the big belly disease. These results suggest that the c- and g-type genes play an important role in resistance to bacterial pathogens in fish. The SNP markers in the two genes associated with the resistance to bacterial pathogens may facilitate the selection of Asian seabass resistant to bacterial diseases.

  1. Analysis of two lysozyme genes and antimicrobial functions of their recombinant proteins in Asian seabass.

    Science.gov (United States)

    Fu, Gui Hong; Bai, Zhi Yi; Xia, Jun Hong; Liu, Feng; Liu, Peng; Yue, Gen Hua

    2013-01-01

    Lysozymes are important proteins of the innate immune system for the defense against bacterial infection. We cloned and analyzed chicken-type (c-type) and goose-type (g-type) lysozymes from Asian seabass (Lates calcarifer). The deduced amino acid sequence of the c-type lysozyme contained 144 residues and possessed typical structure residues, conserved catalytic residues (Glu(50) and Asp(67)) and a "GSTDYGIFQINS" motif. The deduced g-type lysozyme contained 187 residues and possessed a goose egg white lysozyme (GEWL) domain containing three conserved catalytic residues (Glu(71), Asp(84), Asp(95)) essential for catalytic activity. Real time quantitative PCR (qRT-PCR) revealed that the two lysozyme genes were constitutively expressed in all the examined tissues. The c-type lysozyme was most abundant in liver, while the g-type lysozyme was predominantly expressed in intestine and weakly expressed in muscle. The c-type and g-type transcripts were up-regulated in the kidney, spleen and liver in response to a challenge with Vibrio harveyi. The up-regulation of the c-type lysozyme was much stronger than that of the g-type lysozyme in kidney and spleen. The recombinant proteins of the c-type and g-type lysozymes showed lytic activities against the bacterial pathogens Vibrio harveyi and Photobacterium damselae in a dosage-dependent manner. We identified single nucleotide polymorphisms (SNPs) in the two lysozyme genes. There were significant associations of these polymorphisms with resistance to the big belly disease. These results suggest that the c- and g-type genes play an important role in resistance to bacterial pathogens in fish. The SNP markers in the two genes associated with the resistance to bacterial pathogens may facilitate the selection of Asian seabass resistant to bacterial diseases.

  2. Analysis of Two Lysozyme Genes and Antimicrobial Functions of Their Recombinant Proteins in Asian Seabass

    Science.gov (United States)

    Fu, Gui Hong; Bai, Zhi Yi; Xia, Jun Hong; Liu, Feng; Liu, Peng; Yue, Gen Hua

    2013-01-01

    Lysozymes are important proteins of the innate immune system for the defense against bacterial infection. We cloned and analyzed chicken-type (c-type) and goose-type (g-type) lysozymes from Asian seabass (Lates calcarifer). The deduced amino acid sequence of the c-type lysozyme contained 144 residues and possessed typical structure residues, conserved catalytic residues (Glu50 and Asp67) and a “GSTDYGIFQINS” motif. The deduced g-type lysozyme contained 187 residues and possessed a goose egg white lysozyme (GEWL) domain containing three conserved catalytic residues (Glu71, Asp84, Asp95) essential for catalytic activity. Real time quantitative PCR (qRT-PCR) revealed that the two lysozyme genes were constitutively expressed in all the examined tissues. The c-type lysozyme was most abundant in liver, while the g-type lysozyme was predominantly expressed in intestine and weakly expressed in muscle. The c-type and g-type transcripts were up-regulated in the kidney, spleen and liver in response to a challenge with Vibrio harveyi. The up-regulation of the c-type lysozyme was much stronger than that of the g-type lysozyme in kidney and spleen. The recombinant proteins of the c-type and g-type lysozymes showed lytic activities against the bacterial pathogens Vibrio harveyi and Photobacterium damselae in a dosage-dependent manner. We identified single nucleotide polymorphisms (SNPs) in the two lysozyme genes. There were significant associations of these polymorphisms with resistance to the big belly disease. These results suggest that the c- and g-type genes play an important role in resistance to bacterial pathogens in fish. The SNP markers in the two genes associated with the resistance to bacterial pathogens may facilitate the selection of Asian seabass resistant to bacterial diseases. PMID:24244553

  3. Development of a new platform for secretory production of recombinant proteins in Corynebacterium glutamicum.

    Science.gov (United States)

    Yim, Sung Sun; Choi, Jae Woong; Lee, Roo Jin; Lee, Yong Jae; Lee, Se Hwa; Kim, So Young; Jeong, Ki Jun

    2016-01-01

    Corynebacterium glutamicum, which has been for long an industrial producer of various L-amino acids, nucleic acids, and vitamins, is now also regarded as a potential host for the secretory production of recombinant proteins. To harness its potential as an industrial platform for recombinant protein production, the development of an efficient secretion system is necessary. Particularly, regarding protein production in large-scale bioreactors, it would be appropriate to develop a secretory expression system that is specialized for high cell density cultivation conditions. Here we isolated a new signal peptide that mediates the efficient secretion of recombinant proteins under high cell density cultivation conditions. The secretome of C. glutamicum ATCC 13032 under high cell density cultivation conditions was initially investigated, and one major protein was identified as a hypothetical protein encoded by cg1514. Novel secretory production systems were then developed using the Cg1514 signal peptide and its own promoter. Efficient protein secretion was demonstrated using three protein models: endoxylanase, α-amylase, and camelid antibody fragment (VHH). For large-scale production, fed-batch cultivations were also conducted and high yields were successfully achieved--as high as 1.07 g/L (endoxylanase), 782.6 mg/L (α-amylase), and 1.57 g/L (VHH)--in the extracellular medium. From the culture media, all model proteins could be simply purified by one-step column chromatography with high purities and recovery yields. To the best of our knowledge, this is the first report of the development of an efficient secretory expression system by secretome analysis under high cell density cultivation conditions in C. glutamicum. © 2015 Wiley Periodicals, Inc.

  4. High-level expression of soluble recombinant proteins in Escherichia coli using an HE-maltotriose-binding protein fusion tag.

    Science.gov (United States)

    Han, Yingqian; Guo, Wanying; Su, Bingqian; Guo, Yujie; Wang, Jiang; Chu, Beibei; Yang, Guoyu

    2018-02-01

    Recombinant proteins are commonly expressed in prokaryotic expression systems for large-scale production. The use of genetically engineered affinity and solubility enhancing fusion proteins has increased greatly in recent years, and there now exists a considerable repertoire of these that can be used to enhance the expression, stability, solubility, folding, and purification of their fusion partner. Here, a modified histidine tag (HE) used as an affinity tag was employed together with a truncated maltotriose-binding protein (MBP; consisting of residues 59-433) from Pyrococcus furiosus as a solubility enhancing tag accompanying a tobacco etch virus protease-recognition site for protein expression and purification in Escherichia coli. Various proteins tagged at the N-terminus with HE-MBP(Pyr) were expressed in E. coli BL21(DE3) cells to determine expression and solubility relative to those tagged with His6-MBP or His6-MBP(Pyr). Furthermore, four HE-MBP(Pyr)-fused proteins were purified by immobilized metal affinity chromatography to assess the affinity of HE with immobilized Ni 2+ . Our results showed that HE-MBP(Pyr) represents an attractive fusion protein allowing high levels of soluble expression and purification of recombinant protein in E. coli. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Simple and Efficient Purification of Recombinant Proteins Using the Heparin-Binding Affinity Tag.

    Science.gov (United States)

    Jayanthi, Srinivas; Gundampati, Ravi Kumar; Kumar, Thallapuranam Krishnaswamy Suresh

    2017-11-01

    Heparin, a member of the glycosaminoglycan family, is known to interact with more than 400 different types of proteins. For the past few decades, significant progress has been made to understand the molecular details involved in heparin-protein interactions. Based on the structural knowledge available from the FGF1-heparin interaction studies, we have designed a novel heparin-binding peptide (HBP) affinity tag that can be used for the simple, efficient, and cost-effective purification of recombinant proteins of interest. HBP-tagged fusion proteins can be purified by heparin Sepharose affinity chromatography using a simple sodium chloride gradient to elute the bound fusion protein. In addition, owing to the high density of positive charges on the HBP tag, recombinant target proteins are preferably expressed in their soluble forms. The purification of HBP-fusion proteins can also be achieved in the presence of chemical denaturants, including urea. Additionally, polyclonal antibodies raised against the affinity tag can be used to detect HBP-fused target proteins with high sensitivity. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  6. Real-time single-molecule tethered particle motion experiments reveal the kinetics and mechanisms of Cre-mediated site-specific recombination

    Science.gov (United States)

    Fan, Hsiu-Fang

    2012-01-01

    Tyrosine family recombinases (YRs) are widely utilized in genome engineering systems because they can easily direct DNA rearrangement. Cre recombinases, one of the most commonly used types of YRs, catalyze site-specific recombination between two loxP sites without the need for high-energy cofactors, other accessory proteins or a specific DNA target sequence between the loxP sites. Previous structural, analytical ultracentrifuge and electrophoretic analyses have provided details of the reaction kinetics and mechanisms of Cre recombinase activity; whether there are reaction intermediates or side pathways involved has been left unaddressed. Using tethered particle motion (TPM), the Cre-mediated site-specific recombination process has been delineated, from beginning to end, at the single-molecule level, including the formation of abortive complexes and wayward complexes blocking inactive nucleoprotein complexes from entering the recombination process. Reversibility in the strand-cleavage/-ligation process and the formation of a thermally stable Holliday junction intermediate were observed within the Cre-mediated site-specific recombination process. Rate constants for each elementary step, which explain the overall reaction outcomes under various conditions, were determined. Taking the findings of this study together, they demonstrate the potential of single-molecule methodology as an alternative approach for exploring reaction mechanisms in detail. PMID:22467208

  7. Protection against Asiatic Taenia solium induced by a recombinant 45W-4B protein.

    Science.gov (United States)

    Luo, Xuenong; Zheng, Yadong; Hou, Junling; Zhang, Shaohua; Cai, Xuepeng

    2009-02-01

    Taenia solium is a great threat not only to human health but also to the pig-raising industry. Oncospheral stage-specific 45W proteins are good candidates for the development of anticysticercosis vaccines. In this study, a recombinant 45W-4B protein was highly produced and used for vaccination. Two animal trials resulted in a significant reduction in parasite burden induced by the definite protein against Asiatic T. solium, up to 97.0% and 98.4%, respectively. These provide informative results for the development of effective 45W-4B vaccines against cysticercosis caused by both Chinese and Mexican T. solium isolates and even by other isolates.

  8. Recombinant expression of TLR5 proteins by ligand supplementation and a leucine-rich repeat hybrid technique

    Science.gov (United States)

    Wilson, Ian A.

    2013-01-01

    Vertebrate TLR5 directly binds bacterial flagellin proteins and activates innate immune responses against pathogenic flagellated bacteria. Structural and biochemical studies on the TLR5/flagellin interaction have been challenging due to the technical difficulty in obtaining active recombinant proteins of TLR5 ectodomain (TLR5-ECD). We recently succeeded in production of the N-terminal leucine rich repeats (LRRs) of Danio rerio (dr) TLR5-ECD in a hybrid with another LRR protein, hagfish variable lymphocyte receptor (VLR), and determined the crystal structure of its complex with flagellin D1–D2–D3 domains. Although the structure provides valuable information about the interaction, it remains to be revealed how the C-terminal region of TLR5-ECD contributes to the interaction. Here, we present two methods to obtain recombinant TLR5 proteins that contain the C-terminal region in a baculovirus expression system. First, production of biologically active full-length drTLR5-ECD was substantially enhanced by supplementation of expression culture with purified flagellin proteins. Second, we designed TLR5-VLR hybrids using an LRR hybrid technology by single and double LRR fusions and were able to express diverse regions of drTLR5-ECD, allowing us to detect a previously unidentified TLR5/flagellin interaction. The drTLR5-VLR hybrid technique was also successfully applied to human TLR5-ECD whose expression has been highly problematic. These alternative TLR5 expression strategies provide an opportunity to obtain a complete view of the TLR5/flagellin interaction and can be applied to other LRR proteins. PMID:22989748

  9. Highly sensitive detection of mutations in CHO cell recombinant DNA using multi-parallel single molecule real-time DNA sequencing.

    Science.gov (United States)

    Cartwright, Joseph F; Anderson, Karin; Longworth, Joseph; Lobb, Philip; James, David C

    2018-02-10

    High-fidelity replication of biologic-encoding recombinant DNA sequences by engineered mammalian cell cultures is an essential pre-requisite for the development of stable cell lines for the production of biotherapeutics. However, immortalized mammalian cells characteristically exhibit an increased point mutation frequency compared to mammalian cells in vivo, both across their genomes and at specific loci (hotspots). Thus unforeseen mutations in recombinant DNA sequences can arise and be maintained within producer cell populations. These may affect both the stability of recombinant gene expression and give rise to protein sequence variants with variable bioactivity and immunogenicity. Rigorous quantitative assessment of recombinant DNA integrity should therefore form part of the cell line development process and be an essential quality assurance metric for instances where synthetic/multi-component assemblies are utilized to engineer mammalian cells, such as the assessment of recombinant DNA fidelity or the mutability of single-site integration target loci. Based on Pacific Biosciences (Menlo Park, CA) single molecule real-time (SMRT™) circular consensus sequencing (CCS) technology we developed a rDNA sequence analysis tool to process the multi-parallel sequencing of ∼40,000 single recombinant DNA molecules. After statistical filtering of raw sequencing data, we show that this analytical method is capable of detecting single point mutations in rDNA to a minimum single mutation frequency of 0.0042% (sequence. There was no discernable difference between the mutation frequencies of coding and non-coding DNA. The putative ratio of non-synonymous and synonymous changes within the open reading frames (ORFs) in the plasmid sequence indicates that natural selection does not impact upon the prevalence of these mutations. Here we have demonstrated the abundance of mutations that fall outside of the reported range of detection of next generation sequencing (NGS) and second

  10. A novel chimeric protein composed of recombinant Mycoplasma hyopneumoniae antigens as a vaccine candidate evaluated in mice.

    Science.gov (United States)

    de Oliveira, Natasha Rodrigues; Jorge, Sérgio; Gomes, Charles Klazer; Rizzi, Caroline; Pacce, Violetta Dias; Collares, Thais Farias; Monte, Leonardo Garcia; Dellagostin, Odir Antônio

    2017-03-01

    Enzootic Pneumonia (EP) is caused by the Mycoplasma hyopneumoniae pathogenic bacteria, and it represents a significant respiratory disease that is responsible for major economic losses within the pig industry throughout the world. The bacterins that are currently commercially available have been proven to offer only partial protection against M. hyopneumoniae, and the development of more efficient vaccines is required. Several recombinant antigens have been evaluated via different immunization strategies and have been found to be highly immunogenic. This work describes the construction and immunological characterization of a multi-antigen chimera composed of four M. hyopneumoniae antigens: P97R1, P46, P95, and P42. Immunogenic regions of each antigen were selected and combined to encode a single polypeptide. The gene was cloned and expressed in Escherichia coli, and the chimeric protein was recognized by specific antibodies against each subunit, as well as by convalescent pig sera. The immunogenic properties of the chimera were then evaluated in a mice model through two recombinant vaccines that were formulated as follows: (1) purified chimeric protein plus adjuvant or (2) recombinant Escherichia coli bacterin. The immune response induced in BALB/c mice immunized with each formulation was characterized in terms of total IgG levels, IgG1, and IgG2a isotypes against each antigen present in the chimera. The results of the study indicated that novel chimeric protein is a potential candidate for the future development of a more effective vaccine against EP. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Production of Brugia malayi BmSXP Recombinant Protein Expressed in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Khoo, T. K.

    2010-01-01

    Full Text Available A rapid antibody detection test is very useful for detection of lymphatic filariasis, especially for certification and surveillance of post-mass drug administration. One such kit, panLF RapidTM (commercialized by Malaysian BioDiagnostic Research Sdn. Bhd. had been developed in our laboratory for the detection of all species of filarial infections. It is based on the detection of anti-filarial IgG4 antibodies that react with recombinant Brugia malayi antigens, BmR1 and BmSXP. In this study, the growth of recombinant bacteria that produce BmSXP was optimized under shake flask fermentation for high yield of the recombinant antigen. The optimizations involved selection of suitable growth medium, IPTG concentration and induction time. The medium that yielded the highest biomass as well as total protein was Terrific Broth (TB medium, which is an undefined medium. Initiation of induction of protein expression was found to be best at mid-log phase (OD600 = 1.5, with IPTG concentration of 1.0 mM, and harvest time at 9 h post-induction. This study showed that under the optimized conditions, the shake flask culture produced 4 g/L biomass (dry cell weight of recombinant Escherichia coli BmSXP/pPROEXHTa/TOP10F’, which yielded 2.42 mg/L of purified BmSXP recombinant antigen. The purified antigen was analyzed by SDS-PAGE and the antigenicity of protein was confirmed by Western blot.

  12. Beyond the 12/23 rule of VDJ recombination independent of the Rag proteins.

    Science.gov (United States)

    Olaru, Alexandru; Petrie, Howard T; Livák, Ferenc

    2005-05-15

    The combinatorial repertoire of AgRs is established through somatic recombination of V, D, and J gene segments during lymphocyte development. Incorporation of D segments into IgH, TCRbeta, and TCRdelta chains also contributes to junctional diversification by substantially extending the length of the third CDR. The V, D, and J gene segments are flanked by recombination signals (RS) of 12- or 23-mer spacer length that direct recombination according to the 12/23 rule. D genes in the TCRbeta and TCRdelta loci are flanked by a 12RS and 23RS, and their incorporation is controlled by mechanisms "beyond the 12/23 rule." In the TCRbeta locus, selective interactions between Rag proteins and the RS flanking the V-D and D-J genes, respectively, are sufficient to enforce D gene usage. In this article, we report that in the TCRdelta locus, the Rag proteins are not the major determinant of D gene incorporation. In developing mouse and human thymocytes, the two Ddelta genes rearrange predominantly to form D-D coding joints. In contrast, when tested in ex vivo transfection assays in a nonlymphoid cell line, the flanking RS mediate deletion, rather than incorporation, of the two D genes on both exogenous recombination substrates and the endogenous locus. These results suggest that selective Rag-RS interactions are not the sole regulators of D gene segment incorporation, and additional, perhaps lymphocyte-specific, mechanisms exist that allow proper shaping of the primary AgR repertoire.

  13. Recombinant GDNF: Tetanus toxin fragment C fusion protein produced from insect cells

    International Nuclear Information System (INIS)

    Li, Jianhong; Chian, Ru-Ju; Ay, Ilknur; Celia, Samuel A.; Kashi, Brenda B.; Tamrazian, Eric; Matthews, Jonathan C.; Remington, Mary P.; Pepinsky, R. Blake; Fishman, Paul S.; Brown, Robert H.; Francis, Jonathan W.

    2009-01-01

    Glial cell line-derived neurotrophic factor (GDNF) has potent survival-promoting effects on CNS motor neurons in experimental animals. Its therapeutic efficacy in humans, however, may have been limited by poor bioavailability to the brain and spinal cord. With a view toward improving delivery of GDNF to CNS motor neurons in vivo, we generated a recombinant fusion protein comprised of rat GDNF linked to the non-toxic, neuron-binding fragment of tetanus toxin. Recombinant GDNF:TTC produced from insect cells was a soluble homodimer like wild-type GDNF and was bi-functional with respect to GDNF and TTC activity. Like recombinant rat GDNF, the fusion protein increased levels of immunoreactive phosphoAkt in treated NB41A3-hGFRα-1 neuroblastoma cells. Like TTC, GDNF:TTC bound to immobilized ganglioside GT1b in vitro with high affinity and selectivity. These results support further testing of recombinant GDNF:TTC as a non-viral vector to improve delivery of GDNF to brain and spinal cord in vivo.

  14. Recombinant GDNF: Tetanus toxin fragment C fusion protein produced from insect cells

    Energy Technology Data Exchange (ETDEWEB)

    Li, Jianhong; Chian, Ru-Ju; Ay, Ilknur; Celia, Samuel A.; Kashi, Brenda B.; Tamrazian, Eric; Matthews, Jonathan C. [Cecil B. Day Laboratory for Neuromuscular Research, Department of Neurology, Massachusetts General Hospital, Charlestown, MA 02129 (United States); Remington, Mary P. [Research Service, Baltimore Veterans Affairs Medical Center, Baltimore, MD 21201 (United States); Pepinsky, R. Blake [BiogenIdec, Inc., 14 Cambridge Center, Cambridge, MA 02142 (United States); Fishman, Paul S. [Research Service, Baltimore Veterans Affairs Medical Center, Baltimore, MD 21201 (United States); Department of Neurology, University of Maryland School of Medicine, Baltimore, MD 21201 (United States); Brown, Robert H. [Cecil B. Day Laboratory for Neuromuscular Research, Department of Neurology, Massachusetts General Hospital, Charlestown, MA 02129 (United States); Francis, Jonathan W., E-mail: jwfrancisby@gmail.com [Cecil B. Day Laboratory for Neuromuscular Research, Department of Neurology, Massachusetts General Hospital, Charlestown, MA 02129 (United States)

    2009-07-31

    Glial cell line-derived neurotrophic factor (GDNF) has potent survival-promoting effects on CNS motor neurons in experimental animals. Its therapeutic efficacy in humans, however, may have been limited by poor bioavailability to the brain and spinal cord. With a view toward improving delivery of GDNF to CNS motor neurons in vivo, we generated a recombinant fusion protein comprised of rat GDNF linked to the non-toxic, neuron-binding fragment of tetanus toxin. Recombinant GDNF:TTC produced from insect cells was a soluble homodimer like wild-type GDNF and was bi-functional with respect to GDNF and TTC activity. Like recombinant rat GDNF, the fusion protein increased levels of immunoreactive phosphoAkt in treated NB41A3-hGFR{alpha}-1 neuroblastoma cells. Like TTC, GDNF:TTC bound to immobilized ganglioside GT1b in vitro with high affinity and selectivity. These results support further testing of recombinant GDNF:TTC as a non-viral vector to improve delivery of GDNF to brain and spinal cord in vivo.

  15. Towards a more precise serological diagnosis of human tegumentary leishmaniasis using Leishmania recombinant proteins.

    Science.gov (United States)

    Souza, Ana Paula; Soto, Manuel; Costa, Jackson M L; Boaventura, Viviane S; de Oliveira, Camila I; Cristal, Juqueline R; Barral-Netto, Manoel; Barral, Aldina

    2013-01-01

    Exposure to Leishmania induces a humoral immune response that can be used as a marker of parasite exposure. Herein, ELISA was used to screen sera from patients with Tegumentary Leishmaniasis (TL) against different L. infantum-chagasi-derived recombinant proteins (rHSP70, rH2A, rH2B, rH3, rH4 and rKMP11). Among the recombinant proteins, rHSP70 and rH2A showed the best reactivity against human sera obtained from endemic areas of TL. Receiver-Operator Characteristics (ROC) curve analysis was used to identify the effectiveness of these proteins for serodiagnosis of TL. ROC curves confirmed the superior performance of rHSP70 and rH2A, in comparison to the other tested recombinant proteins. Additionally, we evaluated the specificity of the response to rHSP70 and rH2A by testing sera obtained from patients with Chagas' disease, Tuberculosis, Leprosy or Systemic Lupus Erythematosus. In this case, rHSP70 displayed an increased ability to discriminate diseases, in comparison to SLA. Our results raise possibility of using rHSP70 for the serodiagnosis of TL.

  16. Towards a more precise serological diagnosis of human tegumentary leishmaniasis using Leishmania recombinant proteins.

    Directory of Open Access Journals (Sweden)

    Ana Paula Souza

    Full Text Available BACKGROUND: Exposure to Leishmania induces a humoral immune response that can be used as a marker of parasite exposure. METHODOLOGY/PRINCIPAL FINDINGS: Herein, ELISA was used to screen sera from patients with Tegumentary Leishmaniasis (TL against different L. infantum-chagasi-derived recombinant proteins (rHSP70, rH2A, rH2B, rH3, rH4 and rKMP11. Among the recombinant proteins, rHSP70 and rH2A showed the best reactivity against human sera obtained from endemic areas of TL. Receiver-Operator Characteristics (ROC curve analysis was used to identify the effectiveness of these proteins for serodiagnosis of TL. ROC curves confirmed the superior performance of rHSP70 and rH2A, in comparison to the other tested recombinant proteins. Additionally, we evaluated the specificity of the response to rHSP70 and rH2A by testing sera obtained from patients with Chagas' disease, Tuberculosis, Leprosy or Systemic Lupus Erythematosus. In this case, rHSP70 displayed an increased ability to discriminate diseases, in comparison to SLA. CONCLUSION: Our results raise possibility of using rHSP70 for the serodiagnosis of TL.

  17. Escherichia coli MW005: lambda Red-mediated recombineering and copy-number induction of oriV-equipped constructs in a single host

    Directory of Open Access Journals (Sweden)

    Hope Ian A

    2010-03-01

    Full Text Available Abstract Background Escherichia coli strain EL350 contains chromosomally integrated phage lambda Red recombinase genes enabling this strain to be used for modifying the sequence of resident clones via recombineering. BAC and fosmid clones are highly suitable for modification by recombineering but, because they are present at low (1-2 copies per cell, the DNA is difficult to isolate in high yield and purity. To overcome this limitation vectors, e.g. pCC1FOS, have been constructed that contain the additional replication origin, oriV, which permits copy-number to be induced transiently when propagated in a suitable host strain, e.g. EPI300, that supplies the cognate trans-replication protein TrfA. Previously, we used EL350 and EPI300 sequentially to recombineer oriV-equipped fosmid genomic clones and, subsequently, to induce copy-number of the resulting recombinant clone. To eliminate these intervening DNA isolation and transformation steps we retrofitted EL350 with a PBAD-driven trfA gene generating strain MW005 that supports, independently, both recombineering and copy-number induction. Results The PBAD-driven copy of cre in EL350 was replaced seamlessly with a copy of trfA, PCR-amplified from EPI300 chromosomal DNA, to generate MW005. This new strain has been used to both generate, via recombineering, a number of reporter gene fusions directly from pCC1FOS-based Caenorhabditis elegans genomic clones and to transiently induce copy-number of fosmid and BAC clones prior to DNA preparation. Conclusions By retrofitting EL350, an established 'recombineering' E. coli strain, with a tightly regulated copy of trfA we have produced a new strain, MW005, which combines recombineering capacity with the useful ability to transiently induce copy-number of oriV-equipped clones. By coupling these two steps in a single strain, use of MW005 will enable the more rapid recombineering-mediated production of recombinant clones in the yield and quality necessary for

  18. Production and Application of Polyclonal Antibodies Against Recombinant Capsid Protein of Extra Small Virus of Macrobrachium rosenbergii

    OpenAIRE

    Neethi, V.; Sivakumar, N.; Kumar, Kundan; Rajendran, K. V.; Makesh, M.

    2012-01-01

    Macrobrachium rosenbergii nodavirus along with a satellite virus, extra small virus (XSV) causes white tail disease (WTD) in the giant freshwater prawn M. rosenbergii. Infected M. rosenbergii postlarvae were collected from a hatchery in Kakinada, Andhra Pradesh. The gene coding the capsid protein of XSV was cloned in a bacterial expression vector pRSET A and the recombinant protein was expressed in Escherichia coli BL21(DE3)pLysS cells. The recombinant protein was purified by Nickel affinity ...

  19. Analysis of direct immobilized recombinant protein G on a gold surface

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Hyunhee [Department of Chemical and Biomolecular Engineering, Sogang University, Shinsu-dong, Mapo-gu, Seoul 121-742 (Korea, Republic of); Research Institute for Applied Science and Technology, Sogang University , Shinsu-dong, Mapo-gu, Seoul 121-742 (Korea, Republic of); Kang, Da-Yeon [Department of Chemical and Biomolecular Engineering, Sogang University, Shinsu-dong, Mapo-gu, Seoul 121-742 (Korea, Republic of); Goh, Hyun-Jeong [Interdisciplinary Program of Integrated Biotechnology, Sogang University, Shinsu-dong, Mapo-gu, Seoul 121-742 (Korea, Republic of); Oh, Byung-Keun [Department of Chemical and Biomolecular Engineering, Sogang University, Shinsu-dong, Mapo-gu, Seoul 121-742 (Korea, Republic of); Interdisciplinary Program of Integrated Biotechnology, Sogang University, Shinsu-dong, Mapo-gu, Seoul 121-742 (Korea, Republic of); Singh, Ravindra P. [Interdisciplinary Program of Integrated Biotechnology, Sogang University, Shinsu-dong, Mapo-gu, Seoul 121-742 (Korea, Republic of); Oh, Soo-Min [Department of Chemical and Biomolecular Engineering, Sogang University, Shinsu-dong, Mapo-gu, Seoul 121-742 (Korea, Republic of); Research Institute for Applied Science and Technology, Sogang University , Shinsu-dong, Mapo-gu, Seoul 121-742 (Korea, Republic of); Choi, Jeong-Woo [Department of Chemical and Biomolecular Engineering, Sogang University, Shinsu-dong, Mapo-gu, Seoul 121-742 (Korea, Republic of); Interdisciplinary Program of Integrated Biotechnology, Sogang University, Shinsu-dong, Mapo-gu, Seoul 121-742 (Korea, Republic of)], E-mail: jwchoi@sogang.ac.kr

    2008-09-15

    Abstact: For the immobilization of IgG, various techniques such as chemical linker, thiolated protein G methods, and fragmentation of antibodies have been reported [Y.M. Bae, B.K. Oh, W. Lee, W.H. Lee, J.W. Choi, Biosensors Bioelectron. 21 (2005) 103; W. Lee, B.K. Oh, W.H. Lee, J.W. Choi, Colloids Surf. B-Biointerfaces, 40 (2005) 143; A.A. Karyakin, G.V. Presnova, M.Y. Rubtsova, A.M. Egorov, Anal. Chem. 72 (2000) 3805]. Here, we modified the immunoglobulin Fc-binding B-domain of protein G to contain two cysteine residues at its C-terminus by a genetic engineering technique. The resulting recombinant protein, RPGcys, retained IgG-binding activity in the same manner as native protein G. RPGcys was immobilized on a gold surface by strong affinity between thiol of cysteine and gold. The orientations of both IgG layers immobilized on the base recombinant protein Gs were analyzed by fluorescence microscope, atomic force microscope (AFM), and surface plasmon resonance (SPR). Our data revealed that IgG-binding activity of RPGcys on gold surface significantly increased in comparison to wild type of protein G (RPGwild), which was physically adsorbed due to absence of cysteine residue. Immobilization of highly oriented antibodies based on cysteine-modified protein G could be useful for the fabrication of immunosensor systems.

  20. Vaccination using recombinants influenza and adenoviruses encoding amastigote surface protein-2 are highly effective on protection against Trypanosoma cruzi infection.

    Science.gov (United States)

    Barbosa, Rafael Polidoro Alves; Filho, Bruno Galvão; Dos Santos, Luara Isabela; Junior, Policarpo Ademar Sales; Marques, Pedro Elias; Pereira, Rafaela Vaz Sousa; Cara, Denise Carmona; Bruña-Romero, Oscar; Rodrigues, Maurício Martins; Gazzinelli, Ricardo Tostes; Machado, Alexandre Vieira

    2013-01-01

    In the present study we evaluated the protection raised by immunization with recombinant influenza viruses carrying sequences coding for polypeptides corresponding to medial and carboxi-terminal moieties of Trypanosoma cruzi ´s amastigote surface protein 2 (ASP2). Those viruses were used in sequential immunization with recombinant adenovirus (heterologous prime-boost immunization protocol) encoding the complete sequence of ASP2 (Ad-ASP2) in two mouse strains (C57BL/6 and C3H/He). The CD8 effector response elicited by this protocol was comparable to that observed in mice immunized twice with Ad-ASP2 and more robust than that observed in mice that were immunized once with Ad-ASP2. Whereas a single immunization with Ad-ASP2 sufficed to completely protect C57BL/6 mice, a higher survival rate was observed in C3H/He mice that were primed with recombinant influenza virus and boosted with Ad-ASP2 after being challenged with T. cruzi. Analyzing the phenotype of CD8+ T cells obtained from spleen of vaccinated C3H/He mice we observed that heterologous prime-boost immunization protocol elicited more CD8+ T cells specific for the immunodominant epitope as well as a higher number of CD8+ T cells producing TNF-α and IFN-γ and a higher mobilization of surface marker CD107a. Taken together, our results suggest that immunodominant subpopulations of CD8+ T elicited after immunization could be directly related to degree of protection achieved by different immunization protocols using different viral vectors. Overall, these results demonstrated the usefulness of recombinant influenza viruses in immunization protocols against Chagas Disease.

  1. Effect of the conserved oligosaccharides of recombinant monoclonal antibodies on the separation by protein A and protein G chromatography.

    Science.gov (United States)

    Gaza-Bulseco, Georgeen; Hickman, Keith; Sinicropi-Yao, Sara; Hurkmans, Karen; Chumsae, Chris; Liu, Hongcheng

    2009-03-20

    Glycosylation of the conserved asparagine residue in CH2 domains of IgG molecules is an important post-translational modification. The presence of oligosaccharides is critical for structure, stability and biological function of IgG antibodies. Effect of the glycosylation states of recombinant monoclonal antibodies on protein A and protein G chromatography was evaluated. Antibodies lacking oligosaccharides eluted later from protein A and earlier from protein G columns than antibodies with oligosaccharides using a gradient of decreasing pH. Interestingly, different types of oligosaccharides also affected the elution of the antibodies. Antibodies with high mannose type oligosaccharides were enriched in later eluting fractions from protein A and earlier eluting fractions from protein G. While antibodies with more mature oligosaccharides, such as core fucosylated biantennary complex oligosaccharides with zero (Gal 0), one (Gal 1) or two (Gal 2) terminal galactoses, were enriched in earlier eluting fractions from protein A and in the later eluting fractions from protein G. However, analysis by enzyme-linked immunosorbent assay (ELISA) revealed that antibody binding affinity to protein A and protein G was not affected by the absence or presence of oligosaccharides. It was thus concluded that the elution difference of antibodies with or without oligosaccharides and antibodies with different types of oligosaccharides were due to differential structural changes around the CH2-CH3 domain interface under the low pH conditions used for protein A and protein G chromatography.

  2. Recombination-stable multimeric green fluorescent protein for characterization of weak promoter outputs in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Rugbjerg, Peter; Knuf, Christoph; Förster, Jochen

    2015-01-01

    Green fluorescent proteins (GFPs) are widely used for visualization of proteins to track localization and expression dynamics. However, phenotypically important processes can operate at too low expression levels for routine detection, i.e. be overshadowed by autofluorescence noise. While GFP...... functions well in translational fusions, the use of tandem GFPs to amplify fluorescence signals is currently avoided in Saccharomyces cerevisiae and many other microorganisms due to the risk of loop-out by direct-repeat recombination. We increased GFP fluorescence by translationally fusing three different...... cultured for 25 generations under strong and slightly toxic expression after which only limited reduction in fluorescence was detectable. Such non-recombinogenic GFPs can help quantify intracellular responses operating a low copy number in recombination-prone organisms....

  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...... optimization of metabolic facilities towards enhanced product yields. We have shown that by permeabilizing cyanobacteria cells with a commercially available B-PER reagent, cells can be directly used for measurement of intracellular enzymes in an easy, efficient and scalable manner....... biotechnology hosts including safety, metabolic diversity, scalability, sustainability and low production cost. Over the past decades, considerable improvement has been made to express and secrete recombinant proteins in high levels: however current yields are still low. The first research project presented...

  4. Altering the ribosomal subunit ratio in yeast maximizes recombinant protein yield

    Directory of Open Access Journals (Sweden)

    Poyner David R

    2009-01-01

    Full Text Available Abstract Background The production of high yields of recombinant proteins is an enduring bottleneck in the post-genomic sciences that has yet to be addressed in a truly rational manner. Typically eukaryotic protein production experiments have relied on varying expression construct cassettes such as promoters and tags, or culture process parameters such as pH, temperature and aeration to enhance yields. These approaches require repeated rounds of trial-and-error optimization and cannot provide a mechanistic insight into the biology of recombinant protein production. We published an early transcriptome analysis that identified genes implicated in successful membrane protein production experiments in yeast. While there has been a subsequent explosion in such analyses in a range of production organisms, no one has yet exploited the genes identified. The aim of this study was to use the results of our previous comparative transcriptome analysis to engineer improved yeast strains and thereby gain an understanding of the mechanisms involved in high-yielding protein production hosts. Results We show that tuning BMS1 transcript levels in a doxycycline-dependent manner resulted in optimized yields of functional membrane and soluble protein targets. Online flow microcalorimetry demonstrated that there had been a substantial metabolic change to cells cultured under high-yielding conditions, and in particular that high yielding cells were more metabolically efficient. Polysome profiling showed that the key molecular event contributing to this metabolically efficient, high-yielding phenotype is a perturbation of the ratio of 60S to 40S ribosomal subunits from approximately 1:1 to 2:1, and correspondingly of 25S:18S ratios from 2:1 to 3:1. This result is consistent with the role of the gene product of BMS1 in ribosome biogenesis. Conclusion This work demonstrates the power of a rational approach to recombinant protein production by using the results of

  5. Bacterial-based systems for expression and purification of recombinant Lassa virus proteins of immunological relevance

    Directory of Open Access Journals (Sweden)

    Cashman Kathleen A

    2008-06-01

    Full Text Available Abstract Background There is a significant requirement for the development and acquisition of reagents that will facilitate effective diagnosis, treatment, and prevention of Lassa fever. In this regard, recombinant Lassa virus (LASV proteins may serve as valuable tools in diverse antiviral applications. Bacterial-based systems were engineered for expression and purification of recombinant LASV nucleoprotein (NP, glycoprotein 1 (GP1, and glycoprotein 2 (GP2. Results Full-length NP and the ectodomains of GP1 and GP2 were generated as maltose-binding protein (MBP fusions in the Rosetta strains of Escherichia coli (E. coli using pMAL-c2x vectors. Average fusion protein yields per liter of culture for MBP-NP, MBP-GP1, and MBP-GP2 were 10 mg, 9 mg, and 9 mg, respectively. Each protein was captured from cell lysates using amylose resin, cleaved with Factor Xa, and purified using size-exclusion chromatography (SEC. Fermentation cultures resulted in average yields per liter of 1.6 mg, 1.5 mg, and 0.7 mg of purified NP, GP1 and GP2, respectively. LASV-specific antibodies in human convalescent sera specifically detected each of the purified recombinant LASV proteins, highlighting their utility in diagnostic applications. In addition, mouse hyperimmune ascitic fluids (MHAF against a panel of Old and New World arenaviruses demonstrated selective cross reactivity with LASV proteins in Western blot and enzyme-linked immunosorbent assay (ELISA. Conclusion These results demonstrate the potential for developing broadly reactive immunological assays that employ all three arenaviral proteins individually and in combination.

  6. Monitoring of protein profiles for the optimization of recombinant fermentation processes using public domain databases.

    Science.gov (United States)

    Dürrschmid, Karin; Marzban, Gorji; Dürrschmid, Eberhard; Striedner, Gerald; Clementschitsch, Franz; Cserjan-Puschmann, Monika; Bayer, Karl

    2003-01-01

    The expression of human superoxide dismutase in fed-batch fermentation of E. coli HMS174(DE3)(pET3ahSOD) was studied as model system. Due to the frequently used strong T7 promoter system a high metabolic load is exerted, which triggers stress response mechanisms and finally leads to the differentiation of the host cell. As a consequence, host cell metabolism is partly shifted from growth to survival accompanied by significant alterations of the protein pattern. In terms of process optimization two-dimensional electrophoresis deserves as a powerful tool to monitor these changes on protein level. For the analysis of samples derived from different states of recombinant protein production wide-range Immobiline Dry Strips pH 3-10 were used. In order to establish an efficient procedure for accelerated process optimization and to avoid costly and time-consuming analysis like mass spectrometry (MS), a database approach for the identification of significant changes of the protein pattern was evaluated. On average, 935 spots per gel were detected, whereby 50 are presumably stress-relevant. Out of these, 24 proteins could be identified by using the SWISS-2DPAGE database (www.expasy.ch/ch2d/). The identified proteins are involved in regulatory networks, energy metabolism, purine and pyrimidine nucleotide synthesis and translation. By this database approach, significant fluctuations of individual proteins in relation to recombinant protein production could be identified. Seven proteins show strong alterations (>100%) directly after induction and can therefore be stated as reliable marker proteins for the assessment of stress response. For distinctive interpretation of this highly specific information, a bioinformatic and statistic tool would be essential in order to perceive the role and contribution of individual proteins in stress response.

  7. Profiling of external metabolites during production of hantavirus nucleocapsid protein with recombinant Saccharomyces cerevisiae

    OpenAIRE

    Antoniukas, Linas; Grammel, Hartmut; Sasnauskas, Kestutis; Reichl, Udo

    2007-01-01

    Recombinant strains of Saccharomyces cerevisiae, producing hantavirus Puumala nucleocapsid protein for diagnostics and as a candidate vaccine were analyzed for uptake and excretion of intermediary metabolites during process optimization studies of fed-batch bioreactor cultures. Concentrations of glucose, maltose, galactose, pyruvate, acetaldehyde, ethanol, acetate, succinate and formaldehyde (used as a selection agent) were measured in the culture medium in order to find a metabolite pattern,...

  8. Localization of recombination proteins and Srs2 reveals anti-recombinase function in vivo

    DEFF Research Database (Denmark)

    Burgess, Rebecca C; Lisby, Michael; Altmannova, Veronika

    2009-01-01

    Homologous recombination (HR), although an important DNA repair mechanism, is dangerous to the cell if improperly regulated. The Srs2 "anti-recombinase" restricts HR by disassembling the Rad51 nucleoprotein filament, an intermediate preceding the exchange of homologous DNA strands. Here, we...... removes Rad51 indiscriminately from DNA, while the Rad52 protein coordinates appropriate filament reformation. This constant breakdown and rebuilding of filaments may act as a stringent quality control mechanism during HR....

  9. Production, purification and oxidative folding of the mouse recombinant prion protein

    Czech Academy of Sciences Publication Activity Database

    Pavlíček, A.; Bednárová, Lucie; Holada, K.

    2007-01-01

    Roč. 52, č. 4 (2007), s. 391-397 ISSN 0015-5632 R&D Projects: GA ČR GD310/05/H533 Grant - others:GA ČR(CZ) GA310/04/0419 Institutional research plan: CEZ:AV0Z40550506 Keywords : recombinant prion protein * production * purification * folding Subject RIV: CE - Biochemistry Impact factor: 0.989, year: 2007 http://www.biomed.cas.cz/mbu/folia/

  10. Activities of Fluoroquinolones against Streptococcus pneumoniae Type II Topoisomerases Purified as Recombinant Proteins

    OpenAIRE

    Morrissey, Ian; George, John

    1999-01-01

    Streptococcus pneumoniae topoisomerase IV and DNA gyrase have been purified from a fluoroquinolone-susceptible Streptococcus pneumoniae strain, from first-step mutants showing low-level resistance to ciprofloxacin, sparfloxacin, levofloxacin, and ofloxacin, and from two clinical isolates showing intermediate- and high-level fluoroquinolone resistance by a gene cloning method that produces recombinant proteins from Escherichia coli. The concentrations of ciprofloxacin, sparfloxacin, levofloxac...

  11. Growth and recombinant protein expression with Escherichia coli in different batch cultivation media.

    Science.gov (United States)

    Hortsch, Ralf; Weuster-Botz, Dirk

    2011-04-01

    Parallel operated milliliter-scale stirred tank bioreactors were applied for recombinant protein expression studies in simple batch experiments without pH titration. An enzymatic glucose release system (EnBase), a complex medium, and the frequently used LB and TB media were compared with regard to growth of Escherichia coli and recombinant protein expression (alcohol dehydrogenase (ADH) from Lactobacillus brevis and formate dehydrogenase (FDH) from Candida boidinii). Dissolved oxygen and pH were recorded online, optical densities were measured at-line, and the activities of ADH and FDH were analyzed offline. Best growth was observed in a complex medium with maximum dry cell weight concentrations of 14 g L(-1). EnBase cultivations enabled final dry cell weight concentrations between 6 and 8 g L(-1). The pH remained nearly constant in EnBase cultivations due to the continuous glucose release, showing the usefulness of this glucose release system especially for pH-sensitive bioprocesses. Cell-specific enzyme activities varied considerably depending on the different media used. Maximum specific ADH activities were measured with the complex medium, 6 h after induction with IPTG, whereas the highest specific FDH activities were achieved with the EnBase medium at low glucose release profiles 24 h after induction. Hence, depending on the recombinant protein, different medium compositions, times for induction, and times for cell harvest have to be evaluated to achieve efficient expression of recombinant proteins in E. coli. A rapid experimental evaluation can easily be performed with parallel batch operated small-scale stirred tank bioreactors.

  12. [Characterization of a recombinant Listeria monocytogenes strain containing the fusion protein gene of Newcastle disease virus].

    Science.gov (United States)

    Xu, Jing-Jing; Jiang, Ling-Li; Chen, Ning; Shuai, Jiang-Bing; Fang, Wei-Huan

    2006-06-01

    Homologous recombination was utilized for construction of a recombinant strain of L. monocytogenes carrying a gene from the Newcastle diseases virus by insertional mutation targeting its listeriolysin O gene (hly). The gene encoding fusion protein of the Newcastle disease virus (NDV-F) was used as the model heterologous gene. The F gene was inserted into hly downstream to its promoter and signal sequence by overlapping extension polymerase chain reaction, which was then subcloned into the shuttle plasmid pKSV7 for allelic exchange with L. monocytogenes chromosome. PCR amplification of the target genes indicated insertion of the F gene into the chromosome DNA of L. monocytogenes. RT-PCR showed transcription of F gene from the recombinant L. monocytogenes strain. Comparisons were then made between the recombinant strain and its wild parent strain in terms of the hemolytic activity, adhesion and invasiveness to cultured HeLa cells, virulence to mice and chicken embryos, and growth kinetics in broth medium as well as its stability upon repeated subculturing and serial passages in mice. The recombinant L. monocytogenes lost its hemolytic activity on the blood agar and had no hemolytic titer from its culture supernatants as compared with the titer of 24 in the supernatant from the wild parent strain. The recombinant strain also had lower adhesiveness (P > 0.05) and significantly lower relative invasiveness to the HeLa cells than its wild type strain (P gene NDV-F from its genomic DNA after subculturing in BHI broth or in mice for 5 times.

  13. Structural characterization by transmission electron microscopy and immunoreactivity of recombinant Hendra virus nucleocapsid protein expressed and purified from Escherichia coli.

    Science.gov (United States)

    Pearce, Lesley A; Yu, Meng; Waddington, Lynne J; Barr, Jennifer A; Scoble, Judith A; Crameri, Gary S; McKinstry, William J

    2015-12-01

    Hendra virus (family Paramyxoviridae) is a negative sense single-stranded RNA virus (NSRV) which has been found to cause disease in humans, horses, and experimentally in other animals, e.g. pigs and cats. Pteropid bats commonly known as flying foxes have been identified as the natural host reservoir. The Hendra virus nucleocapsid protein (HeV N) represents the most abundant viral protein produced by the host cell, and is highly immunogenic with naturally infected humans and horses producing specific antibodies towards this protein. The purpose of this study was to express and purify soluble, functionally active recombinant HeV N, suitable for use as an immunodiagnostic reagent to detect antibodies against HeV. We expressed both full-length HeV N, (HeV NFL), and a C-terminal truncated form, (HeV NCORE), using a bacterial heterologous expression system. Both HeV N constructs were engineered with an N-terminal Hisx6 tag, and purified using a combination of immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC). Purified recombinant HeV N proteins self-assembled into soluble higher order oligomers as determined by SEC and negative-stain transmission electron microscopy. Both HeV N proteins were highly immuno-reactive with sera from animals and humans infected with either HeV or the closely related Nipah virus (NiV), but displayed no immuno-reactivity towards sera from animals infected with a non-pathogenic paramyxovirus (CedPV), or animals receiving Equivac® (HeV G glycoprotein subunit vaccine), using a Luminex-based multiplexed microsphere assay. Crown Copyright © 2015. Published by Elsevier Inc. All rights reserved.

  14. Expression and Characterisation of Recombinant Rhodocyclus tenuis High Potential Iron-Sulphur Protein

    DEFF Research Database (Denmark)

    Caspersen, Michael Bjerg; Bennet, K.; Christensen, Hans Erik Mølager

    2000-01-01

    The high potential iron-sulfur protein (HiPIP) from Rhodocyclus tenuis strain 2761 has been overproduced in Escherichia coli from its structural gene, purified to apparent homogeneity, and then characterized by an array of methods. UV-visible spectra of the reduced and oxidized recombinant protein...... of the apoprotein was 6296.6 Da compared to the expected average molecular mass of 6297.2 Da of the apoprotein, The reduction potential was determined using cyclic and square-wave voltammetry to be 321 and 314 mV versus NHE, respectively. All the observed properties of the recombinant protein parallel those...... were similar to those of the native protein. EPR of the oxidized protein shows g values of 2.11, 2.03, and 2.03. ESI-MS gave a mass difference of 350 Da between the holoprotein and acid-treated protein, consistent with incorporation of a [Fe4S4] cluster in the holoprotein, The observed mass...

  15. Quantifying the Effects of Frequency and Amplitude of Periodic Oxygen-Related Stress on Recombinant Protein Production in Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Mohammadhadi Jazini

    2013-12-01

    Full Text Available Pichia pastoris is an attractive candidate platform for recombinant protein production. Dissolved oxygen is one of the most important factors in the cultivation of P. pastoris. However, the effect of oxygen on triggering productivity led to ambivalent results. In our earlier work, a two-compartment system, consisting of a single reactor coupled with a plug flow reactor (PFR, has been proposed as a tool to improve protein quantity and quality. The goal of this work was to investigate the effects of frequency (the residence time of broth in the PFR and amplitude (the dissolved oxygen level in the reactor of the stress on productivity, titer and physiology. A recombinant P. pastoris strain, which expressed horseradish peroxidase, was used as the model system. Thirteen experiments were performed. Multivariate data analysis was done and the results showed that the residence time did not influence titer, productivity and physiology over the range of residence time studied while dissolved oxygen influenced titer and specific productivity in a quadratic function. In other words, an intermediate level of dissolved oxygen (25% showed the highest specific productivity and titter, irrespective of the residence time in the PFR. In turn, the variation of the residence time and dissolved oxygen did not influence growth physiology, as quantified in biomass and carbon dioxide yields.

  16. Dunaliella salina as a novel host for the production of recombinant proteins.

    Science.gov (United States)

    Feng, Shuying; Li, Xuebing; Xu, Zhengshun; Qi, Jingjiao

    2014-05-01

    Although several expression systems are currently available for the production of recombinant proteins, they still have some inherent disadvantages, thereby resulting in the desire to explore a novel expression system for producing recombinant proteins. Dunaliella salina (D. salina) has been exploited as a novel expression system for the field of genetic engineering because of its distinct advantages, including low production cost, fast growth, easy culture, ease of transgenic manipulation, and modified abilities of transcription and translation. Thus far, studies on D. salina host have made great development and significant progress. This paper presents a comprehensive summary of the achievements of D. salina host from the following aspects: the advantages of D. salina cells, transformation methods, cloning of D. salina genes, and expression of exogenous genes into D. salina. Furthermore, the authors identified the current main obstacles and future application prospects for the recombinant proteins produced by D. salina, which could be used as a basis for the future maturation of D. salina expression system.

  17. Monitoring EDTA process residuals in recombinant protein manufacturing using liquid chromatography.

    Science.gov (United States)

    Lin, Miao-Fang; Royal, Mabel; Hayenga, Kirk; Conn, Greg

    2003-07-25

    We have developed a chromatographic method for the high sensitivity quantitation of EDTA process residuals in recombinant protein manufacturing validation studies. The reversed-phase HPLC method is based upon the detection of Cu(2+)/EDTA complexes at 254 nm, and has been qualified for use on intermediates from a purification process for a recombinant protein expressed in E. coli. Quantitation of EDTA in recombinant protein process intermediates is linear in the range of 0.2 to 64 microM with LOD/LOQ values below 2.0 microM. The assay is suitable for use in process backgrounds containing Tris, HEPES, MES, NaCl, hexanediol, NH(4)SO(4), and PEG. EDTA spike recovery values in all process samples tested were greater than 90% at the 4.0 microM concentration. System suitability parameters for the chromatographic method were developed based upon peak area and retention time precision, column efficiency and USP tailing. Peak area precision and intermediate precision values across the linear range of the assay exhibited C.V. values less than 15% at any concentration tested in all sample backgrounds. The assay robustness was tested by transfer of the assay to a second laboratory and analyst with use of multiple process intermediate lots, reagent/column lots, and HPLC systems.

  18. High-throughput recombinant protein expression in Escherichia coli: current status and future perspectives

    Science.gov (United States)

    2016-01-01

    The ease of genetic manipulation, low cost, rapid growth and number of previous studies have made Escherichia coli one of the most widely used microorganism species for producing recombinant proteins. In this post-genomic era, challenges remain to rapidly express and purify large numbers of proteins for academic and commercial purposes in a high-throughput manner. In this review, we describe several state-of-the-art approaches that are suitable for the cloning, expression and purification, conducted in parallel, of numerous molecules, and we discuss recent progress related to soluble protein expression, mRNA folding, fusion tags, post-translational modification and production of membrane proteins. Moreover, we address the ongoing efforts to overcome various challenges faced in protein expression in E. coli, which could lead to an improvement of the current system from trial and error to a predictable and rational design. PMID:27581654

  19. High-throughput recombinant protein expression in Escherichia coli: current status and future perspectives.

    Science.gov (United States)

    Jia, Baolei; Jeon, Che Ok

    2016-08-01

    The ease of genetic manipulation, low cost, rapid growth and number of previous studies have made Escherichia coli one of the most widely used microorganism species for producing recombinant proteins. In this post-genomic era, challenges remain to rapidly express and purify large numbers of proteins for academic and commercial purposes in a high-throughput manner. In this review, we describe several state-of-the-art approaches that are suitable for the cloning, expression and purification, conducted in parallel, of numerous molecules, and we discuss recent progress related to soluble protein expression, mRNA folding, fusion tags, post-translational modification and production of membrane proteins. Moreover, we address the ongoing efforts to overcome various challenges faced in protein expression in E. coli, which could lead to an improvement of the current system from trial and error to a predictable and rational design. © 2016 The Authors.

  20. Generation of Recombinant Schmallenberg Virus Nucleocapsid Protein in Yeast and Development of Virus-Specific Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Justas Lazutka

    2014-01-01

    Full Text Available Schmallenberg virus (SBV, discovered in continental Europe in late 2011, causes mild clinical signs in adult ruminants, including diarrhoea and reduced milk yield. However, fetal infection can lead to severe malformation in newborn offspring. To develop improved reagents for SBV serology, a high-level yeast expression system was employed to produce recombinant SBV nucleocapsid (N protein. Recombinant SBV N protein was investigated as an antigen in SBV-specific IgG enzyme immunoassay and used for generation of monoclonal antibodies (MAbs. Yeast-expressed SBV N protein was reactive with anti-SBV IgG-positive cow serum specimens collected from different farms of Lithuania. After immunization of mice with recombinant SBV N protein, four MAbs were generated. The MAbs raised against recombinant SBV N protein reacted with native viral nucleocapsids in SBV-infected BHK cells by immunofluorescence assay. The reactivity of recombinant N protein with SBV-positive cow serum specimens and the ability of the MAbs to recognize virus-infected cells confirm the antigenic similarity between yeast-expressed SBV N protein and native viral nucleocapsids. Our study demonstrates that yeast expression system is suitable for high-level production of recombinant SBV N protein and provides the first evidence on the presence of SBV-specific antibodies in cow serum specimens collected in Lithuania.

  1. Transgenic silkworms secrete the recombinant glycosylated MRJP1 protein of Chinese honeybee, Apis cerana cerana.

    Science.gov (United States)

    You, Zhengying; Qian, Qiujie; Wang, Yiran; Che, Jiaqian; Ye, Lupeng; Shen, Lirong; Zhong, Boxiong

    2017-10-01

    Major royal jelly protein-1 (MRJP1) is the most abundant glycoprotein of royal jelly (RJ) and is considered a potential component of functional foods. In this study, we used silkworm transgenic technology to obtain five transgenic silkworm lineages expressing the exogenous recombinant Chinese honeybee, Apis cerana cerana, protein-1 (rAccMRJP1) under the control of a fibroin light chain (Fib-L) promoter in the posterior silk glands. The protein was successfully secreted into cocoons; specifically, the highest rAccMRJP1 protein content was 0.78% of the dried cocoons. Our results confirmed that the protein band of the exogenous rAccMRJP1 protein expressed in the transgenic silkworm lineages was a glycosylated protein. Therefore, this rAccMRJP1 protein could be used as an alternative standard protein sample to measure the freshness of RJ. Moreover, we also found that the overall trend between the expression of the endogenous and exogenous genes was that the expression level of the endogenous Fib-L gene declined as the expression of the exogenous rAccMRJP1 gene increased in the transgenic silkworm lineages. Thus, by employing genome editing technology to reduce silk protein expression levels, a silkworm bioreactor expression system could be developed as a highly successful system for producing various valuable heterologous proteins, potentially broadening the applications of the silkworm.

  2. Target-selective homologous recombination cloning for high-throughput generation of monoclonal antibodies from single plasma cells.

    Science.gov (United States)

    Kurosawa, Nobuyuki; Yoshioka, Megumi; Isobe, Masaharu

    2011-04-13

    Molecular cloning of functional immunoglobulin genes from single plasma cells is one of the most promising technologies for the rapid development of monoclonal antibody drugs. However, the proper insertion of PCR-amplified immunoglobulin genes into expression vectors remains an obstacle to the high-throughput production of recombinant monoclonal antibodies. We developed a single-step cloning method, target-selective homologous recombination (TS-HR), in which PCR-amplified immunoglobulin variable genes were selectively inserted into vectors, even in the presence of nonspecifically amplified DNA. TS-HR utilizes Red/ET-mediated homologous recombination with a target-selective vector (TS-vector) with unique homology arms on its termini. Using TS-HR, immunoglobulin variable genes were cloned directly into expression vectors by co-transforming unpurified PCR products and the TS-vector into E. coli. Furthermore, the high cloning specificity of TS-HR allowed plasmids to be extracted from pools of transformed bacteria without screening single colonies for correct clones. We present a one-week protocol for the production of recombinant mouse monoclonal antibodies from large numbers of single plasma cells. The time requirements and limitations of traditional cloning procedures for the production of recombinant immunoglobulins have been significantly reduced with the development of the TS-HR cloning technique. © 2011 Kurosawa et al; licensee BioMed Central Ltd.

  3. Target-selective homologous recombination cloning for high-throughput generation of monoclonal antibodies from single plasma cells

    Directory of Open Access Journals (Sweden)

    Isobe Masaharu

    2011-04-01

    Full Text Available Abstract Background Molecular cloning of functional immunoglobulin genes from single plasma cells is one of the most promising technologies for the rapid development of monoclonal antibody drugs. However, the proper insertion of PCR-amplified immunoglobulin genes into expression vectors remains an obstacle to the high-throughput production of recombinant monoclonal antibodies. Results We developed a single-step cloning method, target-selective homologous recombination (TS-HR, in which PCR-amplified immunoglobulin variable genes were selectively inserted into vectors, even in the presence of nonspecifically amplified DNA. TS-HR utilizes Red/ET-mediated homologous recombination with a target-selective vector (TS-vector with unique homology arms on its termini. Using TS-HR, immunoglobulin variable genes were cloned directly into expression vectors by co-transforming unpurified PCR products and the TS-vector into E. coli. Furthermore, the high cloning specificity of TS-HR allowed plasmids to be extracted from pools of transformed bacteria without screening single colonies for correct clones. We present a one-week protocol for the production of recombinant mouse monoclonal antibodies from large numbers of single plasma cells. Conclusion The time requirements and limitations of traditional cloning procedures for the production of recombinant immunoglobulins have been significantly reduced with the development of the TS-HR cloning technique.

  4. Electronic fine structure and recombination dynamics in single InAs quantum dots

    International Nuclear Information System (INIS)

    Seguin, R.

    2008-01-01

    In the work at hand single InAs/GaAs quantum dots (QDs) are examined via cathodoluminescence spectroscopy. A thorough analysis of the spectra leads to an unambiguous assignment of the lines to the decay of specific excitonic complexes. A special aspect of the Coulomb interaction, the exchange interaction, gives rise to a fine structure in the initial and final states of an excitonic decay. This leads to a fine structure in the emission spectra that again is unique for every excitonic complex. The exchange interaction is discussed in great detail in this work.QDs of different sizes are investigated and the influence on the electronic properties is monitored. Additionally, the structure is modified ex situ by a thermal annealing process. The changes of the spectra under different annealing temperatures are traced. Finally, recombination dynamics of different excitonic complexes are examined by performing time-resolved cathodoluminescence spectroscopy. (orig.)

  5. Electronic fine structure and recombination dynamics in single InAs quantum dots

    Energy Technology Data Exchange (ETDEWEB)

    Seguin, R.

    2008-01-28

    In the work at hand single InAs/GaAs quantum dots (QDs) are examined via cathodoluminescence spectroscopy. A thorough analysis of the spectra leads to an unambiguous assignment of the lines to the decay of specific excitonic complexes. A special aspect of the Coulomb interaction, the exchange interaction, gives rise to a fine structure in the initial and final states of an excitonic decay. This leads to a fine structure in the emission spectra that again is unique for every excitonic complex. The exchange interaction is discussed in great detail in this work.QDs of different sizes are investigated and the influence on the electronic properties is monitored. Additionally, the structure is modified ex situ by a thermal annealing process. The changes of the spectra under different annealing temperatures are traced. Finally, recombination dynamics of different excitonic complexes are examined by performing time-resolved cathodoluminescence spectroscopy. (orig.)

  6. HUMAN PAPILLOMA VIRUS IMMUNOGEN CREATION ON THE BASE OF CHIMERIC RECOMBINANT PROTEIN L2E7

    Directory of Open Access Journals (Sweden)

    I. S. Malakhov

    2016-01-01

    Full Text Available The cervical cancer is one of the most common diseases in world. This malignancy is the seventh highest prevalence oncological disease worldwide and the second highest prevalence oncological disease of women in the world. Meanwhile women need to be infected by human papilloma virus (HPV is absolutely necessary for it further evolution, HPV DNA was found in 99.97% cases of disease. Except cervical cancer, HPV cause 85% of rectal cancer, 50% of the vulva, vagina and penis cancers, 20% of oropharyngeal cancer and 10% of larynx and esophagus cancers. In 2009, 14 000 women were diagnosed with cervical cancer in Russia. The growth in morbidity was 19% (in comparison with 1999. The most effective recognised measure for almost each infection prophylaxis is a vaccination. Two human papilloma virus vaccines are available in Russia nowadays — Gardasil and Cervarix, produced in Belgium and the Netherlands respectively. Cervarix is a bivalent vaccine based on virus-like particles (VLP of two types. Recombinant major capsid proteins L1 HPV 16 and HPV 18 express in baculovirus expression system and self-assembled into virus-like particles (about 70 percent of cervical cancers are caused by HPV 16 and HPV 18. VLP of each strain produced in different baculovirus vectors and then combined in single drug. Gardasil is like Cervarix with few exceptions. Producing organisms are fungi S. cerevisiae in this case, and this vaccine contains low-risk HPV 6 and HPV 11 VLP. Thus, Gardasil is quadrivalent HPV-6/11/16/18 vaccine. These vaccines are very effective in averting infection of disease and don’t have significant side-effects, however they have some disadvantages. Firstly, they have a high price because of necessity of their expression in eukaryotic cells. Secondly, they are strain-specific, so vaccines are completely effective only for virus’s strains which are represented in the vaccine. Thirdly, it`s the absence of therapeutic (treatment of established

  7. Recombinant Plasmodium falciparum glutamate rich protein; purification and use in enzyme-linked immunosorbent assay

    DEFF Research Database (Denmark)

    Dziegiel, Morten Hanefeld; Borre, M B; Jepsen, S

    1991-01-01

    New Guinea (MAD20) and Honduras (HB3) completely absorbed specific antibodies, indicating the presence of conserved epitopes produced by all isolates of P. falciparum. Recombinant GLURP489-1271 ELISA is sensitive and rapid, and therefore well-suited for sero-epidemiological studies, and for control...... GLURP489-1271 was expressed as a chimeric protein, fused with E. coli beta-galactosidase. However, antibodies in sera were directed only against the malaria part of the fusion protein and not against beta-galactosidase. Antigen from in vitro P. falciparum cultures of isolates from Tanzania (F32), Papua...

  8. Helicobacter pylori recombinant UreG protein: cloning, expression, and assessment of its seroreactivity.

    Science.gov (United States)

    Khalilpour, Akbar; Osman, Sabariah; Yunus, Muhammad Hafiznur; Santhanam, Amutha; Vellasamy, Nagarajan; Noordin, Rahmah

    2014-11-18

    Helicobacter pylori is a human pathogen and during the process of infection, antigens from the bacterium elicit strong host humoral immune responses. In our previous report, native H. pylori UreG protein showed good reactivity with sera from H. pylori patients. This study was aimed at producing the recombinant form of the protein (rUreG) and determining its seroreactivities. The coding sequence of H. pylori UreG was cloned and the recombinant protein expressed and purified by affinity chromatography using nickel nitrilotriacetic acid (Ni-NTA) resin. The antigenicity of rUreG to detect H. pylori specific antibodies was determined by western blot, using HRP-conjugated anti-human IgG and IgA antibodies as probes. A total of 70 sera, comprising 30 positive and 40 control serum samples, were used. The positive sera were from culture-positive H. pylori-infected patients with duodenal ulcers, gastric ulcers, or gastritis. The control sera comprised three types of samples without detectable H. pylori antibodies, i.e. healthy individuals (with no history of gastric disorders) (n = 10); patients who attended an endoscopy clinic (because of gastrointestinal complaints) but were H. pylori culture negative (n = 20); and people with other diseases (n = 10). Additionally, hyperimmune mice serum against rUreG was raised and tested with the native and recombinant UreG protein. The ureG gene fragment was successfully cloned and expressed in both soluble and insoluble forms. Western blots on rUreG protein showed 70% (21/30) and 60% (18/30) reactivity with patients' sera when probed with HRP-conjugated anti-human IgG and IgA antibodies, respectively; and the combination of the IgG and IgA western blots showed reactivity of 83.3% (25/30). By comparison, 97.5% and 92.5% of the control sera showed no reactivity when probed with HRP-conjugated anti-human IgG and IgA antibodies, respectively. Both the H. pylori lysate antigen and rUreG protein displayed a distinctive band at the expected

  9. Characterization of a mitochondrially targeted single-stranded DNA-binding protein in Arabidopsis thaliana.

    Science.gov (United States)

    Edmondson, Andrew C; Song, Daqing; Alvarez, Luis A; Wall, Melisa K; Almond, David; McClellan, David A; Maxwell, Anthony; Nielsen, Brent L

    2005-04-01

    A gene encoding a predicted mitochondrially targeted single-stranded DNA binding protein (mtSSB) was identified in the Arabidopsis thaliana genome sequence. This gene (At4g11060) codes for a protein of 201 amino acids, including a 28-residue putative mitochondrial targeting transit peptide. Protein sequence alignment shows high similarity between the mtSSB protein and single-stranded DNA binding proteins (SSB) from bacteria, including residues conserved for SSB function. Phylogenetic analysis indicates a close relationship between this protein and other mitochondrially targeted SSB proteins. The predicted targeting sequence was fused with the GFP coding region, and the organellar localization of the expressed fusion protein was determined. Specific targeting to mitochondria was observed in in-vitro import experiments and by transient expression of a GFP fusion construct in Arabidopsis leaves after microprojectile bombardment. The mature mtSSB coding region was overexpressed in Escherichia coli and the protein was purified for biochemical characterization. The purified protein binds single-stranded, but not double-stranded, DNA. MtSSB stimulates the homologous strand-exchange activity of E. coli RecA. These results indicate that mtSSB is a functional homologue of the E. coli SSB, and that it may play a role in mitochondrial DNA recombination.

  10. Outlook in the application of Chlamydomonas reinhardtii chloroplast as a platform for recombinant protein production.

    Science.gov (United States)

    Shamriz, Shabnam; Ofoghi, Hamideh

    Microalgae, also called microphytes, are a vast group of microscopic photosynthetic organisms living in aquatic ecosystems. Microalgae have attracted the attention of biotechnology industry as a platform for extracting natural products with high commercial value. During last decades, microalgae have been also used as cost-effective and easily scalable platform for the production of recombinant proteins with medical and industrial applications. Most progress in this field has been made with Chlamydomonas reinhardtii as a model organism mainly because of its simple life cycle, well-established genetics and ease of cultivation. However, due to the scarcity of existing infrastructure for commercial production and processing together with relatively low product yields, no recombinant products from C. reinhardtii have gained approval for commercial production and most of them are still in research and development. In this review, we focus on the chloroplast of C. reinhardtii as an algal recombinant expression platform and compare its advantages and disadvantages to other currently used expression systems. We then discuss the strategies for engineering the chloroplast of C. reinhardtii to produce recombinant cells and present a comprehensive overview of works that have used this platform for the expression of high-value products.

  11. Structural and functional characterization of recombinant napin-like protein of Momordica charantia expressed in methylotrophic yeast Pichia pastoris.

    Science.gov (United States)

    Yadav, Shailesh Kumar R; Sahu, Tejram; Dixit, Aparna

    2016-08-01

    Napin and napin-like proteins belong to the 2S albumin seed storage family of proteins and have been shown to display a variety of biological activities. However, due to a high degree of polymorphism, purification of a single napin or napin-like protein exhibiting biological activity is extremely difficult. In the present study, we have produced the napin-like protein of Momordica charantia using the methylotrophic Pichia pastoris expression system. The recombinant napin-like protein (rMcnapin) secreted in the extracellular culture supernatant was enriched by ammonium sulfate precipitation, and purified using size exclusion chromatography at a yield of ∼290 mg/L of culture. Secondary structure analysis of the purified rMcnapin revealed it to be predominantly α-helical with minimal β strand content. CD spectroscopic and fluorescence spectroscopic analyses revealed the rMcnapin to be stable at a wide range of temperatures and pH. The rMcnapin exhibited antifungal activity against Trichoderma viride with an IC50 of ∼3.7 μg/ml and trypsin inhibitor activity with an IC50 of 4.2 μM. Thus, large amounts of homogenous preparations of the biologically active rMcnapin could be obtained at shake flask level, which is otherwise difficult from its natural source.

  12. Leishmania infantum: gene cloning of the GRP94 homologue, its expression as recombinant protein, and analysis of antigenicity.

    Science.gov (United States)

    Larreta, R; Soto, M; Alonso, C; Requena, J M

    2000-10-01

    The complete nucleotide sequence for the Leishmania infantum homologue to the glucose-regulated protein 94 (GRP94) gene was determined from the isolation and characterization of a genomic clone. Like the mammalian and plant GRP94s, the L. infantum GRP94 sequence possesses both an N-terminal signal peptide and a putative endoplasmic reticulum retention signal, consisting of the C-terminal tetrapeptide EDDL. Thus, L. infantum is the first protozoan organism in which GRP94 has been identified. Southern blot analysis has indicated that this protein is encoded by a single-copy gene. The L. infantum GRP94 gene was expressed in Escherichia coli and the recombinant protein used to evaluate its antigenicity and immunogenicity. Eighty-four percent of sera from dogs with visceral leishmaniasis reacted with the protein, indicating that GRP94 is a potent immunogen during Leishmania infection. Given the immunogenic and antigenic properties shown by the L. infantum GRP94, we think that this protein constitutes a valuable molecule for diagnostic purposes and a potential candidate for studies of protective immunogenicity. Copyright 2000 Academic Press.

  13. Biophysics of DNA-Protein Interactions From Single Molecules to Biological Systems

    CERN Document Server

    Williams, Mark C

    2011-01-01

    This book presents a concise overview of current research on the biophysics of DNA-protein interactions. A wide range of new and classical methods are presented by authors investigating physical mechanisms by which proteins interact with DNA. For example, several chapters address the mechanisms by which proteins search for and recognize specific binding sites on DNA, a process critical for cellular function. Single molecule methods such as force spectroscopy as well as fluorescence imaging and tracking are described in these chapters as well as other parts of the book that address the dynamics of protein-DNA interactions. Other important topics include the mechanisms by which proteins engage DNA sequences and/or alter DNA structure. These simple but important model interactions are then placed in the broader biological context with discussion of larger protein-DNA complexes . Topics include replication forks, recombination complexes, DNA repair interactions, and ultimately, methods to understand the chromatin...

  14. Sequencing of Single Pollen Nuclei Reveals Meiotic Recombination Events at Megabase Resolution and Circumvents Segregation Distortion Caused by Postmeiotic Processes

    Directory of Open Access Journals (Sweden)

    Steven Dreissig

    2017-09-01

    Full Text Available Meiotic recombination is a fundamental mechanism to generate novel allelic combinations which can be harnessed by breeders to achieve crop improvement. The recombination landscape of many crop species, including the major crop barley, is characterized by a dearth of recombination in 65% of the genome. In addition, segregation distortion caused by selection on genetically linked loci is a frequent and undesirable phenomenon in double haploid populations which hampers genetic mapping and breeding. Here, we present an approach to directly investigate recombination at the DNA sequence level by combining flow-sorting of haploid pollen nuclei of barley with single-cell genome sequencing. We confirm the skewed distribution of recombination events toward distal chromosomal regions at megabase resolution and show that segregation distortion is almost absent if directly measured in pollen. Furthermore, we show a bimodal distribution of inter-crossover distances, which supports the existence of two classes of crossovers which are sensitive or less sensitive to physical interference. We conclude that single pollen nuclei sequencing is an approach capable of revealing recombination patterns in the absence of segregation distortion.

  15. Vaccination with recombinant heat shock protein 60 from Histoplasma capsulatum protects mice against pulmonary histoplasmosis.

    Science.gov (United States)

    Gomez, F J; Allendoerfer, R; Deepe, G S

    1995-07-01

    HIS-62 is a glycoprotein that has been isolated from the cell wall and cell membrane fraction of the pathogenic fungus Histoplasma capsulatum. It is a target of the cellular immune response to this fungus, and it protects mice against a lethal intravenous inoculum of H. capsulatum yeast cells. In this study, we cloned the gene encoding this antigen to reveal its biological nature and studied the immunological activity of recombinant antigen. The amino acid sequences of the NH2 terminus and internal peptides were obtained by Edman degradation. Degenerate oligonucleotides were used to isolate a gene fragment of HIS-62 by PCR. One 680-bp segment that corresponded to the known peptide sequence was amplified from H. capsulatum DNA. This DNA was used to screen a genomic library, and the full-length gene was isolated and sequenced. The deduced amino acid sequence of the gene demonstrated approximately 70 and approximately 50% identity to heat shock protein 60 (hsp 60) from Saccharomyces cerevisiae and hsp 60 from Escherichia coli, respectively. A cDNA was synthesized by reverse transcription PCR and was expressed in E. coli. Recombinant protein reacted with a monospecific polyclonal rabbit antiserum raised against native HIS-62, with monoclonal HIS-62-reactive T cells, and with splenocytes from mice immunized with viable yeast cells. Moreover, vaccination with the recombinant protein conferred protection in mice against a lethal intranasal inoculation with yeast cells. Thus, HIS-62 is a member of the hsp 60 family, and the recombinant hsp 60 is protective against pulmonary histoplasmosis in mice.

  16. The Study of Posttranslational Modifications of Tau Protein by Nuclear Magnetic Resonance Spectroscopy: Phosphorylation of Tau Protein by ERK2 Recombinant Kinase and Rat Brain Extract, and Acetylation by Recombinant Creb-Binding Protein.

    Science.gov (United States)

    Qi, Haoling; Despres, Clément; Prabakaran, Sudhakaran; Cantrelle, François-Xavier; Chambraud, Béatrice; Gunawardena, Jeremy; Lippens, Guy; Smet-Nocca, Caroline; Landrieu, Isabelle

    2017-01-01

    Nuclear magnetic resonance (NMR) spectroscopy can be used as an analytical tool to investigate posttranslational modifications of protein. NMR is a valuable tool to map the interaction regions of protein partners. Here, we present protocols that have been developed in the course of our studies of the neuronal Tau protein. Tau is found aggregated in the neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, abnormal phosphorylation and acetylation of Tau. We have used NMR to investigate how these posttranslational modifications of Tau affect the interactions with its partners. We present here detailed protocols of in vitro phosphorylation of Tau by recombinant kinase, ERK2, or kinase activity of rat brain extracts, and acetylation by recombinant Creb-binding protein (CBP) acetyltransferase. The analytical characterization of the modified Tau by NMR spectroscopy is additionally described.

  17. Protein fusion tags for efficient expression and purification of recombinant proteins in the periplasmic space of E. coli.

    Science.gov (United States)

    Malik, Ajamaluddin

    2016-06-01

    Disulfide bonds occurred in majority of secreted protein. Formation of correct disulfide bonds are must for achieving native conformation, solubility and activity. Production of recombinant proteins containing disulfide bond for therapeutic, diagnostic and various other purposes is a challenging task of research. Production of such proteins in the reducing cytosolic compartment of E. coli usually ends up in inclusion bodies formation. Refolding of inclusion bodies can be difficult, time and labor consuming and uneconomical. Translocation of these proteins into the oxidative periplasmic compartment provides correct environment to undergo proper disulfide bonds formation and thus achieving native conformation. However, not all proteins can be efficiently translocated to the periplasm with the help of bacterial signal peptides. Therefore, fusion to a small well-folded and stable periplasmic protein is more promising for periplasmic production of disulfide bonded proteins. In the past decades, several full-length proteins or domains were used for enhancing translocation and solubility. Here, protein fusion tags that significantly increase the yields of target proteins in the periplasmic space are reviewed.

  18. Production of recombinant proteins in Mycobacterium smegmatis for structural and functional studies.

    Science.gov (United States)

    Bashiri, Ghader; Baker, Edward N

    2015-01-01

    Protein production using recombinant DNA technology has a fundamental impact on our understanding of biology through providing proteins for structural and functional studies. Escherichia coli (E. coli) has been traditionally used as the default expression host to over-express and purify proteins from many different organisms. E. coli does, however, have known shortcomings for obtaining soluble, properly folded proteins suitable for downstream studies. These shortcomings are even more pronounced for the mycobacterial pathogen Mycobacterium tuberculosis, the bacterium that causes tuberculosis, with typically only one third of proteins expressed in E. coli produced as soluble proteins. Mycobacterium smegmatis (M. smegmatis) is a closely related and non-pathogenic species that has been successfully used as an expression host for production of proteins from various mycobacterial species. In this review, we describe the early attempts to produce mycobacterial proteins in alternative expression hosts and then focus on available expression systems in M. smegmatis. The advantages of using M. smegmatis as an expression host, its application in structural biology and some practical aspects of protein production are also discussed. M. smegmatis provides an effective expression platform for enhanced understanding of mycobacterial biology and pathogenesis and for developing novel and better therapeutics and diagnostics. © 2014 The Protein Society.

  19. 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.

  20. Low levels of aflatoxin B1, ricin, and milk enhance recombinant protein production in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Reuven Rasooly

    Full Text Available Gene expression in transduced mammalian cells correlates with virus titer, but high doses of vector for gene therapy leads to toxicity in humans and in animals. Changing the optimal tissue culture medium by adding low levels of environmental stressors, such as 1 µM of the fungal toxin aflatoxin B1 (AFB1, 1 ng of the castor bean protein toxin ricin, or 1% reconstituted milk, enhances transcription and increases production of proteins in transduced mammalian cells as demonstrated by production of the following three recombinant proteins: firefly luciferase, β-galactosidase, and green fluorescent protein (GFP. Higher concentrations of the stress-producing substances damage the cells beyond recovery, resulting in inhibited gene expression and cell death. We also evaluated the effect of the stressor substances on the enhanced infectivity of virus. The presented findings extend methods for large-scale transient recombinant protein production in mammalian cells and suggest that it may be possible to reduce the cytotoxicity of the adenovirus by reducing the virus titer without adversely affecting gene expression levels.

  1. Low levels of aflatoxin B1, ricin, and milk enhance recombinant protein production in mammalian cells.

    Science.gov (United States)

    Rasooly, Reuven; Hernlem, Bradley; Friedman, Mendel

    2013-01-01

    Gene expression in transduced mammalian cells correlates with virus titer, but high doses of vector for gene therapy leads to toxicity in humans and in animals. Changing the optimal tissue culture medium by adding low levels of environmental stressors, such as 1 µM of the fungal toxin aflatoxin B1 (AFB1), 1 ng of the castor bean protein toxin ricin, or 1% reconstituted milk, enhances transcription and increases production of proteins in transduced mammalian cells as demonstrated by production of the following three recombinant proteins: firefly luciferase, β-galactosidase, and green fluorescent protein (GFP). Higher concentrations of the stress-producing substances damage the cells beyond recovery, resulting in inhibited gene expression and cell death. We also evaluated the effect of the stressor substances on the enhanced infectivity of virus. The presented findings extend methods for large-scale transient recombinant protein production in mammalian cells and suggest that it may be possible to reduce the cytotoxicity of the adenovirus by reducing the virus titer without adversely affecting gene expression levels.

  2. PRODUCTION OF MYCOBACTERIUM TUBERCULOSIS TB10.4 RECOMBINANT PROTEIN IN ESCHERICHIA COLI

    Directory of Open Access Journals (Sweden)

    I. V. Dukhovlinov

    2015-01-01

    Full Text Available Nowadays tuberculosis is considered one of the most dangerous infectious diseases occurring everywhere, and it remains a cause of death of millions of people around the world. According to the World Health Organization data, in 2013 tuberculosis caused more than 9 million cases worldwide and about 1.5 million of infected people died. The causative agent of tuberculosis in most cases is Mycobacterium tuberculosis. But sometimes it can be Mycobacterium bovis or Mycobacterium africanum. Mainly as a result of infection, a bacterial infection affects the lungs, but the disease may develop in other organs and tissues. Now for the prevention of tuberculosis vaccination of newborns with attenuated vaccine BCG is widely used. The production of this vaccine is cheap and it is safe to use. Thus today, vaccination is the primary means of prevention of tuberculosis. However dubious efficacy and a number of side effects observed after vaccination, makes the scientific community to develop new effective methods for the treatment of tuberculosis. One of the ways to develop new vaccines against tuberculosis is to provide a subunit vaccine based on recombinant proteins. Advantages of subunit vaccines are that the preparation containing the purified protein is stable and secure, its chemical properties are known, it does not contain additional proteins and nucleic acids, which could cause undesirable effects in the human body. One of the most promising antigens for use as components in new vaccines is considered a low molecular weight secreted protein TB10.4. TB10.4 protein is recognized at an early stage of tuberculous infection and contributes to the proliferation of lymphocytes responsible for the production of IFNγ. TB10.4 protein also possesses an adjuvant effect when administered in combination with mycobacterial proteins. Given these properties, the recombinant protein TB10.4 can be used to generate new candidate vaccines against tuberculosis. During the

  3. Novel Recombinant Multiepitope Proteins for the Diagnosis of Asymptomatic Leishmania infantum-Infected Dogs

    Science.gov (United States)

    Faria, Angélica Rosa; de Castro Veloso, Luciano; Coura-Vital, Wendel; Reis, Alexandre Barbosa; Damasceno, Leonardo Miranda; Gazzinelli, Ricardo T.; Andrade, Hélida M.

    2015-01-01

    Background Visceral leishmaniasis is the most severe form of leishmaniasis. Worldwide, approximately 20% of zoonotic human visceral leishmaniasis is caused by Leishmania infantum, also known as Leishmania chagasi in Latin America. Current diagnostic methods are not accurate enough to identify Leishmania-infected animals and may compromise the effectiveness of disease control. Therefore, we aimed to produce and test two recombinant multiepitope proteins as a means to improve and increase accuracy in the diagnosis of canine visceral leishmaniasis (CVL). Methodology/Principal Findings Ten antigenic peptides were identified by CVL ELISA in previous work. In the current proposal, the coding sequences of these ten peptides were assembled into a synthetic gene. Furthermore, other twenty peptides were selected from work by our group where good B and T cell epitopes were mapped. The coding sequences of these peptides were also assembled into a synthetic gene. Both genes have been cloned and expressed in Escherichia coli, producing two multiepitope recombinant proteins, PQ10 and PQ20. These antigens have been used in CVL ELISA and were able to identify asymptomatic dogs (80%) more effectively than EIE-LVC kit, produced by Bio-Manguinhos (0%) and DPP kit (10%). Moreover, our recombinant proteins presented an early detection (before PCR) of infected dogs, with positivities ranging from 23% to 65%, depending on the phase of infection in which sera were acquired. Conclusions/Significance Our study shows that ELISA using the multiepitope proteins PQ10 and PQ20 has great potential in early CVL diagnosis. The use of these proteins in other methodologies, such as immunochromatographic tests, could be beneficial mainly for the detection of asymptomatic dogs. PMID:25569685

  4. A Novel Recombinant DNA System for High Efficiency Affinity Purification of Proteins in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Brian H. Carrick

    2016-03-01

    Full Text Available Isolation of endogenous proteins from Saccharomyces cerevisiae has been facilitated by inserting encoding polypeptide affinity tags at the C-termini of chromosomal open reading frames (ORFs using homologous recombination of DNA fragments. Tagged protein isolation is limited by a number of factors, including high cost of affinity resins for bulk isolation and low concentration of ligands on the resin surface, leading to low isolation efficiencies and trapping of contaminants. To address this, we have created a recombinant “CelTag” DNA construct from which PCR fragments can be created to easily tag C-termini of S. cerevisiae ORFs using selection for a nat1 marker. The tag has a C-terminal cellulose binding module to be used in the first affinity step. Microgranular cellulose is very inexpensive and has an effectively continuous ligand on its surface, allowing rapid, highly efficient purification with minimal background. Cellulose-bound proteins are released by specific cleavage of an included site for TEV protease, giving nearly pure product. The tag can be lifted from the recombinant DNA construct either with or without a 13x myc epitope tag between the target ORF and the TEV protease site. Binding of CelTag protein fusions to cellulose is stable to high salt, nonionic detergents, and 1 M urea, allowing stringent washing conditions to remove loosely associated components, as needed, before specific elution. It is anticipated that this reagent could allow isolation of protein complexes from large quantities of yeast extract, including soluble, membrane-bound, or nucleic acid-associated assemblies.

  5. Accumulation and processing of a recombinant protein designed as a cleavable fusion to the endogenous Rubisco LSU protein in Chlamydomonas chloroplast

    Directory of Open Access Journals (Sweden)

    Henry Ryan E

    2009-03-01

    Full Text Available Abstract Background Expression of recombinant proteins in green algal chloroplast holds substantial promise as a platform for the production of human therapeutic proteins. A number of proteins have been expressed in the chloroplast of Chlamydomonas reinhardtii, including complex mammalian proteins, but many of these proteins accumulate to significantly lower levels than do endogenous chloroplast proteins. We examined if recombinant protein accumulation could be enhanced by genetically fusing the recombinant reporter protein, luciferase, to the carboxy-terminal end of an abundant endogenous protein, the large subunit of ribulose bisphosphate carboxylase (Rubisco LSU. Additionally, as recombinant proteins fused to endogenous proteins are of little clinical or commercial value, we explored the possibility of engineering our recombinant protein to be cleavable from the endogenous protein in vivo. This strategy would obviate the need for further in vitro processing steps in order to produce the desired recombinant protein. To achieve this, a native protein-processing site from preferredoxin (preFd was placed between the Rubisco LSU and luciferase coding regions in the fusion protein construct. Results The luciferase from the fusion protein accumulated to significantly higher levels than luciferase expressed alone. By eliminating the endogenous Rubisco large subunit gene (rbcL, we achieved a further increase in luciferase accumulation with respect to luciferase expression in the WT background. Importantly, near-wild type levels of functional Rubisco holoenzyme were generated following the proteolytic removal of the fused luciferase, while luciferase activity for the fusion protein was almost ~33 times greater than luciferase expressed alone. These data demonstrate the utility of using fusion proteins to enhance recombinant protein accumulation in algal chloroplasts, and also show that engineered proteolytic processing sites can be used to liberate the

  6. Single Molecule Spectroscopy of Fluorescent Proteins

    NARCIS (Netherlands)

    Blum, Christian; Subramaniam, Vinod

    2009-01-01

    The discovery and use of fluorescent proteins has revolutionized cellular biology. Despite the widespread use of visible fluorescent proteins as reporters and sensors in cellular environments the versatile photophysics of fluorescent proteins is still subject to intense research. Understanding the

  7. Profiling of external metabolites during production of hantavirus nucleocapsid protein with recombinant Saccharomyces cerevisiae.

    Science.gov (United States)

    Antoniukas, Linas; Grammel, Hartmut; Sasnauskas, Kestutis; Reichl, Udo

    2008-03-01

    Recombinant strains of Saccharomyces cerevisiae, producing hantavirus Puumala nucleocapsid protein for diagnostics and as a candidate vaccine were analyzed for uptake and excretion of intermediary metabolites during process optimization studies of fed-batch bioreactor cultures. Concentrations of glucose, maltose, galactose, pyruvate, acetaldehyde, ethanol, acetate, succinate and formaldehyde (used as a selection agent) were measured in the culture medium in order to find a metabolite pattern, indicative for the physiological state of the producer culture. When the inducer galactose was employed as a growth substrate, the metabolite profile of recombinant yeast cells was different from those of the non-recombinant original strain which excreted considerable amounts of metabolites with this substrate. In contrast, galactose-induced heterologous gene expression was indicated by the absence of excreted intermediary metabolites, except succinate. A model strain expressing a GFP fusion of hantavirus nucleocapsid protein differed in the excretion of metabolites from strains without GFP. In addition, the influence of alkali ions, employed for pH control is also demonstrated.

  8. Vaccinia virus recombinants expressing chimeric proteins of human immunodeficiency virus and gamma interferon are attenuated for nude mice.

    OpenAIRE

    Giavedoni, L D; Jones, L; Gardner, M B; Gibson, H L; Ng, C T; Barr, P J; Yilma, T

    1992-01-01

    We have developed a method for attenuating vaccinia virus recombinants by expressing a fusion protein of a lymphokine and an immunogen. Chimeric genes were constructed that coded for gamma interferon (IFN-gamma) and structural proteins of the human immunodeficiency virus type 1 (HIV-1). In this study, we describe the biological and immunological properties of vaccinia virus recombinants expressing chimeric genes of murine or human IFN-gamma with glycoprotein gp120, gag, and a fragment of gp41...

  9. Non-peptide guided auto-secretion of recombinant proteins by super-folder green fluorescent protein in Escherichia coli.

    Science.gov (United States)

    Zhang, Zhen; Tang, Rongxing; Zhu, Dewu; Wang, Wenfeng; Yi, Li; Ma, Lixin

    2017-08-01

    Protein secretion in Escherichia coli is usually led by a signal peptide that targets the protein to specific secretory pathways. In this study, we demonstrated that the superfolder green fluorescent protein (sfGFP) could be served as a non-signal peptide to guide protein auto-secretion in E. coli. This auto-secretion was characterized as a three-step process through the sub-cellular localization analysis: inner membrane trans-location followed by anchoring at outer membrane, and then being released into culture media. We further determined that the beta-barrel structure and net negative charges of sfGFP played important roles in its auto-extracellular secretion property. Using sfGFP as a carrier, heterologous proteins ranging from peptide to complex protein, including antibacterial peptide PG4, endo-beta-N-acethylglucosamindase H (Endo H), human arginase-1 (ARG1), and glutamate decarboxylase (GAD) were all successfully expressed and secreted extracellularly when fused to the carboxyl end of sfGFP. Besides facilitating the extracellular secretion, sfGFP fusion proteins can also be correctly folded and formed the active complex protein structure, including the trimetric human ARG1 and homo-hexametric GAD. This is the first report that sfGFP can guide the secretion of recombinant proteins out of the cells from cytoplasm in E. coli without affecting their conformation and function.

  10. 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...

  11. Case study on human α1-antitrypsin: Recombinant protein titers obtained by commercial ELISA kits are inaccurate

    DEFF Research Database (Denmark)

    Hansen, Henning Gram; Kildegaard, Helene Faustrup; Min Lee, Gyun

    2016-01-01

    Accurate titer determination of recombinant proteins is crucial for evaluating protein production cell lines and processes. Even though enzyme-linked immunosorbent assay (ELISA) is the most widely used assay for determining protein titer, little is known about the accuracy of commercially availab...

  12. Construction and characterisation of infectious recombinant HIV-1 clones containing CTL epitopes from structural proteins in Nef.

    NARCIS (Netherlands)

    C. Guillon (Christophe); P.H.M. Boers (Patrick); E.J. Verschuren (Esther); R.A. Gruters (Rob); A.D.M.E. Osterhaus (Albert); C.A. van Baalen (Carel)

    2002-01-01

    textabstractIn this study the construction is described of HIV-1 molecular clones in which CTL epitopes from RT or Env late proteins were inserted into the Nef early protein. The ectopic epitopes were efficiently processed from the recombinant Nef proteins, were recognized by their cognate CTL in

  13. Reassessment of inclusion body-based production as a versatile opportunity for difficult-to-express recombinant proteins.

    Science.gov (United States)

    Hoffmann, Daniel; Ebrahimi, Mehrdad; Gerlach, Doreen; Salzig, Denise; Czermak, Peter

    2017-11-10

    The production of recombinant proteins in the microbial host Escherichia coli often results in the formation of cytoplasmic protein inclusion bodies (IBs). Proteins forming IBs are often branded as difficult-to-express, neglecting that IBs can be an opportunity for their production. IBs are resistant to proteolytic degradation and contain up to 90% pure recombinant protein, which does not interfere with the host metabolism. This is especially advantageous for host-toxic proteins like antimicrobial peptides (AMPs). IBs can be easily isolated by cell disruption followed by filtration and/or centrifugation, but conventional techniques for the recovery of soluble proteins from IBs are laborious. New approaches therefore simplify protein recovery by optimizing the production process conditions, and often include mild resolubilization methods that either increase the yield after refolding or avoid the necessity of refolding all together. For the AMP production, the IB-based approach is ideal, because these peptides often have simple structures and are easy to refold. The intentional IB production of almost every protein can be achieved by fusing recombinant proteins to pull-down tags. This review discusses the techniques available for IB-based protein production before considering technical approaches for the isolation of IBs from E. coli lysates followed by efficient protein resolubilization which ideally omits further refolding. The techniques are evaluated in terms of their suitability for the process-scale production and downstream processing of recombinant proteins and are discussed for AMP production as an example.

  14. Stochastic single-molecule dynamics of synaptic membrane protein domains

    Science.gov (United States)

    Kahraman, Osman; Li, Yiwei; Haselwandter, Christoph A.

    2016-09-01

    Motivated by single-molecule experiments on synaptic membrane protein domains, we use a stochastic lattice model to study protein reaction and diffusion processes in crowded membranes. We find that the stochastic reaction-diffusion dynamics of synaptic proteins provide a simple physical mechanism for collective fluctuations in synaptic domains, the molecular turnover observed at synaptic domains, key features of the single-molecule trajectories observed for synaptic proteins, and spatially inhomogeneous protein lifetimes at the cell membrane. Our results suggest that central aspects of the single-molecule and collective dynamics observed for membrane protein domains can be understood in terms of stochastic reaction-diffusion processes at the cell membrane.

  15. Cancer risk after use of recombinant bone morphogenetic protein-2 for spinal arthrodesis.

    Science.gov (United States)

    Carragee, Eugene J; Chu, Gilbert; Rohatgi, Rajat; Hurwitz, Eric L; Weiner, Bradley K; Yoon, S Tim; Comer, Garet; Kopjar, Branko

    2013-09-04

    Recombinant human bone morphogenetic protein-2 (rhBMP-2) is a growth factor known to have in vitro effects on the growth and invasiveness of cancer. It has been approved by the U.S. Food and Drug Administration in limited doses for single-level anterior spinal arthrodesis, but it is commonly used off-label and at high doses. The effect of rhBMP-2 on the risk of cancer has been a concern. We sought to evaluate the risk of new cancers in patients receiving high-dose rhBMP-2. We used publicly available data from a pivotal, multicenter, randomized controlled trial of patients with degenerative lumbar spine conditions who underwent a single-level instrumented posterolateral arthrodesis with either high-dose rhBMP-2 in a compression-resistant matrix (CRM) (rhBMP-2/CRM; n = 239) or autogenous bone graft (control group; n = 224). We compared the risks of new cancers in the rhBMP-2/CRM and control groups at two and five years after surgery. At two years, with 86% follow-up, there were fifteen new cancer events in eleven patients in the rhBMP-2/CRM group compared with two new cancer events in two patients in the control group treated with autogenous bone graft. The incidence rate of new cancer events per 100 person-years was 3.37 (95% confidence interval [CI], 1.89 to 5.56) in the rhBMP-2/CRM group at two years compared with 0.50 (95% CI, 0.06 to 1.80) in the control group. The incidence rate ratio was 6.75 (95% CI, 1.57 to 60.83; p = 0.0026) at two years. Calculated in terms of the number of patients with one or more cancer events two years after the surgery, the incidence rate per 100 person-years was 2.54 (95% CI, 1.27 to 4.54) in the rhBMP-2/CRM group compared with 0.50 (95% CI, 0.06 to 1.82) in the control group at two years; the incidence rate ratio was 5.04 (95% CI, 1.10 to 46.82; p = 0.0194). At five years, there was a 37% loss of follow-up, but a significantly greater incidence of cancer events was still observed in the rhBMP-2/CRM group. A high dose of 40 mg of rh

  16. Tender coconut water an economical growth medium for the production of recombinant proteins in Escherichia coli

    Science.gov (United States)

    2013-01-01

    Background Escherichia coli is most widely used prokaryotic expression system for the production of recombinant proteins. Several strategies have been employed for expressing recombinant proteins in E.coli. This includes the development of novel host systems, expression vectors and cost effective media. In this study, we exploit tender coconut water (TCW) as a natural and cheaper growth medium for E.coli and Pichia pastoris. Result E.coli and P.pastoris were cultivated in TCW and the growth rate was monitored by measuring optical density at 600 nm (OD600nm), where 1.55 for E.coli and 8.7 for P.pastoris was obtained after 12 and 60 hours, respectively. However, variation in growth rate was observed among TCW when collected from different localities (0.15-2.5 at OD600nm), which is attributed to the varying chemical profile among samples. In this regard, we attempted the supplementation of TCW with different carbon and nitrogen sources to attain consistency in growth rate. Here, supplementation of TCW with 25 mM ammonium sulphate (TCW-S) was noted efficient for the normalization of inconsistency, which further increased the biomass of E.coli by 2 to 10 folds, and 1.5 to 2 fold in P.pastoris. These results indicate that nitrogen source is the major limiting factor for growth. This was supported by total nitrogen and carbon estimation where, nitrogen varies from 20 to 60 mg/100 ml while carbohydrates showed no considerable variation (2.32 to 3.96 g/100 ml). In this study, we also employed TCW as an expression media for recombinant proteins by demonstrating successful expression of maltose binding protein (MBP), MBP-TEV protease fusion and a photo switchable fluorescent protein (mEos2) using TCW and the expression level was found to be equivalent to Luria Broth (LB). Conclusion This study highlights the possible application of TCW-S as a media for cultivation of a variety of microorganisms and recombinant protein expression. PMID:24004578

  17. Construction of a recombinant single chain antibody recognizing nonreducing terminal mannose residues applicable to immunohistochemistry.

    Science.gov (United States)

    Yuasa, Noriyuki; Iida, Noriko; Sakaue, Hiroyuki; Zhang, Wei; Wilczynski, Sharon; Fujita-Yamaguchi, Yoko

    2007-10-01

    We recently reported characterization of 25 clones isolated from a phage library displaying human scFvs using a neoglycolipid Man3-DPPE, which was synthesized from mannotriose (Man3) and dipalmitoylphosphatidylethanolamine (DPPE). Of those, 5A3 scFv was successfully expressed and purified as a humanized scFv-Fc form (Sakai et al., Biochemistry 46:253, 2007, Zhang et al. ibid 263). To carry out immunohistochemistry (IHC) in human tissues, a HA tag sequence was introduced to the 5A3 scFv-Fc gene and the resulting construct was transfected to murine myeloma NS0 cells. The 5A3 scFv-Fc protein expressed was affinity-purified. Sodium dodecyl sulfate polyacrylamide gel electrophoresis under nonreducing and reducing conditions and enzyme-linked immunosorbent assay confirmed that 5A3 scFv-Fc protein is dimeric and retained the ability to recognize nonreducing terminal mannose residues. IHC staining of non-neoplastic tissues by this recombinant antibody revealed that no immunoreactivity was detectable in most of 16 tissues examined. Exceptions were found in IHC staining of kidney and pancreas, which demonstrated clear staining of proximal tubules and islet of Langerhans, respectively. These results demonstrated that nonreducing terminal mannose residues are not usually present under normal physiological conditions. This study thus provided a potentially useful tool for examination of the nonreducing terminal mannose residues, which may become exposed under certain pathophysiologycal conditions.

  18. Recombinant Protein Production from TPO Gen Cloning and Expression for Early Detection of Autoimmune Thyroid Diseases

    Science.gov (United States)

    Aulanni'am, Aulanni'am; Kinasih Wuragil, Dyah; Wahono Soeatmadji, Djoko; Zulkarnain; Marhendra, Agung Pramana W.

    2018-01-01

    Autoimmune Thyroid Disease (AITD) is an autoimmune disease that has many clinical symptoms but is difficult to detect at the onset of disease progression. Most thyroid autoimmune disease patients are positive with high titre of thyroid autoantibodies, especially thyroid peroxidase (TPO). The detection AITD are still needed because these tests are extremely high cost and have not regularly been performed in most of clinical laboratories. In the past, we have explored the autoimmune disease marker and it has been developed as source of polyclonal antibodies from patient origin. In the current study, we develop recombinant protein which resulted from cloning and expression of TPO gene from normal person and AITD patients. This work flows involves: DNA isolation and PCR to obtain TPO gene from human blood, insertion of TPO gene to plasmid and transformation to E. coli BL21, Bacterial culture to obtain protein product, protein purification and product analysis. This products can use for application to immunochromatography based test. This work could achieved with the goal of producing autoimmune markers with a guaranteed quality, sensitive, specific and economically. So with the collaboration with industries these devices could be used for early detection. Keywords: recombinant protein, TPO gene, Autoimmune thyroid diseases (AITD)ction of the diseases in the community.

  19. Evaluation of the immunodiagnostic potential of a recombinant surface protein domain from Acanthamoeba castellanii.

    Science.gov (United States)

    Sánchez, Alemao G Carpinteyro; Virginio, Veridiana Gomes; Maschio, Vinicius José; Ferreira, Henrique Bunselmeyer; Rott, Marilise Brittes

    2016-10-01

    Acanthamoeba spp. are free-living protists widely distributed in environment, able to cause keratitis, encephalitis and skin lesions in humans and animals. Acanthamoeba spp. exist in two forms: an infective trophozoite and a dormant cyst. Several factors contribute to the pathogenesis of Acanthamoeba spp. The parasite adhesion to the host cell is the primary step for infection and is mediated by a mannose binding-protein, expressed in the surface and considered the main pathogenicity factor in Acanthamoeba spp. So far, there was no evidence of another surface protein of Acanthamoeba spp. relevant for host invasion or infection by these organisms. The aims of this study were to identify and characterize an Acanthamoeba castellanii surface protein and to evaluate its diagnostic potential. In silico predictions of surface proteins allowed to identify the A. castellanii calreticulin as a possible surface antigen. The coding sequence of a predicted extracellular domain of A. castellanii calreticulin was cloned by in vivo homologous recombination and the recombinant polypeptide (AcCRT29-130) was produced. Its immunodiagnostic potential was assessed in a recombinant antigen-based ELISA with sera from experimentally infected rats that developed keratitis and encephalitis, and sera from patients with encephalitis. The AcCRT29-130 was significantly more recognized by sera from encephalitis infected rats in comparison with the non-infected controls. Human sera from encephalitis patients, however presented no significant response. These results showed the AcCRT29-130 potential for A. castellanii infection immunodiagnosis in animals, with further studies being required for assessment of its use for human infections.

  20. Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography.

    Science.gov (United States)

    Wang, Hua-zhen; Chu, Zhi-zhan; Chen, Chang-chao; Cao, Ao-cheng; Tong, Xin; Ouyang, Can-bin; Yuan, Qi-hang; Wang, Mi-nan; Wu, Zhong-kun; Wang, Hai-hong; Wang, Sheng-bin

    2015-01-01

    Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP) could greatly increase the soluble expression level of Glucokinase (GlcK), α-Amylase (Amy) and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases.

  1. Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography.

    Directory of Open Access Journals (Sweden)

    Hua-zhen Wang

    Full Text Available Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP could greatly increase the soluble expression level of Glucokinase (GlcK, α-Amylase (Amy and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases.

  2. Expression Screening of Fusion Partners from an E. coli Genome for Soluble Expression of Recombinant Proteins in a Cell-Free Protein Synthesis System

    OpenAIRE

    Ahn, Jin Ho; Keum, Jung-Won; Kim, Dong-Myung

    2011-01-01

    While access to soluble recombinant proteins is essential for a number of proteome studies, preparation of purified functional proteins is often limited by the protein solubility. In this study, potent solubility-enhancing fusion partners were screened from the repertoire of endogenous E. coli proteins. Based on the presumed correlation between the intracellular abundance and folding efficiency of proteins, PCR-amplified ORFs of a series of highly abundant E. coli proteins were fused with agg...

  3. Simultaneous expression of recombinant proteins in the insect cell-baculovirus system: production of virus-like particles.

    Science.gov (United States)

    Palomares, Laura A; Mena, Jimmy A; Ramírez, Octavio T

    2012-03-01

    The insect cell-baculovirus system (IC-BEVS) is widely used for the production of recombinant viral proteins for vaccine applications. It is especially suitable for the production of virus-like particles, which often require the simultaneous production of several recombinant proteins. Here, the available tools and process requirements for the simultaneous production of several recombinant proteins using the IC-BEVS are discussed. The production of double-layered rotavirus like particles is used as a specific example for the simultaneous production of two recombinant proteins. Methods to quantify VLP in small samples are described. The multiplicity and time of infection are presented as tools to manipulate protein concentration, and the effect on protein concentration ratios on the assembly efficiency of double-layered rotavirus like particles is discussed. It was found that not only the ratio between the recombinant proteins is determinant of VLP assembly efficiency, but also that assembly efficiency is related to the characteristics of the assembled proteins. This is the first time that kinetics of VLP production are followed during cultures, and that the assembly efficiency is quantitatively determined. Copyright © 2012 Elsevier Inc. All rights reserved.

  4. Aequorea green fluorescent protein. Expression of the gene and fluorescence characteristics of the recombinant protein.

    Science.gov (United States)

    Inouye, S; Tsuji, F I

    1994-03-21

    Expression of the cDNA for Aequorea green fluorescent protein in E. coli yielded a fused protein with fluorescence excitation and emission spectra virtually identical to those of the native green fluorescent protein. Further, a solution of the protein, when mixed with aequorin and calcium ion, emitted a greenish luminescence characteristic of the in vivo luminescence of the animal, indicating a radiationless energy transfer to the protein.

  5. Metabolic flux profiling of recombinant protein secreting Pichia pastoris growing on glucose:methanol mixtures

    Science.gov (United States)

    2012-01-01

    Background The methylotrophic yeast Pichia pastoris has emerged as one of the most promising yeast hosts for the production of heterologous proteins. Mixed feeds of methanol and a multicarbon source instead of methanol as sole carbon source have been shown to improve product productivities and alleviate metabolic burden derived from protein production. Nevertheless, systematic quantitative studies on the relationships between the central metabolism and recombinant protein production in P. pastoris are still rather limited, particularly when growing this yeast on mixed carbon sources, thus hampering future metabolic network engineering strategies for improved protein production. Results The metabolic flux distribution in the central metabolism of P. pastoris growing on a mixed feed of glucose and methanol was analyzed by Metabolic Flux Analysis (MFA) using 13C-NMR-derived constraints. For this purpose, we defined new flux ratios for methanol assimilation pathways in P. pastoris cells growing on glucose:methanol mixtures. By using this experimental approach, the metabolic burden caused by the overexpression and secretion of a Rhizopus oryzae lipase (Rol) in P. pastoris was further analyzed. This protein has been previously shown to trigger the unfolded protein response in P. pastoris. A series of 13C-tracer experiments were performed on aerobic chemostat cultivations with a control and two different Rol producing strains growing at a dilution rate of 0.09 h−1 using a glucose:methanol 80:20 (w/w) mix as carbon source. The MFA performed in this study reveals a significant redistristribution of carbon fluxes in the central carbon metabolism when comparing the two recombinant strains vs the control strain, reflected in increased glycolytic, TCA cycle and NADH regeneration fluxes, as well as higher methanol dissimilation rates. Conclusions Overall, a further 13C-based MFA development to characterise the central metabolism of methylotrophic yeasts when growing on mixed

  6. Metabolic flux profiling of recombinant protein secreting Pichia pastoris growing on glucose:methanol mixtures

    Directory of Open Access Journals (Sweden)

    Jordà Joel

    2012-05-01

    Full Text Available Abstract Background The methylotrophic yeast Pichia pastoris has emerged as one of the most promising yeast hosts for the production of heterologous proteins. Mixed feeds of methanol and a multicarbon source instead of methanol as sole carbon source have been shown to improve product productivities and alleviate metabolic burden derived from protein production. Nevertheless, systematic quantitative studies on the relationships between the central metabolism and recombinant protein production in P. pastoris are still rather limited, particularly when growing this yeast on mixed carbon sources, thus hampering future metabolic network engineering strategies for improved protein production. Results The metabolic flux distribution in the central metabolism of P. pastoris growing on a mixed feed of glucose and methanol was analyzed by Metabolic Flux Analysis (MFA using 13C-NMR-derived constraints. For this purpose, we defined new flux ratios for methanol assimilation pathways in P. pastoris cells growing on glucose:methanol mixtures. By using this experimental approach, the metabolic burden caused by the overexpression and secretion of a Rhizopus oryzae lipase (Rol in P. pastoris was further analyzed. This protein has been previously shown to trigger the unfolded protein response in P. pastoris. A series of 13C-tracer experiments were performed on aerobic chemostat cultivations with a control and two different Rol producing strains growing at a dilution rate of 0.09 h−1 using a glucose:methanol 80:20 (w/w mix as carbon source. The MFA performed in this study reveals a significant redistristribution of carbon fluxes in the central carbon metabolism when comparing the two recombinant strains vs the control strain, reflected in increased glycolytic, TCA cycle and NADH regeneration fluxes, as well as higher methanol dissimilation rates. Conclusions Overall, a further 13C-based MFA development to characterise the central metabolism of methylotrophic

  7. Molecular characterization of the recombinant protein RmLTI-BmCG-LTB: Protective immunity against Rhipicephalus (Boophilus microplus.

    Directory of Open Access Journals (Sweden)

    Bárbara Guimarães Csordas

    Full Text Available The bovine tick Rhipicephalus (Boophilus microplus is found in several tropical and subtropical regions of the world. This parasite transmits pathogens that cause disease, such as babesiosis (Babesia bovis and B. bigemina and anaplasmosis (Anaplasma marginale. Tick infestations cause enormous livestock losses, and controlling tick infestations and the transmission of tick-borne diseases remains a challenge for the livestock industry. Because the currently available commercial vaccines offer only partial protection against R. (B. microplus, there is a need for more efficient vaccines. Several recombinant antigens have been evaluated using different immunization strategies, and they show great promise. This work describes the construction and immunological characterization of a multi-antigen chimera composed of two R. (B. microplus antigens (RmLTI and BmCG and one Escherichia coli antigen (B subunit, LTB. The immunogenic regions of each antigen were selected and combined to encode a single polypeptide. The gene was cloned and expressed in E. coli. For all of the experiments, two groups (treated and control of four Angus heifers (3-6 months old were used. The inoculation was performed via intramuscular injection with 200 μg of purified recombinant chimeric protein and adjuvated. The chimeric protein was recognized by specific antibodies against each subunit and by sera from cattle inoculated with the chimera. Immunization of RmLTI-BmCG-LTB cattle reduced the number of adult female ticks by 6.29% and vaccination of cattle with the chimeric antigen provided 55.6% efficacy against R. (B. microplus infestation. The results of this study indicate that the novel chimeric protein is a potential candidate for the future development of a more effective vaccine against R. (B. microplus.

  8. Molecular characterization of the recombinant protein RmLTI-BmCG-LTB: Protective immunity against Rhipicephalus (Boophilus) microplus.

    Science.gov (United States)

    Csordas, Bárbara Guimarães; Cunha, Rodrigo Casquero; Garcia, Marcos Valério; da Silva, Sérgio Silva; Leite, Fábio Leivas; Andreotti, Renato

    2018-01-01

    The bovine tick Rhipicephalus (Boophilus) microplus is found in several tropical and subtropical regions of the world. This parasite transmits pathogens that cause disease, such as babesiosis (Babesia bovis and B. bigemina) and anaplasmosis (Anaplasma marginale). Tick infestations cause enormous livestock losses, and controlling tick infestations and the transmission of tick-borne diseases remains a challenge for the livestock industry. Because the currently available commercial vaccines offer only partial protection against R. (B.) microplus, there is a need for more efficient vaccines. Several recombinant antigens have been evaluated using different immunization strategies, and they show great promise. This work describes the construction and immunological characterization of a multi-antigen chimera composed of two R. (B.) microplus antigens (RmLTI and BmCG) and one Escherichia coli antigen (B subunit, LTB). The immunogenic regions of each antigen were selected and combined to encode a single polypeptide. The gene was cloned and expressed in E. coli. For all of the experiments, two groups (treated and control) of four Angus heifers (3-6 months old) were used. The inoculation was performed via intramuscular injection with 200 μg of purified recombinant chimeric protein and adjuvated. The chimeric protein was recognized by specific antibodies against each subunit and by sera from cattle inoculated with the chimera. Immunization of RmLTI-BmCG-LTB cattle reduced the number of adult female ticks by 6.29% and vaccination of cattle with the chimeric antigen provided 55.6% efficacy against R. (B.) microplus infestation. The results of this study indicate that the novel chimeric protein is a potential candidate for the future development of a more effective vaccine against R. (B.) microplus.

  9. Enhanced production of recombinant proteins by a small molecule protein synthesis enhancer in combination with an antioxidant in recombinant Chinese hamster ovary cells.

    Science.gov (United States)

    Camire, Joseph; Kim, Dongjoo; Kwon, Soonjo

    2017-07-01

    The improvement in the production of recombinant proteins has been linked in a number of small molecules such as carboxylic acids to the inhibition of histone deacetylase, leading to increased transcription of genes. However, carboxylic acids such as pentanoic acid and butanoic acid have been shown to promote an apoptotic response in Chinese hamster ovary (CHO) cell culture. Supplementation of cultures with antioxidants has shown the ability to reduce the apoptotic response of carboxylic acid supplementation, leading to increased therapeutic protein production. In this study, we showed that pentanoic acid reduced the number of cells entering early apoptosis relative to butanoic acid by 15.4%. Additionally, supplementation of butanoic acid- and pentanoic acid-treated cultures with N-acetyl cysteine (NAC) reduced the population of cells entering early apoptosis by 5.3 and 10.0%, respectively, while increasing productivity by 19.5% in the presence of pentanoic acid and NAC. Conversely, a decrease of 5.7% in production was observed in response to combined butanoic acid and N-acetyl cysteine treatment. The results presented herein provide evidence that a culture supplementation method is critical for optimization of biopharmaceutical manufacturing processes.

  10. Homologous Recombination and Human Health: The Roles of BRCA1, BRCA2, and Associated Proteins

    Science.gov (United States)

    Prakash, Rohit; Zhang, Yu; Feng, Weiran; Jasin, Maria

    2015-01-01

    Homologous recombination (HR) is a major pathway for the repair of DNA double-strand breaks in mammalian cells, the defining step of which is homologous strand exchange directed by the RAD51 protein. The physiological importance of HR is underscored by the observation of genomic instability in HR-deficient cells and, importantly, the association of cancer predisposition and developmental defects with mutations in HR genes. The tumor suppressors BRCA1 and BRCA2, key players at different stages of HR, are frequently mutated in familial breast and ovarian cancers. Other HR proteins, including PALB2 and RAD51 paralogs, have also been identified as tumor suppressors. This review summarizes recent findings on BRCA1, BRCA2, and associated proteins involved in human disease with an emphasis on their molecular roles and interactions. PMID:25833843

  11. A single-loop recombinant pseudotyped-virus-based assay to detect HIV-1 phenotypic resistance.

    Science.gov (United States)

    Wu, Shouli; Yan, Pingping; Yan, Yansheng; Qiu, Lijun; Xie, Meirong

    2015-06-01

    HIV/AIDS is a leading public health concern throughout the world. Currently, treatment of HIV/AIDS still depends on highly active antiretroviral therapy (HAART); however, there is increasing evidence showing the emergence of resistance to antiretroviral drugs in HIV-1 strains, making ART less effective over time. Intensive monitoring of HIV-1 drug resistance is therefore of great importance to evaluate the current sensitivity of antiretroviral agents and is urgently needed. The aim of this study was to develop a single-loop recombinant pseudotyped-virus-based assay to detect phenotypic resistance in clinical HIV-1 strains. HIV-1 RNA was extracted from HIV-1-infected human plasma samples, and an approximately 3-kb fragment containing p7/p1/p6 cleavage sites and full-length protease (PR), reverse transcriptase (RT), thermonuclease (TNase), and integrase (1-280 aa) genes was amplified by nested RT-PCR. A retroviral vector was constructed using the HIV-1 infectious molecular clone pLWJ to test antiretroviral drug susceptibility. pLWJ-SV40-Luc contained a luciferase expression cassette inserted within a deleted region of the envelope (env) gene as an indicator gene. Resistance test vectors (RTVs) were constructed by incorporating amplified target genes into pLWJ-SV40-Luc by using ApaI or AgeI and AarI restriction sites and conventional cloning methods. The virus stocks used for drug susceptibility test were produced by co-transfecting 293T cells with RTVs and a plasmid that provided vesicular stomatitis virus glycoprotein (VSV-G). Viral replication was monitored by measuring luciferase activity in infected target cells at approximately 48 h postinfection. A total of 35 clinical plasma samples from HIV-1-infected humans were tested, and target fragments were successfully amplified from 34 samples (97.1 %) and 33 RTVs were successfully constructed by directional cloning, with an overall success rate of 94.3 %. A clear-cut dose-dependent relationship was detected between

  12. Protective efficacy of six immunogenic recombinant proteins of Vibrio anguillarum and evaluation them as vaccine candidate for flounder (Paralichthys olivaceus).

    Science.gov (United States)

    Xing, Jing; Xu, Hongsen; Wang, Yang; Tang, Xiaoqian; Sheng, Xiuzhen; Zhan, Wenbin

    2017-06-01

    Vibrio anguillarum is a severe bacterium that causes terminal haemorrhagic septicaemia in freshwater and marine fish. Virulence-associated proteins play an important role in bacterial pathogenicity and could be applied for immunoprophylaxis. In this study, six antigenic proteins from V. anguillarum were selected and the immune protective efficacy of their recombinant proteins was investigated. VirA, CheR, FlaC, OmpK, OmpR and Hsp33 were recombinantly produced and the reactions of recombinant proteins to flounder-anti-V. anguillarum antibodies (fV-ab) were detected, respectively. Then the recombinant proteins were injected to fish, after immunization, the percentages of surface membrane immunoglobulin-positive (sIg+) cell in lymphocytes, total antibodies, antibodies against V. anguillarum, antibodies against recombinant proteins and relative percent survival (RPS) were analyzed, respectively. The results showed that all the recombinant proteins could react to fV-ab, proliferate sIg + cells in lymphocytes and induce production of total antibodies, specific antibodies against V. anguillarum or the recombinant proteins; the RPS of rVirA, rCheR, rFlaC, rOmpK, rOmpR and rHsp33 against V. anguillarum was 70.27%, 27.03%, 16.22%, 62.16%, 45.95% and 81.08%, respectively. The results revealed that rHsp33, rVirA and rOmpK have good protections against V. anguillarum and could be vaccine candidates against V. anguillarum. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. The recombinant globular head domain of the measles virus hemagglutinin protein as a subunit vaccine against measles.

    Science.gov (United States)

    Lobanova, Liubov M; Eng, Nelson F; Satkunarajah, Malathy; Mutwiri, George K; Rini, James M; Zakhartchouk, Alexander N

    2012-04-26

    Despite the availability of live attenuated measles virus (MV) vaccines, a large number of measles-associated deaths occur among infants in developing countries. The development of a measles subunit vaccine may circumvent the limitations associated with the current live attenuated vaccines and eventually contribute to global measles eradication. Therefore, the goal of this study was to test the feasibility of producing the recombinant globular head domain of the MV hemagglutinin (H) protein by stably transfected human cells and to examine the ability of this recombinant protein to elicit MV-specific immune responses. The recombinant protein was purified from the culture supernatant of stably transfected HEK293T cells secreting a tagged version of the protein. Two subcutaneous immunizations with the purified recombinant protein alone resulted in the production of MV-specific serum IgG and neutralizing antibodies in mice. Formulation of the protein with adjuvants (polyphosphazene or alum) further enhanced the humoral immune response and in addition resulted in the induction of cell-mediated immunity as measured by the production of MV H-specific interferon gamma (IFN-γ) and interleukin 5 (IL-5) by in vitro re-stimulated splenocytes. Furthermore, the inclusion of polyphosphazene into the vaccine formulation induced a mixed Th1/Th2-type immune response. In addition, the purified recombinant protein retained its immunogenicity even after storage at 37°C for 2 weeks. Copyright © 2012 Elsevier Ltd. All rights reserved.

  14. Cell culture media supplementation of uncommonly used sugars sucrose and tagatose for the targeted shifting of protein glycosylation profiles of recombinant protein therapeutics.

    Science.gov (United States)

    Hossler, Patrick; McDermott, Sean; Racicot, Christopher; Chumsae, Christopher; Raharimampionona, Haly; Zhou, Yu; Ouellette, David; Matuck, Joseph; Correia, Ivan; Fann, John; Li, Jianmin

    2014-01-01

    Protein glycosylation is an important post-translational modification toward the structure and function of recombinant therapeutics. The addition of oligosaccharides to recombinant proteins has been shown to greatly influence the overall physiochemical attributes of many proteins. It is for this reason that protein glycosylation is monitored by the developer of a recombinant protein therapeutic, and why protein glycosylation is typically considered a critical quality attribute. In this work, we highlight a systematic study toward the supplementation of sucrose and tagatose into cell culture media for the targeted modulation of protein glycosylation profiles on recombinant proteins. Both sugars were found to affect oligosaccharide maturation resulting in an increase in the percentage of high mannose N-glycan species, as well as a concomitant reduction in fucosylation. The latter effect was demonstrated to increase antibody-dependent cell-mediated cytotoxicity for a recombinant antibody. These aforementioned results were found to be reproducible at different scales, and across different Chinese hamster ovary cell lines. Through the selective supplementation of these described sugars, the targeted modulation of protein glycosylation profiles is demonstrated, as well as yet another tool in the cell culture toolbox for ensuring product comparability. © 2014 American Institute of Chemical Engineers.

  15. Comparative evaluation of heterologous production systems for recombinant pulmonary surfactant protein D

    Directory of Open Access Journals (Sweden)

    Daniela eSalgado

    2014-12-01

    Full Text Available Commercial surfactant products derived from animal lungs are used for the treatment of respiratory diseases in premature neonates. These products contain lipids and the hydrophobic surfactant proteins B and C, which help to lower the surface tension in the lungs. Surfactant products are less effective when pulmonary diseases involve inflammatory complications because two hydrophilic surfactant proteins (A and D are lost during the extraction process, yet surfactant protein D (SP-D is a component of the innate immune system that helps to reduce lung inflammation. The performance of surfactant products could therefore be improved by supplementing them with an additional source of SP-D. Recombinant SP-D is produced in mammalian cells and bacteria (Escherichia coli, and also experimentally in the yeast Pichia pastoris. Mammalian cells produce full-size SP-D, but the yields are low and the cost of production is high. In contrast, bacteria produce a truncated form of SP-D, which is active in vitro and in vivo, and higher yields can be achieved at a lower cost. We compare the efficiency of production of recombinant SP-D in terms of the total yields achieved in each system and the amount of SP-D needed to meet the global demand for the treatment of pulmonary diseases, using respiratory distress syndrome as a case study.

  16. Recombinant VSV G proteins reveal a novel raft-dependent endocytic pathway in resorbing osteoclasts

    International Nuclear Information System (INIS)

    Mulari, Mika T.K.; Nars, Martin; Laitala-Leinonen, Tiina; Kaisto, Tuula; Metsikkoe, Kalervo; Sun Yi; Vaeaenaenen, H. Kalervo

    2008-01-01

    Transcytotic membrane flow delivers degraded bone fragments from the ruffled border to the functional secretory domain, FSD, in bone resorbing osteoclasts. Here we show that there is also a FSD-to-ruffled border trafficking pathway that compensates for the membrane loss during the matrix uptake process and that rafts are essential for this ruffled border-targeted endosomal pathway. Replacing the cytoplasmic tail of the vesicular stomatitis virus G protein with that of CD4 resulted in partial insolubility in Triton X-100 and retargeting from the peripheral non-bone facing plasma membrane to the FSD. Recombinant G proteins were subsequently endosytosed and delivered from the FSD to the peripheral fusion zone of the ruffled border, which were both rich in lipid rafts as suggested by viral protein transport analysis and visualizing the rafts with fluorescent recombinant cholera toxin. Cholesterol depletion by methyl-β-cyclodextrin impaired the ruffled border-targeted vesicle trafficking pathway and inhibited bone resorption dose-dependently as quantified by measuring the CTX and TRACP 5b secreted to the culture medium and by measuring the resorbed area visualized with a bi-phasic labeling method using sulpho-NHS-biotin and WGA-lectin. Thus, rafts are vital for membrane recycling from the FSD to the late endosomal/lysosomal ruffled border and bone resorption

  17. Method for rapid optimization of recombinant GPCR protein expression and stability using virus-like particles.

    Science.gov (United States)

    Ho, Thao T; Nguyen, Jasmine T; Liu, Juping; Stanczak, Pawel; Thompson, Aaron A; Yan, Yingzhuo G; Chen, Jasmine; Allerston, Charles K; Dillard, Charles L; Xu, Hao; Shoger, Nicholas J; Cameron, Jill S; Massari, Mark E; Aertgeerts, Kathleen

    2017-05-01

    Recent innovative approaches to stabilize and crystallize GPCRs have resulted in an unprecedented breakthrough in GPCR crystal structures as well as application of the purified receptor protein in biophysical and biochemical ligand binding assays. However, the protein optimization process to enable these technologies is lengthy and requires iterative overexpression, solubilization, purification and functional analysis of tens to hundreds of protein variants. Here, we report a new and versatile method to screen in parallel hundreds of GPCR variants in HEK293 produced virus-like particles (VLPs) for protein yield, stability, functionality and ligand binding. This approach reduces the time and resources during GPCR construct optimization by eliminating lengthy protein solubilization and purification steps and by its adaptability to many binding assay formats (label or label-free detection). We exemplified the robustness of our VLP method by screening 210 GALR3-VLP variants in a radiometric agonist-based binding assay and a subset of 88 variants in a label-free antagonist-based assay. The resulting GALR3 agonist or antagonist stabilizing variants were then further used for recombinant protein expression in transfected insect cells. The final purified protein variants were successfully immobilized on a biosensor chip and used in a surface plasmon resonance binding assay. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Organization of DNA partners and strand exchange mechanisms during Flp site-specific recombination analyzed by difference topology, single molecule FRET and single molecule TPM.

    Science.gov (United States)

    Ma, Chien-Hui; Liu, Yen-Ting; Savva, Christos G; Rowley, Paul A; Cannon, Brian; Fan, Hsiu-Fang; Russell, Rick; Holzenburg, Andreas; Jayaram, Makkuni

    2014-02-20

    Flp site-specific recombination between two target sites (FRTs) harboring non-homology within the strand exchange region does not yield stable recombinant products. In negatively supercoiled plasmids containing head-to-tail sites, the reaction produces a series of knots with odd-numbered crossings. When the sites are in head-to-head orientation, the knot products contain even-numbered crossings. Both types of knots retain parental DNA configuration. By carrying out Flp recombination after first assembling the topologically well defined Tn3 resolvase synapse, it is possible to determine whether these knots arise by a processive or a dissociative mechanism. The nearly exclusive products from head-to-head and head-to-tail oriented "non-homologous" FRT partners are a 4-noded knot and a 5-noded knot, respectively. The corresponding products from a pair of native (homologous) FRT sites are a 3-noded knot and a 4-noded catenane, respectively. These results are consistent with non-homology-induced two rounds of dissociative recombination by Flp, the first to generate reciprocal recombinants containing non-complementary base pairs and the second to produce parental molecules with restored base pairing. Single molecule fluorescence resonance energy transfer (smFRET) analysis of geometrically restricted FRTs, together with single molecule tethered particle motion (smTPM) assays of unconstrained FRTs, suggests that the sites are preferentially synapsed in an anti-parallel fashion. This selectivity in synapse geometry occurs prior to the chemical steps of recombination, signifying early commitment to a productive reaction path. The cumulative topological, smFRET and smTPM results have implications for the relative orientation of DNA partners and the directionality of strand exchange during recombination mediated by tyrosine site-specific recombinases. Copyright © 2013. Published by Elsevier Ltd.

  19. Comparison of different sites in recombinant Marek's disease virus for the expression of green fluorescent protein.

    Science.gov (United States)

    Liu, Yongzhen; Li, Kai; Cui, Hongyu; Gao, Li; Liu, Changjun; Zhang, Yanping; Gao, Yulong; Wang, Xiaomei

    2017-05-02

    Marek's disease virus (MDV) is a preferred vector for recombinant vaccine construction, and insertion site is the main factor influencing foreign gene expression and vaccine efficacy. In this study, recombinant MDVs inserted with the enhanced green fluorescent protein (eGFP) gene at different sites in its genome were generated from overlapping fosmid DNAs and the eGFP expression was compared. The results showed that the eGFP expression levels from the sites in the unique long (UL) region (within UL41, between UL45 and UL46, and between UL55 and LORF10) were comparable, which were significantly higher than those from the sites in the unique short (US) region (US2 and US10), and the eGFP expression level from US2 was significantly higher than that from US10. The identification and comparison of the insertion sites in MDV genome could help elevate the protection efficacy of the recombinant MDVs expressing desired antigens. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway.

    Science.gov (United States)

    Krainer, Florian W; Dietzsch, Christian; Hajek, Tanja; Herwig, Christoph; Spadiut, Oliver; Glieder, Anton

    2012-02-13

    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. 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. Co-overexpressing enzymes of the

  1. 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

  2. Comparison of Recombinant Proteins of Kinesin 39, Heat Shock Protein 70, Heat Shock Protein 83, and Glycoprotein 63 for Antibody Detection of Leishmania martiniquensis Infection.

    Science.gov (United States)

    Siripattanapipong, Suradej; Kato, Hirotomo; Tan-Ariya, Peerapan; Mungthin, Mathirut; Leelayoova, Saovanee

    2017-11-01

    Leishmania martiniquensis, a zoonotic hemoflagellate, is a causative agent of cutaneous (CL) and visceral leishmaniasis (VL) among humans and animals. This organism, first reported in Martinique Island, now has become an emerging infectious agent in Thailand. Symptomatic cases of L. martiniquensis infection among humans have continuously increased. In the meantime, asymptomatic infection of this novel species has seriously created national public health awareness and concern to prevent and control disease transmission. The unsuccessful serological test using the commercial rK39 dipstick based on antigen from Leishmania donovani to detect the antibodies against VL among infected Thai patients has encouraged us to further explore a new sensitive and specific antigenic epitope. In this study, we determined the sequences and expressed recombinant proteins of kinesin 39 (k39), heat shock protein 70 (hsp70), heat shock protein 83 (hsp83), and glycoprotein 63 (gp63) of L. martiniquensis to evaluate the diagnostic efficiency to detect antibodies against L. martiniquensis in patient sera. The preliminary results from western blot analysis have suggested that K39 is the most sensitive recombinant protein to detect L. martiniquensis. Moreover, this recombinant protein reacts with antibodies against L. donovani and Leishmania infantum, making it a promising antigen for further development of a universal rapid diagnostic tool for VL. © 2017 The Author(s) Journal of Eukaryotic Microbiology © 2017 International Society of Protistologists.

  3. Effective non-denaturing purification method for improving the solubility of recombinant actin-binding proteins produced by bacterial expression.

    Science.gov (United States)

    Chung, Jeong Min; Lee, Sangmin; Jung, Hyun Suk

    2017-05-01

    Bacterial expression is commonly used to produce recombinant and truncated mutant eukaryotic proteins. However, heterologous protein expression may render synthesized proteins insoluble. The conventional method used to express a poorly soluble protein, which involves denaturation and refolding, is time-consuming and inefficient. There are several non-denaturing approaches that can increase the solubility of recombinant proteins that include using different bacterial cell strains, altering the time of induction, lowering the incubation temperature, and employing different detergents for purification. In this study, we compared several non-denaturing protocols to express and purify two insoluble 34 kDa actin-bundling protein mutants. The solubility of the mutant proteins was not affected by any of the approaches except for treatment with the detergent sarkosyl. These results indicate that sarkosyl can effectively improve the solubility of insoluble proteins during bacterial expression. Copyright © 2016. Published by Elsevier Inc.

  4. Homologous recombination in mammalian cells: effect of p53 and Bcl-2 proteins, replication inhibition and ionizing radiations

    International Nuclear Information System (INIS)

    Saintigny, Yannick

    1999-01-01

    The control of cell cycle, associated with the mechanisms of replication, DNA repair/recombination allows the cells to maintain their genetic integrity. The p53 protein ensures the control of G1/S transition. Its inactivation would allow to initial replication on damaged matrix and lead to the block of replication forks followed by DNA strand breaks, good substrates for recombination. This work shows that the expression of mutant p53 protein stimulates both spontaneous and radio-induced homologous recombination, independently of the control of cell cycle. Moreover, the use of a set of replication inhibitors show that inhibition of the replication elongation stimulates recombination more strongly than the initiation inhibition. Replication arrest by these inhibitors also significantly increases the number of DNA strand breaks. These results highlighted a point of action of p53 protein on the ultimate stages of the homologous recombination mechanism. Lastly, the expression of Bcl-2 protein inhibits apoptosis and increases survival, but specifically inhibits conservative recombination, after radiation as well as in absence of apoptotic stress. The extinction of this mechanism of DNA repair is associated with an increase of mutagenesis. Taken together, these results allow ta consider the maintenance of the genetic stability as a cellular network involving different pathways. A multiple stages model for tumoral progression can be deduced. (author) [fr

  5. A recombinant fusion protein containing a spider toxin specific for the insect voltage-gated sodium ion channel shows oral toxicity towards insects of different orders

    Science.gov (United States)

    Yang, Sheng; Pyati, Prashant; Fitches, Elaine; Gatehouse, John A.

    2014-01-01

    Recombinant fusion protein technology allows specific insecticidal protein and peptide toxins to display activity in orally-delivered biopesticides. The spider venom peptide δ-amaurobitoxin-PI1a, which targets insect voltage-gated sodium channels, was fused to the “carrier” snowdrop lectin (GNA) to confer oral toxicity. The toxin itself (PI1a) and an amaurobitoxin/GNA fusion protein (PI1a/GNA) were produced using the yeast Pichia pastoris as expression host. Although both proteins caused mortality when injected into cabbage moth (Mamestra brassicae) larvae, the PI1a/GNA fusion was approximately 6 times as effective as recombinant PI1a on a molar basis. PI1a alone was not orally active against cabbage moth larvae, but a single 30 μg dose of the PI1a/GNA fusion protein caused 100% larval mortality within 6 days when fed to 3rd instar larvae, and caused significant reductions in survival, growth and feeding in 4th – 6th instar larvae. Transport of fusion protein from gut contents to the haemolymph of cabbage moth larvae, and binding to the nerve chord, was shown by Western blotting. The PI1a/GNA fusion protein also caused mortality when delivered orally to dipteran (Musca domestica; housefly) and hemipteran (Acyrthosiphon pisum; pea aphid) insects, making it a promising candidate for development as a biopesticide. PMID:24486516

  6. Air filter devices including nonwoven meshes of electrospun recombinant spider silk proteins.

    Science.gov (United States)

    Lang, Gregor; Jokisch, Stephan; Scheibel, Thomas

    2013-05-08

    Based on the natural sequence of Araneus diadematus Fibroin 4 (ADF4), the recombinant spider silk protein eADF4(C16) has been engineered. This highly repetitive protein has a molecular weight of 48kDa and is soluble in different solvents (hexafluoroisopropanol (HFIP), formic acid and aqueous buffers). eADF4(C16) provides a high potential for various technical applications when processed into morphologies such as films, capsules, particles, hydrogels, coatings, fibers and nonwoven meshes. Due to their chemical stability and controlled morphology, the latter can be used to improve filter materials. In this protocol, we present a procedure to enhance the efficiency of different air filter devices, by deposition of nonwoven meshes of electrospun recombinant spider silk proteins. Electrospinning of eADF4(C16) dissolved in HFIP results in smooth fibers. Variation of the protein concentration (5-25% w/v) results in different fiber diameters (80-1,100 nm) and thus pore sizes of the nonwoven mesh. Post-treatment of eADF4(C16) electrospun from HFIP is necessary since the protein displays a predominantly α-helical secondary structure in freshly spun fibers, and therefore the fibers are water soluble. Subsequent treatment with ethanol vapor induces formation of water resistant, stable β-sheet structures, preserving the morphology of the silk fibers and meshes. Secondary structure analysis was performed using Fourier transform infrared spectroscopy (FTIR) and subsequent Fourier self-deconvolution (FSD). The primary goal was to improve the filter efficiency of existing filter substrates by adding silk nonwoven layers on top. To evaluate the influence of electrospinning duration and thus nonwoven layer thickness on the filter efficiency, we performed air permeability tests in combination with particle deposition measurements. The experiments were carried out according to standard protocols.

  7. Recombinant production and solution structure of lipid transfer protein from lentil Lens culinaris

    International Nuclear Information System (INIS)

    Gizatullina, Albina K.; Finkina, Ekaterina I.; Mineev, Konstantin S.; Melnikova, Daria N.; Bogdanov, Ivan V.; Telezhinskaya, Irina N.; Balandin, Sergey V.; Shenkarev, Zakhar O.; Arseniev, Alexander S.; Ovchinnikova, Tatiana V.

    2013-01-01

    Highlights: •Lipid transfer protein from lentil seeds (Lc-LTP2) was overexpressed in E. coli. •Antimicrobial activity and spatial structure of the recombinant Lc-LTP2 were examined. •Internal tunnel-like lipid-binding cavity occupies ∼7% of the total Lc-LTP2 volume. •Binding of DMPG lipid induces moderate rearrangements in the Lc-LTP2 structure. •Lc-LTP2/DMPG complex has limited lifetime and dissociates within tens of hours. -- Abstract: Lipid transfer protein, designated as Lc-LTP2, was isolated from seeds of the lentil Lens culinaris. The protein has molecular mass 9282.7 Da, consists of 93 amino acid residues including 8 cysteines forming 4 disulfide bonds. Lc-LTP2 and its stable isotope labeled analogues were overexpressed in Escherichia coli and purified. Antimicrobial activity of the recombinant protein was examined, and its spatial structure was studied by NMR spectroscopy. The polypeptide chain of Lc-LTP2 forms four α-helices (Cys4-Leu18, Pro26-Ala37, Thr42-Ala56, Thr64-Lys73) and a long C-terminal tail without regular secondary structure. Side chains of the hydrophobic residues form a relatively large internal tunnel-like lipid-binding cavity (van der Waals volume comes up to ∼600 Å 3 ). The side-chains of Arg45, Pro79, and Tyr80 are located near an assumed mouth of the cavity. Titration with dimyristoyl phosphatidylglycerol (DMPG) revealed formation of the Lc-LTP2/lipid non-covalent complex accompanied by rearrangements in the protein spatial structure and expansion of the internal cavity. The resultant Lc-LTP2/DMPG complex demonstrates limited lifetime and dissociates within tens of hours

  8. Air Filter Devices Including Nonwoven Meshes of Electrospun Recombinant Spider Silk Proteins

    Science.gov (United States)

    Lang, Gregor; Jokisch, Stephan; Scheibel, Thomas

    2013-01-01

    Based on the natural sequence of Araneus diadematus Fibroin 4 (ADF4), the recombinant spider silk protein eADF4(C16) has been engineered. This highly repetitive protein has a molecular weight of 48kDa and is soluble in different solvents (hexafluoroisopropanol (HFIP), formic acid and aqueous buffers). eADF4(C16) provides a high potential for various technical applications when processed into morphologies such as films, capsules, particles, hydrogels, coatings, fibers and nonwoven meshes. Due to their chemical stability and controlled morphology, the latter can be used to improve filter materials. In this protocol, we present a procedure to enhance the efficiency of different air filter devices, by deposition of nonwoven meshes of electrospun recombinant spider silk proteins. Electrospinning of eADF4(C16) dissolved in HFIP results in smooth fibers. Variation of the protein concentration (5-25% w/v) results in different fiber diameters (80-1,100 nm) and thus pore sizes of the nonwoven mesh. Post-treatment of eADF4(C16) electrospun from HFIP is necessary since the protein displays a predominantly α-helical secondary structure in freshly spun fibers, and therefore the fibers are water soluble. Subsequent treatment with ethanol vapor induces formation of water resistant, stable β-sheet structures, preserving the morphology of the silk fibers and meshes. Secondary structure analysis was performed using Fourier transform infrared spectroscopy (FTIR) and subsequent Fourier self-deconvolution (FSD). The primary goal was to improve the filter efficiency of existing filter substrates by adding silk nonwoven layers on top. To evaluate the influence of electrospinning duration and thus nonwoven layer thickness on the filter efficiency, we performed air permeability tests in combination with particle deposition measurements. The experiments were carried out according to standard protocols. PMID:23685883

  9. Transactivator protein: An alternative for delivery of recombinant proteins for safer reprogramming of induced Pluripotent Stem Cell.

    Science.gov (United States)

    Nordin, Fazlina; Ahmad, Raja Norazireen Raja; Farzaneh, Farzin

    2017-05-02

    Induced pluripotent stem cells (iPSC) are somatic cells reprogrammed to pluripotency by forced expression of pluripotency factors. These cells are shown to have the same pluripotent potential as embryonic stem cells (ESC) and considered as an alternative to the much controversial usage of ESC which involved human embryos. However, the traditional method in reprogramming cells into iPSC using genome-integrating retro- or lenti- viruses remains an obstacle for its application in clinical setting. Although numerous studies have been conducted for a safer DNA-based reprogramming, reprogramming of iPSC by genetic modifications may raise the possibility of malignant transformation and has been a major limitation for its usage in clinical applications. Therefore, there is a need for an alternative method to reprogram the cells without the use of gene editing and a much safer way to deliver transcription factors to induce pluripotency on target cells. Using protein transduction approach, a number of studies have demonstrated the generation of human iPSCs from human fibroblasts and mouse embryonic fibroblasts by direct delivery of reprogramming proteins. In this review, the definition and mechanism of HIV-TAT protein (a type of protein transduction domain) in delivering recombinant proteins, including the potential of protein-based delivery to induce iPSC were further discussed. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. A recombinant fusion protein-based, fluorescent protease assay for high throughput-compatible substrate screening.

    Science.gov (United States)

    Bozóki, Beáta; Gazda, Lívia; Tóth, Ferenc; Miczi, Márió; Mótyán, János András; Tőzsér, József

    2018-01-01

    In connection with the intensive investigation of proteases, several methods have been developed for analysis of the substrate specificity. Due to the great number of proteases and the expected target molecules to be analyzed, time- and cost-efficient high-throughput screening (HTS) methods are preferred. Here we describe the development and application of a separation-based HTS-compatible fluorescent protease assay, which is based on the use of recombinant fusion proteins as substrates of proteases. The protein substrates used in this assay consists of N-terminal (hexahistidine and maltose binding protein) fusion tags, cleavage sequences of the tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein (mApple or mTurquoise2). The assay is based on the fluorimetric detection of the fluorescent proteins, which are released from the magnetic bead-attached substrates by the proteolytic cleavage. The protease assay has been applied for activity measurements of TEV and HIV-1 proteases to test the suitability of the system for enzyme kinetic measurements, inhibition studies, and determination of pH optimum. We also found that denatured fluorescent proteins can be renatured after SDS-PAGE of denaturing conditions, but showed differences in their renaturation abilities. After in-gel renaturation both substrates and cleavage products can be identified by in-gel UV detection. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Potential of Recombinant Opa Proteins as Vaccine Candidates against Hyperinvasive Meningococci ▿ †

    Science.gov (United States)

    Callaghan, M. J.; Lewis, S.; Sadarangani, M.; Bailey, S. E. S.; Chan, H.; Ferguson, D. J. P.; Derrick, J. P.; Feavers, I.; Maiden, M. C.; Pollard, A. J.

    2011-01-01

    Neisseria meningitidis causes half a million cases of septicemia and meningitis globally each year. The opacity (Opa) integral outer membrane proteins from N. meningitidis are polymorphic and highly immunogenic. Particular combinations of Opa proteins are associated with the hyperinvasive meningococcal lineages that have caused the majority of serogroup B and C meningococcal disease in industrialized countries over the last 60 years. For the first time, this genetic structuring of a diverse outer membrane protein family has been used to select a novel combination of representative antigens for immunogenicity testing. Fourteen recombinant Opa variants were produced and used in murine immunizations inducing an increase in specific antimeningococcal total IgG levels. All 14 Opa proteins elicited bactericidal antibodies against at least one hyperinvasive meningococcal isolate, and most isolates from each hyperinvasive lineage were killed by at least one Opa antiserum at a titer of 1:16 or greater. Cross-reactive bactericidal antibody responses were observed among clonal complexes. A theoretical coverage of 90% can be achieved by using a particular combination of 6 Opa proteins against an isolate collection of 227 recent United Kingdom disease cases. This study indicates the potential of Opa proteins to provide broad coverage against multiple meningococcal hyperinvasive lineages. PMID:21464082

  12. Potential of recombinant opa proteins as vaccine candidates against hyperinvasive meningococci.

    Science.gov (United States)

    Callaghan, M J; Lewis, S; Sadarangani, M; Bailey, S E S; Chan, H; Ferguson, D J P; Derrick, J P; Feavers, I; Maiden, M C; Pollard, A J

    2011-07-01

    Neisseria meningitidis causes half a million cases of septicemia and meningitis globally each year. The opacity (Opa) integral outer membrane proteins from N. meningitidis are polymorphic and highly immunogenic. Particular combinations of Opa proteins are associated with the hyperinvasive meningococcal lineages that have caused the majority of serogroup B and C meningococcal disease in industrialized countries over the last 60 years. For the first time, this genetic structuring of a diverse outer membrane protein family has been used to select a novel combination of representative antigens for immunogenicity testing. Fourteen recombinant Opa variants were produced and used in murine immunizations inducing an increase in specific antimeningococcal total IgG levels. All 14 Opa proteins elicited bactericidal antibodies against at least one hyperinvasive meningococcal isolate, and most isolates from each hyperinvasive lineage were killed by at least one Opa antiserum at a titer of 1:16 or greater. Cross-reactive bactericidal antibody responses were observed among clonal complexes. A theoretical coverage of 90% can be achieved by using a particular combination of 6 Opa proteins against an isolate collection of 227 recent United Kingdom disease cases. This study indicates the potential of Opa proteins to provide broad coverage against multiple meningococcal hyperinvasive lineages.

  13. Medium-throughput production of recombinant human proteins: protein production in E. coli.

    Science.gov (United States)

    Burgess-Brown, Nicola A; Mahajan, Pravin; Strain-Damerell, Claire; Gileadi, Opher; Gräslund, Susanne

    2014-01-01

    In Chapter 4 we described the SGC process for generating multiple constructs of truncated versions of each protein using LIC. In this chapter we provide a step-by-step procedure of our E. coli system for test expressing intracellular (soluble) proteins in a 96-well format that enables us to identify which proteins or truncated versions are expressed in a soluble and stable form suitable for structural studies. In addition, we detail the process for scaling up cultures for large-scale protein purification. This level of production is required to obtain sufficient quantities (i.e., milligram amounts) of protein for further characterization and/or crystallization experiments. Our standard process is purification by immobilized metal affinity chromatography (IMAC) using nickel resin followed by size exclusion chromatography (SEC), with additional procedures arising from the complexity of the protein itself.

  14. Implications of evolutionary engineering for growth and recombinant protein production in methanol-based growth media in the yeast Pichia pastoris.

    Science.gov (United States)

    Moser, Josef W; Prielhofer, Roland; Gerner, Samuel M; Graf, Alexandra B; Wilson, Iain B H; Mattanovich, Diethard; Dragosits, Martin

    2017-03-17

    Pichia pastoris is a widely used eukaryotic expression host for recombinant protein production. Adaptive laboratory evolution (ALE) has been applied in a wide range of studies in order to improve strains for biotechnological purposes. In this context, the impact of long-term carbon source adaptation in P. pastoris has not been addressed so far. Thus, we performed a pilot experiment in order to analyze the applicability and potential benefits of ALE towards improved growth and recombinant protein production in P. pastoris. Adaptation towards growth on methanol was performed in replicate cultures in rich and minimal growth medium for 250 generations. Increased growth rates on these growth media were observed at the population and single clone level. Evolved populations showed various degrees of growth advantages and trade-offs in non-evolutionary growth conditions. Genome resequencing revealed a wide variety of potential genetic targets associated with improved growth performance on methanol-based growth media. Alcohol oxidase represented a mutational hotspot since four out of seven evolved P. pastoris clones harbored mutations in this gene, resulting in decreased Aox activity, despite increased growth rates. Selected clones displayed strain-dependent variations for AOX-promoter based recombinant protein expression yield. One particularly interesting clone showed increased product titers ranging from a 2.5-fold increase in shake flask batch culture to a 1.8-fold increase during fed batch cultivation. Our data indicate a complex correlation of carbon source, growth context and recombinant protein production. While similar experiments have already shown their potential in other biotechnological areas where microbes were evolutionary engineered for improved stress resistance and growth, the current dataset encourages the analysis of the potential of ALE for improved protein production in P. pastoris on a broader scale.

  15. Heat-shock protein fusion vectors for improved expression of soluble recombinant proteins in Escherichia coli.

    Science.gov (United States)

    Kyratsous, Christos A; Panagiotidis, Christos A

    2012-01-01

    Molecular chaperones or heat-shock proteins (HSPs) are protein machines that interact with unfolded or partially folded polypeptides and assist them in attaining their proper conformation. The folding reaction relies on a complex array of scaffolding effects and ATP-driven conformational changes that mediate the temporary unfolding and subsequent refolding of protein substrates. DnaK and GroEL are the two major Escherichia coli chaperones. They belong to the HSP70 and HSP60 families of proteins, respectively, and play a major role in protein folding. Here, we describe a set of bacterial expression vectors that permits the fusion of a protein of interest to DnaK or GroEL and its subsequent quantitative expression in a soluble, easily purifiable form. We also provide a set of compatible co-chaperone expression constructs that permit the simultaneous co-expression of the DnaK and GroEL physiological partners to further increase protein solubility. The system was successfully tested using the murine prion protein (PrP). Although PrP is normally insoluble when expressed in E. coli, we show that utilizing our vectors it can be produced in a soluble form as a DnaK or GroEL fusion. This system is useful for the production of a large array of proteins that fail to fold properly when expressed in E. coli.

  16. Identification of antibody-interacting proteins that contribute to the production of recombinant antibody in mammalian cells.

    Science.gov (United States)

    Nishimiya, Daisuke; Ogura, Yuji; Sakurai, Hidetaka; Takahashi, Tohru

    2012-11-01

    Protein folding and assembly processes are essential for antibody secretion; however, the endogenous proteins involved in these processes remain largely unknown. Therefore, except for some well-known endoplasmic reticulum (ER) chaperones such as GRP78/Bip and protein disulfide isomerase, enhancement of recombinant antibody expression by co-expression of interacting proteins has been largely elusive. Here, in addition to known ER chaperones, we identified additional endogenous proteins that interact with recombinant antibody in mammalian cells by immunoprecipitation coupled with liquid chromatography-tandem mass spectrometry. Most of our identified proteins enhanced antibody production, and furthermore, some of their combinations resulted in greater enhancement. In particular, eukaryotic initiation factor 4A combined with other proteins had approximately fourfold higher effect on antibody production. Identified proteins that could improve antibody expression contain not only ER-resident proteins like GRP78/Bip but also non-ER-resident proteins. These results suggest that this method could be effective in the investigation of novel proteins that are involved in enhancing recombinant antibody production because immunoprecipitation coupled with mass spectroscopy could identify proteins which directly interact with the antibody.

  17. RYBP Is a K63-Ubiquitin-Chain-Binding Protein that Inhibits Homologous Recombination Repair

    Directory of Open Access Journals (Sweden)

    Mohammad A.M. Ali

    2018-01-01

    Full Text Available Summary: Ring1-YY1-binding protein (RYBP is a member of the non-canonical polycomb repressive complex 1 (PRC1, and like other PRC1 members, it is best described as a transcriptional regulator. However, several PRC1 members were recently shown to function in DNA repair. Here, we report that RYBP preferentially binds K63-ubiquitin chains via its Npl4 zinc finger (NZF domain. Since K63-linked ubiquitin chains are assembled at DNA double-strand breaks (DSBs, we examined the contribution of RYBP to DSB repair. Surprisingly, we find that RYBP is K48 polyubiquitylated by RNF8 and rapidly removed from chromatin upon DNA damage by the VCP/p97 segregase. High expression of RYBP competitively inhibits recruitment of BRCA1 repair complex to DSBs, reducing DNA end resection and homologous recombination (HR repair. Moreover, breast cancer cell lines expressing high endogenous RYBP levels show increased sensitivity to DNA-damaging agents and poly ADP-ribose polymerase (PARP inhibition. These data suggest that RYBP negatively regulates HR repair by competing for K63-ubiquitin chain binding. : Ali et al. find that RYBP binds K63-linked ubiquitin chains and is removed from DNA damage sites. This K63-ubiquitin binding allows RYBP to hinder the recruitment of BRCA1 and Rad51 to DNA double-strand breaks, thus inhibiting homologous recombination repair. Accordingly, cancer cells expressing high RYBP are more sensitive to DNA-damaging therapies. Keywords: DNA damage response, homologous recombination, ubiquitylation, RYBP, polycomb proteins, double-strand break repair, chromatin, histone modification

  18. Novel escherichia coli strain allows efficient recombinant protein production using lactose as inducer.

    Science.gov (United States)

    Menzella, Hugo G; Ceccarelli, Eduardo A; Gramajo, Hugo C

    2003-06-30

    An important characteristic of promoters used in recombinant protein production in Escherichi coli is their inducibility in a simple and cost-effective manner. The IPTG inducible promoters lac, tac, and trc are powerful and widely used for basic research. However, the use of IPTG in large-scale production is undesirable due to its high cost and toxicity. The promoters mentioned above can also be induced by the addition of lactose, which has the double role of inducer and carbon and energy source. Nevertheless, the use of this sugar in industrial processes has several drawbacks, which result in low volumetric yields and difficulties in process control. We have genetically engineered a BL21 strain to allow the efficient use of lactose as inducer in fed-batch cultures. Two modifications were introduced, the exchange of the wild-type lac operator by a constitutive one (lacO(c)) and the replacement of the gal alleles to recover the Gal(+) phenotype. The constitutive expression of the lac operon overcame the negative effects that the Lac nongenetic heterogeneity of wild-type E. coli introduces when lactose is used as inducer. The gal(+) genotype allowed the complete use of the lactose as carbon and energy source. The relevance of these two modifications in the efficient utilization of lactose as inducer was demonstrated in fed-batch cultures of the novel recombinant strain MP101 harboring expression vectors containing the calf prochymosin gene or the pccB gene, which encodes for the carboxyltransferase component of a propionyl-CoA carboxylase complex from Streptomyces coelicolor. Similar levels of recombinant protein production (up to 16 g/L) were obtained by using either lactose or IPTG as inducers, which confirmed the success of the genetics modifications introduced. Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 809-817, 2003.

  19. Evaluation of immobilized metal affinity chromatography kits for the purification of histidine-tagged recombinant CagA protein.

    Science.gov (United States)

    Karakus, Cebrail; Uslu, Merve; Yazici, Duygu; Salih, Barik A

    2016-05-15

    Immobilized metal affinity chromatography (IMAC) technique is used for fast and reliable purification of histidine(His)-tagged recombinant proteins. The technique provides purification under native and denaturing conditions. The aim of this study is to evaluate three commercially available IMAC kits (Thermo Scientific, GE Healthcare and Qiagen) for the purification of a 6xHis-tagged recombinant CagA (cytotoxin-associated gene A) protein from IPTG-induced Escherichia coli BL21(DE3) culture. The kits were tested according to the manufacturer instructions and the protein was purified with only GE Healthcare and Qiagen kits under denaturing conditions. 1% (w/v) SDS was used as denaturing agent in PBS instead of extraction reagent of Thermo Scientific kit to lyse bacterial cells from 100ml culture. The 6xHis-tagged recombinant protein was purified by the three kits equally. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Single Strand Annealing Plays a Major Role in RecA-Independent Recombination between Repeated Sequences in the Radioresistant Deinococcus radiodurans Bacterium.

    Directory of Open Access Journals (Sweden)

    Solenne Ithurbide

    2015-10-01

    Full Text Available The bacterium Deinococcus radiodurans is one of the most radioresistant organisms known. It is able to reconstruct a functional genome from hundreds of radiation-induced chromosomal fragments. Our work aims to highlight the genes involved in recombination between 438 bp direct repeats separated by intervening sequences of various lengths ranging from 1,479 bp to 10,500 bp to restore a functional tetA gene in the presence or absence of radiation-induced DNA double strand breaks. The frequency of spontaneous deletion events between the chromosomal direct repeats were the same in recA+ and in ΔrecA, ΔrecF, and ΔrecO bacteria, whereas recombination between chromosomal and plasmid DNA was shown to be strictly dependent on the RecA and RecF proteins. The presence of mutations in one of the repeated sequence reduced, in a MutS-dependent manner, the frequency of the deletion events. The distance between the repeats did not influence the frequencies of deletion events in recA+ as well in ΔrecA bacteria. The absence of the UvrD protein stimulated the recombination between the direct repeats whereas the absence of the DdrB protein, previously shown to be involved in DNA double strand break repair through a single strand annealing (SSA pathway, strongly reduces the frequency of RecA- (and RecO- independent deletions events. The absence of the DdrB protein also increased the lethal sectoring of cells devoid of RecA or RecO protein. γ-irradiation of recA+ cells increased about 10-fold the frequencies of the deletion events, but at a lesser extend in cells devoid of the DdrB protein. Altogether, our results suggest a major role of single strand annealing in DNA repeat deletion events in bacteria devoid of the RecA protein, and also in recA+ bacteria exposed to ionizing radiation.

  1. Optimising expression of the recombinant fusion protein biopesticide ω-hexatoxin-Hv1a/GNA in Pichia pastoris: sequence modifications and a simple method for the generation of multi-copy strains.

    Science.gov (United States)

    Pyati, Prashant; Fitches, Elaine; Gatehouse, John A

    2014-08-01

    Production of recombinant protein bio-insecticides on a commercial scale can only be cost effective if host strains with very high expression levels are available. A recombinant fusion protein containing an arthropod toxin, ω-hexatoxin-Hv1a, (from funnel web spider Hadronyche versuta) linked to snowdrop lectin (Galanthus nivalis agglutinin; GNA) is an effective oral insecticide and candidate biopesticide. However, the fusion protein was vulnerable to proteolysis during production in the yeast Pichia pastoris. To prevent proteolysis, the Hv1a/GNA fusion expression construct was modified by site-directed mutagenesis to remove a potential Kex2 cleavage site at the C-terminus of the Hv1a peptide. To obtain a high expressing clone of P. pastoris to produce recombinant Hv1a/GNA, a straightforward method was used to produce multi-copy expression plasmids, which does not require multiple integrations to give clones of P. pastoris containing high copy numbers of the introduced gene. Removal of the Kex2 site resulted in increased levels of intact fusion protein expressed in wild-type P. pastoris strains, improving levels of intact recombinant protein recoverable. Incorporation of a C-terminal (His)6 tag enabled single step purification of the fusion protein. These modifications did not affect the insecticidal activity of the recombinant toxin towards lepidopteran larvae. Introduction of multiple expression cassettes increased the amount of secreted recombinant fusion protein in a laboratory scale fermentation by almost tenfold on a per litre of culture basis. Simple modifications in the expression construct can be advantageous for the generation of high expressing P. pastoris strains for production of a recombinant protein, without altering its functional properties.

  2. Inhibition of activated protein C by recombinant alpha 1-antitrypsin variants with substitution of arginine or leucine for methionine358

    NARCIS (Netherlands)

    Heeb, M.J.; Bischoff, Rainer; Courtney, M.; Griffin, J.H.

    1990-01-01

    alpha 1-Antitrypsin (alpha 1-AT) was recently identified as a major physiologic plasma inhibitor of activated protein C. The reaction with activated protein C of recombinant alpha 1-AT containing amino acid substitutions at the reactive center was studied. The substitution of Arg358 for Met, as

  3. Immobilized palladium(II) ion affinity chromatography for recovery of recombinant proteins with peptide tags containing histidine and cysteine.

    Science.gov (United States)

    Kikot, Pamela; Polat, Aise; Achilli, Estefania; Fernandez Lahore, Marcelo; Grasselli, Mariano

    2014-11-01

    Fusion of peptide-based tags to recombinant proteins is currently one of the most used tools for protein production. Also, immobilized metal ion affinity chromatography (IMAC) has a huge application in protein purification, especially in research labs. The combination of expression systems of recombinant tagged proteins with this robust chromatographic system has become an efficient and rapid tool to produce milligram-range amounts of proteins. IMAC-Ni(II) columns have become the natural partners of 6xHis-tagged proteins. The Ni(II) ion is considered as the best compromise of selectivity and affinity for purification of a recombinant His-tagged protein. The palladium(II) ion is also able to bind to side chains of amino acids and form ternary complexes with iminodiacetic acid and free amino acids and other sulfur-containing molecules. In this work, we evaluated two different cysteine- and histidine-containing six amino acid tags linked to the N-terminal group of green fluorescent protein (GFP) and studied the adsorption and elution conditions using novel eluents. Both cysteine-containing tagged GFPs were able to bind to IMAC-Pd(II) matrices and eluted successfully using a low concentration of thiourea solution. The IMAC-Ni(II) system reaches less than 20% recovery of the cysteine-containing tagged GFP from a crude homogenate of recombinant Escherichia coli, meanwhile the IMAC-Pd(II) yields a recovery of 45% with a purification factor of 13. Copyright © 2014 John Wiley & Sons, Ltd.

  4. "Recombinant Protein of the Day": Using Daily Student Presentations to Add Real-World Aspects to a Biotechnology Course

    Science.gov (United States)

    Shaffer, Justin F.

    2013-01-01

    To provide a realistic view of the biotechnology industry for students, a novel course focusing on recombinant proteins and their importance in medicine, pharmaceuticals, industry, scientific research, and agriculture was developed. ''Designer Proteins and Society,'' an upper-division elective, was taught in the Fall 2012 semester to 16 junior,…

  5. Recombinant proteins of helminths with immunoregulatory properties and their possible therapeutic use.

    Science.gov (United States)

    Nascimento Santos, Leonardo; Carvalho Pacheco, Luis Gustavo; Silva Pinheiro, Carina; Alcantara-Neves, Neuza Maria

    2017-02-01

    The inverse relationship between helminth infections and the development of immune-mediated diseases is a cornerstone of the hygiene hypothesis and studies were carried out to elucidate the mechanisms by which helminth-derived molecules can suppress immunological disorders. These studies have fostered the idea that parasitic worms may be used as a promising therapeutic alternative for prevention and treatment of immune-mediated diseases. We discuss the current approaches for identification of helminth proteins with potential immunoregulatory properties, including the strategies based on high-throughput technologies. We also explore the methodological approaches and expression systems used for production of the recombinant forms of more than 20 helminth immunomodulatory proteins, besides their performances when evaluated as immunotherapeutic molecules to treat different immune-mediated conditions, including asthma and inflammatory bowel diseases. Finally, we discuss the perspectives of using these parasite-derived recombinant molecules as tools for future immunotherapy and immunoprophylaxis of human inflammatory diseases. Copyright © 2016. Published by Elsevier B.V.

  6. An All-Recombinant Protein-Based Culture System Specifically Identifies Hematopoietic Stem Cell Maintenance Factors.

    Science.gov (United States)

    Ieyasu, Aki; Ishida, Reiko; Kimura, Takaharu; Morita, Maiko; Wilkinson, Adam C; Sudo, Kazuhiro; Nishimura, Toshinobu; Ohehara, Jun; Tajima, Yoko; Lai, Chen-Yi; Otsu, Makoto; Nakamura, Yukio; Ema, Hideo; Nakauchi, Hiromitsu; Yamazaki, Satoshi

    2017-03-14

    Hematopoietic stem cells (HSCs) are considered one of the most promising therapeutic targets for the treatment of various blood disorders. However, due to difficulties in establishing stable maintenance and expansion of HSCs in vitro, their insufficient supply is a major constraint to transplantation studies. To solve these problems we have developed a fully defined, all-recombinant protein-based culture system. Through this system, we have identified hemopexin (HPX) and interleukin-1α as responsible for HSC maintenance in vitro. Subsequent molecular analysis revealed that HPX reduces intracellular reactive oxygen species levels within cultured HSCs. Furthermore, bone marrow immunostaining and 3D immunohistochemistry revealed that HPX is expressed in non-myelinating Schwann cells, known HSC niche constituents. These results highlight the utility of this fully defined all-recombinant protein-based culture system for reproducible in vitro HSC culture and its potential to contribute to the identification of factors responsible for in vitro maintenance, expansion, and differentiation of stem cell populations. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  7. An All-Recombinant Protein-Based Culture System Specifically Identifies Hematopoietic Stem Cell Maintenance Factors

    Directory of Open Access Journals (Sweden)

    Aki Ieyasu

    2017-03-01

    Full Text Available Hematopoietic stem cells (HSCs are considered one of the most promising therapeutic targets for the treatment of various blood disorders. However, due to difficulties in establishing stable maintenance and expansion of HSCs in vitro, their insufficient supply is a major constraint to transplantation studies. To solve these problems we have developed a fully defined, all-recombinant protein-based culture system. Through this system, we have identified hemopexin (HPX and interleukin-1α as responsible for HSC maintenance in vitro. Subsequent molecular analysis revealed that HPX reduces intracellular reactive oxygen species levels within cultured HSCs. Furthermore, bone marrow immunostaining and 3D immunohistochemistry revealed that HPX is expressed in non-myelinating Schwann cells, known HSC niche constituents. These results highlight the utility of this fully defined all-recombinant protein-based culture system for reproducible in vitro HSC culture and its potential to contribute to the identification of factors responsible for in vitro maintenance, expansion, and differentiation of stem cell populations.

  8. Immunization with recombinant enterovirus 71 viral capsid protein 1 fragment stimulated antibody responses in hamsters

    Directory of Open Access Journals (Sweden)

    Ch’ng Wei-Choong

    2012-08-01

    Full Text Available Abstract Enterovirus 71 (EV71 causes severe neurological diseases resulting in high mortality in young children worldwide. Development of an effective vaccine against EV71 infection is hampered by the lack of appropriate animal models for efficacy testing of candidate vaccines. Previously, we have successfully tested the immunogenicity and protectiveness of a candidate EV71 vaccine, containing recombinant Newcastle disease virus capsids that display an EV71 VP1 fragment (NPt-VP11-100 protein, in a mouse model of EV71 infection. A drawback of this system is its limited window of EV71 susceptibility period, 2 weeks after birth, leading to restricted options in the evaluation of optimal dosing regimens. To address this issue, we have assessed the NPt-VP11-100 candidate vaccine in a hamster system, which offers a 4-week susceptibility period to EV71 infection. Results obtained showed that the NPt-VP11-100 candidate vaccine stimulated excellent humoral immune response in the hamsters. Despite the high level of antibody production, they failed to neutralize EV71 viruses or protect vaccinated hamsters in viral challenge studies. Nevertheless, these findings have contributed towards a better understanding of the NPt-VP11-100 recombinant protein as a candidate vaccine in an alternative animal model system.

  9. A recombinant protein based on Trypanosoma cruzi P21 enhances phagocytosis.

    Science.gov (United States)

    Rodrigues, Adele A; Clemente, Tatiana M; Dos Santos, Marlus A; Machado, Fabrício C; Gomes, Rafael G B; Moreira, Heline Hellen T; Cruz, Mário C; Brígido, Paula C; Dos Santos, Paulo C F; Martins, Flávia A; Bahia, Diana; Maricato, Juliana T; Janini, Luiz M R; Reboredo, Eduardo H; Mortara, Renato A; da Silva, Claudio V

    2012-01-01

    P21 is a secreted protein expressed in all developmental stages of Trypanosoma cruzi. The aim of this study was to determine the effect of the recombinant protein based on P21 (P21-His(6)) on inflammatory macrophages during phagocytosis. Our results showed that P21-His(6) acts as a phagocytosis inducer by binding to CXCR4 chemokine receptor and activating actin polymerization in a way dependent onthe PI3-kinase signaling pathway. Thus, our results shed light on the notion that native P21 is a component related to T. cruzi evasion from the immune response and that CXCR4 may be involved in phagocytosis. P21-His(6) represents an important experimental control tool to study phagocytosis signaling pathways of different intracellular parasites and particles.

  10. A recombinant protein based on Trypanosoma cruzi P21 enhances phagocytosis.

    Directory of Open Access Journals (Sweden)

    Adele A Rodrigues

    Full Text Available BACKGROUND: P21 is a secreted protein expressed in all developmental stages of Trypanosoma cruzi. The aim of this study was to determine the effect of the recombinant protein based on P21 (P21-His(6 on inflammatory macrophages during phagocytosis. FINDINGS: Our results showed that P21-His(6 acts as a phagocytosis inducer by binding to CXCR4 chemokine receptor and activating actin polymerization in a way dependent onthe PI3-kinase signaling pathway. CONCLUSIONS: Thus, our results shed light on the notion that native P21 is a component related to T. cruzi evasion from the immune response and that CXCR4 may be involved in phagocytosis. P21-His(6 represents an important experimental control tool to study phagocytosis signaling pathways of different intracellular parasites and particles.

  11. Adeno-associated-virus-mediated transduction of the mammary gland enables sustained production of recombinant proteins in milk

    OpenAIRE

    Stefan Wagner; Rosemary Thresher; Ross Bland; Götz Laible

    2015-01-01

    Biopharming for the production of recombinant pharmaceutical proteins in the mammary gland of transgenic animals is an attractive but laborious alternative compared to mammalian cell fermentation. The disadvantage of the lengthy process of genetically modifying an entire animal could be circumvented with somatic transduction of only the mammary epithelium with recombinant, replication-defective viruses. While other viral vectors offer very limited scope for this approach, vectors based on ade...

  12. Production of functional human insulin-like growth factor binding proteins (IGFBPs) using recombinant expression in HEK293 cells

    DEFF Research Database (Denmark)

    Wanscher, Anne Sofie Molsted; Williamson, Michael; Ebersole, Tasja Wainani

    2015-01-01

    on human proteins with therapeutic relevance is needed to design and process the next generation of protein therapeutics. In order to conduct structural and functional investigations large quantities of recombinant proteins are needed. However, finding a suitable recombinant production system for proteins......Insulin-like growth factor binding proteins (IGFBPs) display many functions in humans including regulation of the insulin-like growth factor (IGF) signaling pathway. The various roles of human IGFBPs make them attractive protein candidates in drug discovery. Structural and functional knowledge...... such as full-length human IGFBPs, still remains a challenge. Here we present a mammalian HEK293 expression method suitable for over-expression of secretory full-length human IGFBP-1 to -7. Protein purification of full-length human IGFBP-1, -2, -3 and -5 was conducted using a two-step chromatography procedure...

  13. Transfection of primary brain capillary endothelial cells for protein synthesis and secretion of recombinant erythropoietin: a strategy to enable protein delivery to the brain

    DEFF Research Database (Denmark)

    Burkhart, Annette; Andresen, Thomas Lars; Aigner, Achim

    2017-01-01

    , as turning BCECs into recombinant protein factories by transfection could result in protein secretion further into the brain. The present study aims to investigate the possibility of transfecting primary rat brain endothelial cells (RBECs) for recombinant protein synthesis and secretion...... of the neuroprotective protein erythropoietin (EPO). We previously showed that 4% of RBECs with BBB properties can be transfected without disrupting the BBB integrity in vitro, but it can be questioned whether this is sufficient to enable protein secretion at therapeutic levels. The present study examined various...... transfection vectors, with regard to increasing the transfection efficiency without disrupting the BBB integrity. Lipofectamine 3000™ was the most potent vector compared to polyethylenimine (PEI) and Turbofect. When co-cultured with astrocytes, the genetically modified RBECs secreted recombinant EPO...

  14. Medium-throughput production of recombinant human proteins: protein production in insect cells.

    Science.gov (United States)

    Mahajan, Pravin; Strain-Damerell, Claire; Gileadi, Opher; Burgess-Brown, Nicola A

    2014-01-01

    This chapter describes the step-by-step methods employed by the Structural Genomics Consortium (SGC) for screening and producing proteins in the baculovirus expression vector system (BEVS). This eukaryotic expression system was selected and a screening process established in 2007 as a measure to tackle the more challenging kinase, RNA-DNA processing and integral membrane protein families on our target list. Here, we discuss our platform for identifying soluble proteins from 3 ml of insect cell culture and describe the procedures involved in producing protein from liter-scale cultures. Although not discussed in this chapter, the same process can also be applied to integral membrane proteins (IMPs) with slight adaptations to the purification procedure.

  15. Antigenic and immunosuppressive properties of a trimeric recombinant transmembrane envelope protein gp41 of HIV-1.

    Directory of Open Access Journals (Sweden)

    Michael Mühle

    Full Text Available The transmembrane envelope (TM protein gp41 of the human immunodeficiency virus-1 (HIV-1 plays an important role during virus infection inducing the fusion of the viral and cellular membranes. In addition, there are indications that the TM protein plays a role in the immunopathogenesis leading to the acquired immunodeficiency syndrome (AIDS. Inactivated virus particles and recombinant gp41 have been reported to inhibit lymphocyte proliferation, as well as to alter cytokine release and gene expression. The same was shown for a peptide corresponding to a highly conserved domain of all retroviral TM proteins, the immunosuppressive domain. Due to its propensity to aggregate and to be expressed at low levels, studies comprising authentic gp41 produced in eukaryotic cells are extremely rare. Here we describe the production of a secreted, soluble recombinant gp41 in 293 cells. The antigen was purified to homogeneity and characterised thoroughly by various biochemical and immunological methods. It was shown that the protein was glycosylated and assembled into trimers. Binding studies by ELISA and surface plasmon resonance using conformation-specific monoclonal antibodies implied a six-helix bundle conformation. The low binding of broadly neutralising antibodies (bnAb directed against the membrane proximal external region (MPER suggested that this gp41 is probably not suited as vaccine to induce such bnAb. Purified gp41 bound to monocytes and to a lesser extent to lymphocytes and triggered the production of specific cytokines when added to normal peripheral blood mononuclear cells. In addition, gp41 expressed on target cells inhibited the antigen-specific response of murine CD8+ T cells by drastically impairing their IFNγ production. To our knowledge, this is the first comprehensive analysis of a gp41 produced in eukaryotic cells including its immunosuppressive properties. Our data provide another line of evidence that gp41 might be directly involved in

  16. Vaccination with Recombinant Microneme Proteins Confers Protection against Experimental Toxoplasmosis in Mice.

    Directory of Open Access Journals (Sweden)

    Camila Figueiredo Pinzan

    Full Text Available Toxoplasmosis, a zoonotic disease caused by Toxoplasma gondii, is an important public health problem and veterinary concern. Although there is no vaccine for human toxoplasmosis, many attempts have been made to develop one. Promising vaccine candidates utilize proteins, or their genes, from microneme organelle of T. gondii that are involved in the initial stages of host cell invasion by the parasite. In the present study, we used different recombinant microneme proteins (TgMIC1, TgMIC4, or TgMIC6 or combinations of these proteins (TgMIC1-4 and TgMIC1-4-6 to evaluate the immune response and protection against experimental toxoplasmosis in C57BL/6 mice. Vaccination with recombinant TgMIC1, TgMIC4, or TgMIC6 alone conferred partial protection, as demonstrated by reduced brain cyst burden and mortality rates after challenge. Immunization with TgMIC1-4 or TgMIC1-4-6 vaccines provided the most effective protection, since 70% and 80% of mice, respectively, survived to the acute phase of infection. In addition, these vaccinated mice, in comparison to non-vaccinated ones, showed reduced parasite burden by 59% and 68%, respectively. The protective effect was related to the cellular and humoral immune responses induced by vaccination and included the release of Th1 cytokines IFN-γ and IL-12, antigen-stimulated spleen cell proliferation, and production of antigen-specific serum antibodies. Our results demonstrate that microneme proteins are potential vaccines against T. gondii, since their inoculation prevents or decreases the deleterious effects of the infection.

  17. Vaccination with Recombinant Microneme Proteins Confers Protection against Experimental Toxoplasmosis in Mice.

    Science.gov (United States)

    Pinzan, Camila Figueiredo; Sardinha-Silva, Aline; Almeida, Fausto; Lai, Livia; Lopes, Carla Duque; Lourenço, Elaine Vicente; Panunto-Castelo, Ademilson; Matthews, Stephen; Roque-Barreira, Maria Cristina

    2015-01-01

    Toxoplasmosis, a zoonotic disease caused by Toxoplasma gondii, is an important public health problem and veterinary concern. Although there is no vaccine for human toxoplasmosis, many attempts have been made to develop one. Promising vaccine candidates utilize proteins, or their genes, from microneme organelle of T. gondii that are involved in the initial stages of host cell invasion by the parasite. In the present study, we used different recombinant microneme proteins (TgMIC1, TgMIC4, or TgMIC6) or combinations of these proteins (TgMIC1-4 and TgMIC1-4-6) to evaluate the immune response and protection against experimental toxoplasmosis in C57BL/6 mice. Vaccination with recombinant TgMIC1, TgMIC4, or TgMIC6 alone conferred partial protection, as demonstrated by reduced brain cyst burden and mortality rates after challenge. Immunization with TgMIC1-4 or TgMIC1-4-6 vaccines provided the most effective protection, since 70% and 80% of mice, respectively, survived to the acute phase of infection. In addition, these vaccinated mice, in comparison to non-vaccinated ones, showed reduced parasite burden by 59% and 68%, respectively. The protective effect was related to the cellular and humoral immune responses induced by vaccination and included the release of Th1 cytokines IFN-γ and IL-12, antigen-stimulated spleen cell proliferation, and production of antigen-specific serum antibodies. Our results demonstrate that microneme proteins are potential vaccines against T. gondii, since their inoculation prevents or decreases the deleterious effects of the infection.

  18. Production, purification and immunogenicity of recombinant Ebola virus proteins - A comparison of Freund's adjuvant and adjuvant system 03.

    Science.gov (United States)

    Melén, Krister; Kakkola, Laura; He, Felix; Airenne, Kari; Vapalahti, Olli; Karlberg, Helen; Mirazimi, Ali; Julkunen, Ilkka

    2017-04-01

    There is an urgent need for Ebola virus (EBOV) proteins, EBOV-specific antibodies and recombinant antigens to be used in diagnostics and as potential vaccine candidates. Our objective was to produce and purify recombinant proteins for immunological assays and for the production of polyclonal EBOV specific antibodies. In addition, a limited comparison of the adjuvant effects of Freund's complete adjuvant (FCA) and adjuvant system 03 (AS03) was carried out. Recombinant EBOV GST-VP24, -VP30, -VP35, -VP40 and -NP were produced in E. coli and purified with affinity chromatography followed by preparative gel electrophoresis. Recombinant EBOV GP-His was produced in Sf9 insect cells and purified by preparative gel electrophoresis. To compare the adjuvant effect of FCA and AS03, 12 rabbits were immunized four times with one of the six recombinant EBOV proteins using FCA or AS03. In addition, three guinea pigs were immunized with EBOV VP24 using FCA. With the exception of sera from two rabbits immunized with GST-VP24, the antisera against all other EBOV proteins showed very high and specific antibody responses after three to four immunizations. The adjuvant effect of AS03 was comparable to that of FCA. The produced antibodies recognized the corresponding EBOV proteins in wild type EBOV-infected cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Biophysical characterization of recombinant proteins: A key to higher structural genomics success

    Science.gov (United States)

    Vedadi, Masoud; Arrowsmith, Cheryl H.; Allali-Hassani, Abdellah; Senisterra, Guillermo; Wasney, Gregory A.

    2010-01-01

    Hundreds of genomes have been successfully sequenced to date, and the data are publicly available. At the same time, the advances in large-scale expression and purification of recombinant proteins have paved the way for structural genomics efforts. Frequently, however, little is known about newly expressed proteins calling for large-scale protein characterization to better understand their biochemical roles and to enable structure–function relationship studies. In the Structural Genomics Consortium (SGC), we have established a platform to characterize large numbers of purified proteins. This includes screening for ligands, enzyme assays, peptide arrays and peptide displacement in a 384-well format. In this review, we describe this platform in more detail and report on how our approach significantly increases the success rate for structure determination. Coupled with high-resolution X-ray crystallography and structure-guided methods, this platform can also be used toward the development of chemical probes through screening families of proteins against a variety of chemical series and focused chemical libraries. PMID:20466062

  20. Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coli

    Directory of Open Access Journals (Sweden)

    L. Avilán

    1997-12-01

    Full Text Available We cloned the streptokinase (STK gene of Streptococcus equisimilis in an expression vector of Escherichia coli to overexpress the profibrinolytic protein under the control of a tac promoter. Almost all the recombinant STK was exported to the periplasmic space and recovered after gentle lysozyme digestion of induced cells. The periplasmic fraction was chromatographed on DEAE Sepharose followed by chromatography on phenyl-agarose. Active proteins eluted between 4.5 and 0% ammonium sulfate, when a linear gradient was applied. Three major STK derivatives of 47.5 kDa, 45 kDa and 32 kDa were detected by Western blot analysis with a polyclonal antibody. The 32-kDa protein formed a complex with human plasminogen but did not exhibit Glu-plasminogen activator activity, as revealed by a zymographic assay, whereas the 45-kDa protein showed a Km = 0.70 µM and kcat = 0.82 s-1, when assayed with a chromogen-coupled substrate. These results suggest that these proteins are putative fragments of STK, possibly derived from partial degradation during the export pathway or the purification steps. The 47.5-kDa band corresponded to the native STK, as revealed by peptide sequencing

  1. Expression and purification of single cysteine-containing mutant variants of the mouse prion protein by oxidative refolding.

    Science.gov (United States)

    Sengupta, Ishita; Udgaonkar, Jayant B

    2017-12-01

    The folding and aggregation of proteins has been studied extensively, using multiple probes. To facilitate such experiments, introduction of spectroscopically-active moieties in to the protein of interest is often necessary. This is commonly achieved by specifically labelling cysteine residues in the protein, which are either present naturally or introduced artificially by site-directed mutagenesis. In the case of the recombinant prion protein, which is normally expressed in inclusion bodies, the presence of the native disulfide bond complicates the correct refolding of single cysteine-containing mutant variants of the protein. To overcome this major bottleneck, a simple purification strategy for single tryptophan, single cysteine-containing mutant variants of the mouse prion protein is presented, with yields comparable to that of the wild type protein. The protein(s) obtained by this method are correctly folded, with a single reduced cysteine, and the native disulfide bond between residues C178 and C213 intact. The β-sheet rich oligomers formed from these mutant variant protein(s) are identical to the wild type protein oligomer. The current strategy facilitates sample preparation for a number of high resolution spectroscopic measurements for the prion protein, which specifically require thiol labelling. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Evaluation of yeast single cell protein (SCP) diets on growth ...

    African Journals Online (AJOL)

    Jane

    An investigation was carried out on the possibility of replacing fishmeal with graded levels of yeast single cell protein (SCP; 10, 20, 30, 40 and 50%) in isonitrogenous feed formulations (30% protein) in the diet of Oreochromis niloticus fingerlings for a period of 12 weeks. The control diet had fishmeal as the primary protein ...

  3. The new pLAI (lux regulon based auto-inducible expression system for recombinant protein production in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Nocadello Salvatore

    2012-01-01

    Full Text Available Abstract Background After many years of intensive research, it is generally assumed that no universal expression system can exist for high-level production of a given recombinant protein. Among the different expression systems, the inducible systems are the most popular for their tight regulation. However, induction is in many cases less favorable due to the high cost and/or toxicity of inducers, incompatibilities with industrial scale-up or detrimental growth conditions. Expression systems using autoinduction (or self-induction prove to be extremely versatile allowing growth and induction of recombinant proteins without the need to monitor cell density or add inducer. Unfortunately, almost all the actual auto inducible expression systems need endogenous or induced metabolic changes during the growth to trigger induction, both frequently linked to detrimental condition to cell growth. In this context, we use a simple modular approach for a cell density-based genetic regulation in order to assemble an autoinducible recombinant protein expression system in E. coli. Result The newly designed pLAI expression system places the expression of recombinant proteins in Escherichia coli under control of the regulatory genes of the lux regulon of Vibrio fischeri's Quorum Sensing (QS system. The pLAI system allows a tight regulation of the recombinant gene allowing a negligible basal expression and expression only at high cell density. Sequence optimization of regulative genes of QS of V. fischeri for expression in E. coli upgraded the system to high level expression. Moreover, partition of regulative genes between the plasmid and the host genome and introduction of a molecular safety lock permitted tighter control of gene expression. Conclusion Coupling gene expression to cell density using cell-to-cell communication provides a promising approach for recombinant protein production. The system allows the control of expression of the target recombinant gene

  4. Refolding in high hydrostatic pressure of recombinant proteins from inclusion bodies in Escherichia Coli

    International Nuclear Information System (INIS)

    Balduino, Keli Nunes

    2009-01-01

    The expression of proteins as inclusion bodies in bacteria is a widely used alternative for production of recombinant protein. However, the aggregation is a problem often encountered during refolding of these proteins. High hydrostatic pressure are able to solubilise the inclusion bodies in the presence of low concentrations of denaturant reagents, encouraging refolding protein with high efficiency and reduce costs. This work aims to refolding of recombinant proteins expressed in Escherichia coli from inclusion bodies using high hydrostatic pressure. Three toxins, all featuring five or more disulfide bonds were studied: NXH8, Natterin 2 and Bothropstoxin 1. Suspensions of inclusion bodies of the three proteins were pressurized to 2000 bars for 16 hours. The buffers were optimized for refolding of the three proteins. The buffer used in the refolding of NXH8 was 50 mM Tris HCl, pH 9.0 with proportion of 1GSH: 4GSSG at a concentration of 6 mM and 2 M GdnHCl. Inclusion bodies were used in O.D. (A600nm) of 0.5. After refolding process, dialysis was performed at pH 7.0. The final yield of obtaining soluble NXH8 was 40% (28,6 mg of soluble NXH8/L of culture medium). The refolding of Bothropstoxin 1 was obtained in refolding buffer of Tris HCl 50 mM, pH 7,5 with proportion of 2 GSH: GSSG 3 and concentration of 3 mM and 1 M GdnHCl. Use with a suspension of O.D. (A600nm) of 0.5. The final yield of recovery of Bothropstoxin 1 refolded was 32% (9,2 mg of refolded Bothropstoxin 1/L of culture medium). The refolding of Natterin 2 was performed in the refolding buffer: 20 mM Tris HCl pH 9.0 at a ratio of 2 GSH: 3GSSG and concentration of 10 mM and 1 M GdnHCl and inclusion bodies O.D. (A600nm) of 6.0. The yield of Natterin 2 refolded was 20% (3,7 mg/L of culture medium). Physico-chemical and biological analysis were performed by SDS-PAGE, western blot, scanning electron microscopy, biological tests in vivo and in vitro and structural. The analysis conducted in NXH8 did not show

  5. ACCUMULATION OF RECOMBINANT FUSION PROTEIN – SECRETORY ANALOG OF Ag85B AND ESAT6 MYCOBACTERIUM TUBERCULOSIS PROTEINS – IN TRANSGENIC Lemna minor L. PLANTS

    Directory of Open Access Journals (Sweden)

    A.A.Peterson

    2015-10-01

    Full Text Available Determination of the presence of the recombinant fusion protein (ESAT6-Ag85B(ΔTMD-6His and its accumulation level in duckweed plants (Lemna minor L. was the aim of the research. ESAT6 and Ag85B are secretory proteins of Mycobacterium tuberculosis and are considered as potential candidates for development of new vaccine against tuberculosis (TB. Transgenic duckweed plants were obtained previously by Agrobacterium rhizogenes-mediated transformation and possessed fusion gene sequence esxA-fbpBΔTMD. Specific polyclonal antibodies were produced in immunized mice to identify levels of accumulation of TB antigens in plants. Recombinant antigen used for mice immunization was obtained in our laboratory by expression in E. coli. Western blot analysis revealed the recombinant tuberculosis antigen ESAT6-Ag85B(ΔTMD-6His in extracts from transgenic L. minor plants. The level of accumulation of the protein corresponds to 0.4-0.5 µg protein per 1 g of fresh weight of plant. Additionally, the accumulation of recombinant protein was investigated in lyophilized transgenic plants after 1.5 year storage. Duckweed plants accumulating a recombinant analogue of M. tuberculosis secretory proteins can be used for development of plant-based edible vaccines.

  6. Expression and Isolation of Recombinant Microneme 3 (MIC3 Protein of Toxoplasma gondii Local Isolate on Eschericia coli (BL21

    Directory of Open Access Journals (Sweden)

    D Indrasanti

    2011-05-01

    Full Text Available Abstract. Toxoplasmosis is a disease that infects all warm-blooded animals, including livestocks and humans caused by Toxoplasma gondii parasites. There are major drugs used for the therapy, though they have some effects to the patients, such as allergy, toxic and teratogenic for fetus. In addition, toxoplasmosis treatment is only effective for tachyzoites T. gondii in acute infection, while tissue cysts cannot be eradicated in chronic toxoplasmosis Tissue cysts of T. gondii contained in meat that are consumed by humans and meat-derived products may be important sources of infection for humans. Microneme protein (MIC is one of proteins that belongs to excretory-secretory antigens (ESAs of Toxoplasma gondii. Microneme 3 protein (MIC3 is the protein that plays an important role in the invasion process during cell infection as a mediator attachment parasite to the host cell. Recombinant MIC3 protein has been already used for the detection of toxoplasmosis and it could induce humoral and cellular immune response in experimental animals. The aim of this research was to express MIC3 recombinant protein of T. gondii from local isolate that was cloned into expression vector and transformed to E. coli BL21. In the future, recombinant protein MIC3 can be used for vaccine candidate and diagnostic tools for toxoplasmosis in animals and humans. Gene of MIC3 T. gondii local isolate (1.2 Kbp was cloned into expression vector pET-32a(+ (5.9 Kbp and transformed to Escherichia coli BL21. Protein from plasmid recombinant (7.1 Kbp was expressed and performed by culturing recombinant bacteria into LB medium containing ampicillin and IPTG. Recombinant protein was isolated by sonication method and identified using SDS-PAGE. Finally, the recombinant protein was analyzed by immunoblotting using anti-ESAs polyclonal antibody. In conclusion, expression of the MIC3 gene with ~108 kDa has been successfully performed by cloning gene encoding for MIC3 protein of T. gondii

  7. Expression and Isolation of Recombinant Microneme 3 (MIC3 Protein of Toxoplasma gondii Local Isolate on Eschericia coli (BL21

    Directory of Open Access Journals (Sweden)

    D Indrasanti

    2011-05-01

    Full Text Available Toxoplasmosis is a disease that infects all warm-blooded animals, including livestocks and humans caused by Toxoplasma gondii parasites. There are major drugs used for the therapy, though they have some effects to the patients, such as allergy, toxic and teratogenic for fetus. In addition, toxoplasmosis treatment is only effective for tachyzoites T. gondii in acute infection, while tissue cysts cannot be eradicated in chronic toxoplasmosis Tissue cysts of T. gondii contained in meat that are consumed by humans and meat-derived products may be important sources of infection for humans. Microneme protein (MIC is one of proteins that belongs to excretory-secretory antigens (ESAs of Toxoplasma gondii. Microneme 3 protein (MIC3 is the protein that plays an important role in the invasion process during cell infection as a mediator attachment parasite to the host cell. Recombinant MIC3 protein has been already used for the detection of toxoplasmosis and it could induce humoral and cellular immune response in experimental animals. The aim of this research was to express MIC3 recombinant protein of T. gondii from local isolate that was cloned into expression vector and transformed to E. coli BL21. In the future, recombinant protein MIC3 can be used for vaccine candidate and diagnostic tools for toxoplasmosis in animals and humans. Gene of MIC3 T. gondii local isolate (1.2 Kbp was cloned into expression vector pET-32a(+ (5.9 Kbp and transformed to Escherichia coli BL21. Protein from plasmid recombinant (7.1 Kbp was expressed and performed by culturing recombinant bacteria into LB medium containing ampicillin and IPTG. Recombinant protein was isolated by sonication method and identified using SDS-PAGE. Finally, the recombinant protein was analyzed by immunoblotting using anti-ESAs polyclonal antibody. In conclusion, expression of the MIC3 gene with ~108 kDa has been successfully performed by cloning gene encoding for MIC3 protein of T. gondii local isolate

  8. Functional characterization and epitope analysis of a recombinant dermonecrotic protein from Loxosceles intermedia spider.

    Science.gov (United States)

    Felicori, L; Araujo, S C; de Avila, R A Machado; Sanchez, E F; Granier, C; Kalapothakis, E; Chávez-Olórtegui, C

    2006-10-01

    In the present study the recombinant form (recLiD1) of a dermonecrotic protein present in the Brazilian brown spider Loxosceles intermedia venom was expressed in Escherichia coli cells and purified by reversed-phase HPLC using a C8 Vydac column. About 25.8mg of purified recLiD1 was produced from a litre of bacterial culture. SDS/PAGE and immunoblot analysis of the recombinant protein revealed an apparent molecular weight of 32-35kDa. The later result was confirmed by mass spectrometry (32,758Da). recLiD1 displayed dermonecrotic and platelet aggregation activities which were qualitatively similar to that displayed by the crude L. intermedia venom. However, very low sphingomyelinase D enzymatic activity and complement-dependent haemolytic activities were observed. recLiD1 immunized BALB/c mice developed an antibody response. Anti-recLiD1 antibodies recognized L. intermedia venom in an indirect enzyme-linked immunosorbent assay (ELISA) and cross-reacted with crude venoms from L. intermedia, L. gaucho and L. laeta. An in vivo protection assay carried out 5 weeks after the end of the immunization protocol showed that 75% of the vaccinated mice could resist the challenge by 2.5LD(50) of L. intermedia venom. To characterize epitopes associated with protective antibodies, we prepare sets of immobilized synthetic 15 mer overlapping peptides covering the complete amino acid sequences of the recLiD1. Antibodies revealed one antigenic region in the N-terminal part of the toxin. The amino acid sequence of this epitope was found in several dermonecrotic proteins and some of its residues have been implicated with the active site of the toxin.

  9. Expression and In Silico Analysis of the Recombinant Bovine Papillomavirus E6 Protein as a Model for Viral Oncoproteins Studies

    Science.gov (United States)

    Mazzuchelli-de-Souza, J.; Carvalho, R. F.; Ruiz, R. M.; Melo, T. C.; Araldi, R. P.; Carvalho, E.; Thompson, C. E.; Sircili, M. P.; Beçak, W.; Stocco, R. C.

    2013-01-01

    Bovine papillomaviruses (BPVs) are recognized as the causal agents of economical relevant diseases in cattle, associated with the development of tumors in skin and mucosa. The oncogenesis process is mainly associated with different viral oncoprotein expressions, which are involved in cell transformation. The expression and characterization of recombinant viral oncoproteins represent an attractive strategy to obtain biotechnological products as antibodies and potential vaccines, Thus, the aim of this work was to clone and express the BPV-1 and BPV-2 E6 recombinant proteins and perform in silico analysis in order to develop a strategy for the systematic study of other papillomaviruses oncoproteins. The results demonstrated that BPV-1 and BPV-2 E6 recombinant proteins were expressed and purified from bacterial system as well as its in silico analysis was performed in order to explore and predict biological characteristics of these proteins. PMID:23878806

  10. Analysis of recombinant, multivalent dengue virus containing envelope (E proteins from serotypes-1, -3 and -4 and expressed in baculovirus

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    Fedik A. Rantam

    2015-01-01

    Full Text Available Dengue virus has four serotypes that cause a public health problem in Indonesia. Currently, there is no preventative vaccine for this disease, but some model vaccines are in development. The envelop (E protein genes from three isolates of dengue virus (DENV-1, -3 and -4 were isolated, cloned into Escherichia coli and then sub-cloned into a baculovirus vector before co-transfection into Sf9 cells. Recombinant E genes were inserted between the Smal and Sacl sites of the plasmid, adjacent to the baculoviral structural gene, polyhedrin. The sequence of recombinant E gene was relatively stable with 97–98% homology, although there were amino acid substitutions in some regions. The recombinant protein was more antigenic when exposed to polyclonal sera from infected humans than sera from immunized mice, but its binding to monoclonal antibodies IgG1a and IgG2b was stronger than other isotopes, including IgM, IgG and Ig1b. Recombinant E protein induced cellular immune responses in immunized mice, as demonstrated by lymphocyte secretion of IL-3. This study indicates that recombinant E protein expressed in a baculovirus system can induce humoral and cellular immune responses.

  11. Serological Assays Based on Recombinant Viral Proteins for the Diagnosis of Arenavirus Hemorrhagic Fevers

    Directory of Open Access Journals (Sweden)

    Masayuki Saijo

    2012-10-01

    Full Text Available The family Arenaviridae, genus Arenavirus, consists of two phylogenetically independent groups: Old World (OW and New World (NW complexes. The Lassa and Lujo viruses in the OW complex and the Guanarito, Junin, Machupo, Sabia, and Chapare viruses in the NW complex cause viral hemorrhagic fever (VHF in humans, leading to serious public health concerns. These viruses are also considered potential bioterrorism agents. Therefore, it is of great importance to detect these pathogens rapidly and specifically in order to minimize the risk and scale of arenavirus outbreaks. However, these arenaviruses are classified as BSL-4 pathogens, thus making it difficult to develop diagnostic techniques for these virus infections in institutes without BSL-4 facilities. To overcome these difficulties, antibody detection systems in the form of an enzyme-linked immunosorbent assay (ELISA and an indirect immunofluorescence assay were developed using recombinant nucleoproteins (rNPs derived from these viruses. Furthermore, several antigen-detection assays were developed. For example, novel monoclonal antibodies (mAbs to the rNPs of Lassa and Junin viruses were generated. Sandwich antigen-capture (Ag-capture ELISAs using these mAbs as capture antibodies were developed and confirmed to be sensitive and specific for detecting the respective arenavirus NPs. These rNP-based assays were proposed to be useful not only for an etiological diagnosis of VHFs, but also for seroepidemiological studies on VHFs. We recently developed arenavirus neutralization assays using vesicular stomatitis virus (VSV-based pseudotypes bearing arenavirus recombinant glycoproteins. The goal of this article is to review the recent advances in developing laboratory diagnostic assays based on recombinant viral proteins for the diagnosis of VHFs and epidemiological studies on the VHFs caused by arenaviruses.

  12. Evaluation of Cocktails with Recombinant Proteins of Mycobacterium bovis for a Specific Diagnosis of Bovine Tuberculosis

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    María Laura Mon

    2014-01-01

    Full Text Available The Delayed type hypersensitivity skin test (DTH and interferon-gamma assay are used for the diagnosis of bovine tuberculosis (TBB. The specificity of these diagnoses, however, is compromised because both are based on the response against purified protein derivative of Mycobacterium bovis (PPD-B. In this study, we assessed the potential of two cocktails containing M. bovis recombinant proteins: cocktail 1 (C1: ESAT-6, CFP-10 and MPB83 and cocktail 2 (C2: ESAT-6, CFP-10, MPB83, HspX, TB10.3, and MPB70. C1, C2, and PPD-B showed similar response by DTH in M. bovis-sensitized guinea pigs. Importantly, C1 induced a lower response than PPD-B in M. avium-sensitized guinea pigs. In cattle, C1 displayed better performance than PPD-B and C2; indeed, C1 showed the least detection of animals either vaccinated or Map-infected. To optimize the composition of the cocktails, we obtained protein fractions from PPD-B and tested their immunogenicity in experimentally M. bovis-infected cattle. In one highly reactive fraction, seven proteins were identified. The inclusion of FixB in C1 enhanced the recognition of M. bovis-infected cattle without compromising specificity. Our data provide a promising basis for the future development of a cocktail for TBB detection without interference by the presence of sensitized or infected animals with other mycobacteria.

  13. Biological protein-resistance layer construction of recombinant hirudin on polymethyl methacrylate IOL surface.

    Science.gov (United States)

    Zheng, Zhiwen; Jiao, Yan; Ren, Li; Wang, Yingjun

    2015-03-01

    In this article, the surface of intraocular len material PMMA was first aminated for activation on which some polar groups generated such as C-N, COO(-), -OH, NH3(+), etc. Then the anticoagulant drugs recombinant hirudin (rH) was grafted with amido bonds to look forward to resist the adsorption of nonspecific protein or cells in tear, even the cataract. The detailed analysis and discussion about the grafting quantity, molography, wettability, electric charges, chemical structure, and the dynamic adsorption of protein Fn on the material surface were carried on by the technology of ultraviolet photometric, contact angle, solid Zeta potential, X-ray photoelectron spectroscopy, and quartz crystal microbalance. The surface with a certain amount of rH modification existed more hydrophilic due to the amphiphilic structure than before, on which the protein adsorption was the most unstable. The results indicated that the rH modification improved the resistance of PMMA to nonspecific adsorption of protein Fn to achieve the expectative effect. © 2014 Wiley Periodicals, Inc.

  14. 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.

  15. Effective delivery of recombinant proteins to rod photoreceptors via lipid nanovesicles

    Energy Technology Data Exchange (ETDEWEB)

    Asteriti, Sabrina [Dept. of Translational Research, University of Pisa, Pisa (Italy); Dal Cortivo, Giuditta [Dept. of Life Sciences and Reproduction, University of Verona, Strada Le Grazie 8, Verona (Italy); Pontelli, Valeria [Dept. of Neurological and Movement Sciences, University of Verona, Strada Le Grazie 8, Verona (Italy); Cangiano, Lorenzo [Dept. of Translational Research, University of Pisa, Pisa (Italy); Buffelli, Mario, E-mail: mario.buffelli@univr.it [Dept. of Neurological and Movement Sciences, University of Verona, Strada Le Grazie 8, Verona (Italy); Center for Biomedical Computing, University of Verona, Strada le Grazie 8, 37134 Verona (Italy); Dell’Orco, Daniele, E-mail: daniele.dellorco@univr.it [Dept. of Life Sciences and Reproduction, University of Verona, Strada Le Grazie 8, Verona (Italy); Center for Biomedical Computing, University of Verona, Strada le Grazie 8, 37134 Verona (Italy)

    2015-06-12

    The potential of liposomes to deliver functional proteins in retinal photoreceptors and modulate their physiological response was investigated by two experimental approaches. First, we treated isolated mouse retinas with liposomes encapsulating either recoverin, an important endogenous protein operating in visual phototransduction, or antibodies against recoverin. We then intravitrally injected in vivo liposomes encapsulating either rhodamin B or recoverin and we investigated the distribution in retina sections by confocal microscopy. The content of liposomes was found to be released in higher amount in the photoreceptor layer than in the other regions of the retina and the functional effects of the release were in line with the current model of phototransduction. Our study sets the basis for quantitative investigations aimed at assessing the potential of intraocular protein delivery via biocompatible nanovesicles, with promising implications for the treatment of retinal diseases affecting the photoreceptor layer. - Highlights: • Recombinant proteins encapsulated in nano-sized liposomes injected intravitreally reach retinal photoreceptors. • The phototransduction cascade in rods is modulated by the liposome content. • Mathematical modeling predicts the alteration of the photoresponses following liposome fusion.

  16. 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

    Inspired by nature, synthetic mineralizing proteins have been developed to synthesize various structures of silica-based nanomaterials under environmentally friendly conditions. However, the development of bioprocesses able to assist in the translation of these new materials has lagged the development of the materials themselves. The development of cost-effective and scalable bioprocesses which minimize reliance on chromatography to recover biomolecules from microbial cell factories remains a significant challenge. This paper reports a simplified purification process for a recently reported recombinant catalytic modular (D4S2) protein (M(DPSMKQLADS-LHQLARQ-VSRLEHA) 4 EPSRKKRKKRKKRKKGGGY; M 13.3 kDa; pI 10.9), which combines a variant of the established designer biosurfactant protein DAMP4 with a new biomimetic sequence (RKKRKKRKKRKKGGGY), providing for a bi-modular functionality (emulsification and biosilicification). The four-helix bundle structure of the protein has been demonstrated to remain stable and soluble under high temperature and high salt conditions, which confers simplified bioprocessing character. However, the high positive charge on the biosilification sequence necessitates removal of DNA contaminants from crude cell-extract at an early stage in the process by adding poly(ethyleneimine) (PEI). In this process, cellular protein contaminants were selectively precipitated by adding Na 2 SO 4 to the protein mixture up to a high concentration (1 M) and mixed at high temperature (90°C, 5 min) where D4S2 remained stable and soluble due to its four-helix bundle structure. Further increase of the Na 2 SO 4 concentration to 1.8 M precipitated, thus separated, D4S2 from residual PEI. The overall yield of the protein D4S2 was 28.8 mg per 800 mL cells (final cultivation OD 600 ∼2) which gives an approximate 79% D4S2-protein yield. In comparison with the previously reported chromatographic purification of D4S2 protein (Wibowo et al., 2015), the

  17. A lower isoelectric point increases signal sequence-mediated secretion of recombinant proteins through a bacterial ABC transporter.

    Science.gov (United States)

    Byun, Hyunjong; Park, Jiyeon; Kim, Sun Chang; Ahn, Jung Hoon

    2017-12-01

    Efficient protein production for industrial and academic purposes often involves engineering microorganisms to produce and secrete target proteins into the culture. Pseudomonas fluorescens has a TliDEF ATP-binding cassette transporter, a type I secretion system, which recognizes C-terminal LARD3 signal sequence of thermostable lipase TliA. Many proteins are secreted by TliDEF in vivo when recombined with LARD3, but there are still others that cannot be secreted by TliDEF even when LARD3 is attached. However, the factors that determine whether or not a recombinant protein can be secreted through TliDEF are still unknown. Here, we recombined LARD3 with several proteins and examined their secretion through TliDEF. We found that the proteins secreted via LARD3 are highly negatively charged with highly-acidic isoelectric points (pI) lower than 5.5. Attaching oligo-aspartate to lower the pI of negatively-charged recombinant proteins improved their secretion, and attaching oligo-arginine to negatively-charged proteins blocked their secretion by LARD3. In addition, negatively supercharged green fluorescent protein (GFP) showed improved secretion, whereas positively supercharged GFP did not secrete. These results disclosed that proteins' acidic pI and net negative charge are major factors that determine their secretion through TliDEF. Homology modeling for TliDEF revealed that TliD dimer forms evolutionarily-conserved positively-charged clusters in its pore and substrate entrance site, which also partially explains the pI dependence of the TliDEF-dependent secretions. In conclusion, lowering the isoelectric point improved LARD3-mediated protein secretion, both widening the range of protein targets for efficient production via secretion and signifying an important aspect of ABC transporter-mediated secretions. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Population Pharmacokinetic Modelling of FE 999049, a Recombinant Human Follicle-Stimulating Hormone, in Healthy Women After Single Ascending Doses

    OpenAIRE

    Rose, Trine H?yer; R?shammar, Daniel; Erichsen, Lars; Grundemar, Lars; Ottesen, Johnny T.

    2016-01-01

    Objective The purpose of this analysis was to develop a population pharmacokinetic model for a novel recombinant human follicle-stimulating hormone (FSH) (FE 999049) expressed from a human cell line of foetal retinal origin (PER.C6?) developed for controlled ovarian stimulation prior to assisted reproductive technologies. Methods Serum FSH levels were measured following a single subcutaneous FE 999049 injection of 37.5, 75, 150, 225 or 450?IU in 27 pituitary-suppressed healthy female subjects...

  19. Mismatch repair protein hMSH2–hMSH6 recognizes mismatches and forms sliding clamps within a D-loop recombination intermediate

    Science.gov (United States)

    Honda, Masayoshi; Okuno, Yusuke; Hengel, Sarah R.; Martín-López, Juana V.; Cook, Christopher P.; Amunugama, Ravindra; Soukup, Randal J.; Subramanyam, Shyamal; Fishel, Richard; Spies, Maria

    2014-01-01

    High fidelity homologous DNA recombination depends on mismatch repair (MMR), which antagonizes recombination between divergent sequences by rejecting heteroduplex DNA containing excessive nucleotide mismatches. The hMSH2–hMSH6 heterodimer is the first responder in postreplicative MMR and also plays a prominent role in heteroduplex rejection. Whether a similar molecular mechanism underlies its function in these two processes remains enigmatic. We have determined that hMSH2–hMSH6 efficiently recognizes mismatches within a D-loop recombination initiation intermediate. Mismatch recognition by hMSH2–hMSH6 is not abrogated by human replication protein A (HsRPA) bound to the displaced single-stranded DNA (ssDNA) or by HsRAD51. In addition, ATP-bound hMSH2–hMSH6 sliding clamps that are essential for downstream MMR processes are formed and constrained within the heteroduplex region of the D-loop. Moreover, the hMSH2–hMSH6 sliding clamps are stabilized on the D-loop by HsRPA bound to the displaced ssDNA. Our findings reveal similarities and differences in hMSH2–hMSH6 mismatch recognition and sliding-clamp formation between a D-loop recombination intermediate and linear duplex DNA. PMID:24395779

  20. Production of Polyclonal Antibodies to the Recombinant Potato virus M (PVM) Non-structural Triple Gene Block Protein 1 and Coat Protein

    Czech Academy of Sciences Publication Activity Database

    Čeřovská, Noemi; Moravec, Tomáš; Plchová, Helena; Hoffmeisterová, Hana; Dědič, P.

    2012-01-01

    Roč. 160, č. 5 (2012), s. 251-254 ISSN 0931-1785 R&D Projects: GA MŠk 1M06030 Institutional research plan: CEZ:AV0Z50380511 Keywords : Potato virus M * recombinant protein * coat protein Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.000, year: 2012

  1. Utility of P19 Gene-Silencing Suppressor for High Level Expression of Recombinant Human Therapeutic Proteins in Plant Cells

    Directory of Open Access Journals (Sweden)

    Maryam Zangi

    2016-07-01

    Full Text Available Background: The potential of plants, as a safe and eukaryotic system, is considered in the production of recombinant therapeutic human protein today; but the expression level of heterologous proteins is limited by the post-transcriptional gene silencing (PTGS response in this new technology. The use of viral suppressors of gene silencing can prevent PTGS and improve transient expression level of foreign proteins. In this study, we investigated the effect of p19 silencing suppressor on recombinant human nerve growth factor expression in Nicotiana benthamiana. Materials and Methods: The p19 coding region was inserted in the pCAMBIA using NcoI and BstEII recognition sites. Also, the cloned synthesized recombinant human NGF (rhNGF fragment was cloned directly into PVX vector by ClaI and SalI restriction enzymes. The co-agroinfiltration of rhNGF with p19 viral suppressor of gene silencing was evaluated by dot-blot and SDS-PAGE. The amount of expressed rhNGF protein was calculated by AlphaEaseFC software. Results: Co-agroinfiltration of hNGF with P19 suppressor showed about forty-fold increase (8% total soluble protein (TSP when compared to the absence of P19 suppressor (0.2%TSP. Conclusion: The results presented here confirmed that the use of P19 gene silencing suppressor derived from tomato bushy stunt virus (TBSV could efficiently increase the transient expression of recombinant proteins in Nicotiana benthamiana manifold.

  2. Efficient Secretion of Recombinant Proteins from Rice Suspension-Cultured Cells Modulated by the Choice of Signal Peptide

    Science.gov (United States)

    Huang, Li-Fen; Tan, Chia-Chun; Yeh, Ju-Fang; Liu, Hsin-Yi; Liu, Yu-Kuo; Ho, Shin-Lon; Lu, Chung-An

    2015-01-01

    Plant-based expression systems have emerged as a competitive platform in the large-scale production of recombinant proteins. By adding a signal peptide, αAmy3sp, the desired recombinant proteins can be secreted outside transgenic rice cells, making them easy to harvest. In this work, to improve the secretion efficiency of recombinant proteins in rice expression systems, various signal peptides including αAmy3sp, CIN1sp, and 33KDsp have been fused to the N-terminus of green fluorescent protein (GFP) and introduced into rice cells to explore the efficiency of secretion of foreign proteins. 33KDsp had better efficiency than αAmy3sp and CIN1sp for the secretion of GFP from calli and suspension-cultured cells. 33KDsp was further applied for the secretion of mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) from transgenic rice suspension-cultured cells; approximately 76%–92% of total rice-derived mGM-CSF (rmGM-CSF) was detected in the culture medium. The rmGM-CSF was bioactive and could stimulate the proliferation of a murine myeloblastic leukemia cell line, NSF-60. The extracellular yield of rmGM-CSF reached 31.7 mg/L. Our study indicates that 33KDsp is better at promoting the secretion of recombinant proteins in rice suspension-cultured cell systems than the commonly used αAmy3sp. PMID:26473722

  3. Simultaneous Multiplexed Measurement of RNA and Proteins in Single Cells

    Directory of Open Access Journals (Sweden)

    Spyros Darmanis

    2016-01-01

    Full Text Available Significant advances have been made in methods to analyze genomes and transcriptomes of single cells, but to fully define cell states, proteins must also be accessed as central actors defining a cell’s phenotype. Methods currently used to analyze endogenous protein expression in single cells are limited in specificity, throughput, or multiplex capability. Here, we present an approach to simultaneously and specifically interrogate large sets of protein and RNA targets in lysates from individual cells, enabling investigations of cell functions and responses. We applied our method to investigate the effects of BMP4, an experimental therapeutic agent, on early-passage glioblastoma cell cultures. We uncovered significant heterogeneity in responses to treatment at levels of RNA and protein, with a subset of cells reacting in a distinct manner to BMP4. Moreover, we found overall poor correlation between protein and RNA at the level of single cells, with proteins more accurately defining responses to treatment.

  4. Extended gene expression by medium exchange and repeated transient transfection for recombinant protein production enhancement.

    Science.gov (United States)

    Cervera, Laura; Gutiérrez-Granados, Sonia; Berrow, Nicholas Simon; Segura, Maria Mercedes; Gòdia, Francesc

    2015-05-01

    Production of recombinant products in mammalian cell cultures can be achieved by stable gene expression (SGE) or transient gene expression (TGE). The former is based on the integration of a plasmid DNA into the host cell genome allowing continuous gene expression. The latter is based on episomal plasmid DNA expression. Conventional TGE is limited to a short production period of usually about 96 h, therefore limiting productivity. A novel gene expression approach termed extended gene expression (EGE) is explored in this study. The aim of EGE is to prolong the production period by the combination of medium exchange and repeated transfection of cell cultures with plasmid DNA to improve overall protein production. The benefit of this methodology was evaluated for the production of three model recombinant products: intracellular GFP, secreted GFP, and a Gag-GFP virus-like particles (VLPs). Productions were carried out in HEK 293 cell suspension cultures grown in animal-derived component free media using polyethylenimine (PEI) as transfection reagent. Transfections were repeated throughout the production process using different plasmid DNA concentrations, intervals of time, and culture feeding conditions in order to identify the best approach to achieve sustained high-level gene expression. Using this novel EGE strategy, the production period was prolonged between 192 and 240 h with a 4-12-fold increase in production levels, depending on the product type considered. © 2014 Wiley Periodicals, Inc.

  5. Production of functional human insulin-like growth factor binding proteins (IGFBPs) using recombinant expression in HEK293 cells.

    Science.gov (United States)

    Wanscher, Anne Sofie Molsted; Williamson, Michael; Ebersole, Tasja Wainani; Streicher, Werner; Wikström, Mats; Cazzamali, Giuseppe

    2015-04-01

    Insulin-like growth factor binding proteins (IGFBPs) display many functions in humans including regulation of the insulin-like growth factor (IGF) signaling pathway. The various roles of human IGFBPs make them attractive protein candidates in drug discovery. Structural and functional knowledge on human proteins with therapeutic relevance is needed to design and process the next generation of protein therapeutics. In order to conduct structural and functional investigations large quantities of recombinant proteins are needed. However, finding a suitable recombinant production system for proteins such as full-length human IGFBPs, still remains a challenge. Here we present a mammalian HEK293 expression method suitable for over-expression of secretory full-length human IGFBP-1 to -7. Protein purification of full-length human IGFBP-1, -2, -3 and -5 was conducted using a two-step chromatography procedure and the final protein yields were between 1 and 12mg protein per liter culture media. The recombinant IGFBPs contained PTMs and exhibited high-affinity interactions with their natural ligands IGF-1 and IGF-2. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Protein Laboratories in Single Location | Poster

    Science.gov (United States)

    By Andrew Stephen, Timothy Veenstra, and Gordon Whiteley, Guest Writers, and Ken Michaels, Staff Writer The Laboratory of Proteomics and Analytical Technologies (LPAT), Antibody Characterization Laboratory (ACL), and Protein Chemistry Laboratory (PCL), previously located on different floors or in different buildings, are now together on the first floor of C wing in the ATRF.

  7. Kosmotropic anions promote conversion of recombinant prion protein into a PrPSc-like misfolded form.

    Directory of Open Access Journals (Sweden)

    Rodrigo Diaz-Espinoza

    Full Text Available Prions are self-propagating proteins involved in transmissible spongiform encephalopaties in mammals. An aberrant conformation with amyloid-like features of a cell surface protein, termed prion protein (PrP, is thought to be the essential component of the infectious particle, though accessory co-factor molecules such as lipids and nucleotides may be involved. The cellular co-factors and environmental conditions implicated in PrP misfolding are not completely understood. To address this issue, several studies have been done inducing misfolding of recombinant PrP (recPrP into classical amyloid structures using partially denaturing conditions. In this work, we report that misfolding of recPrP into PrP(Sc-like aggregates can be induced by simply incubating the protein in the presence of kosmotropic salts at concentrations that are known to retain or increase the stability of the protein. We used a simple experimental reaction (protein, buffer and salts submitted to agitation/incubation cycles at physiological temperature and pH. The formation of protease resistant-recPrP was time and salt-concentration dependent and required the presence of kosmotropic anions such as F(- or SO(4(-2. The molecular weights of the protease resistant recPrP fragments are reminiscent of those found in degradation assays of bona fide PrP(Sc. The aggregates also exhibited PrP(Sc-like ultrastructural features including rod-shape morphology under electron microscope, high beta-sheet content and thioflavin-T positive signal. The formation of recPrP aggregates with PrP(Sc biochemical features under conditions closer to physiological in the absence of organic co-factor molecules provides a simple setup that may prove helpful to understand the molecular mechanism of PrP misfolding.

  8. Kosmotropic anions promote conversion of recombinant prion protein into a PrPSc-like misfolded form.

    Science.gov (United States)

    Diaz-Espinoza, Rodrigo; Mukherjee, Abhisek; Soto, Claudio

    2012-01-01

    Prions are self-propagating proteins involved in transmissible spongiform encephalopaties in mammals. An aberrant conformation with amyloid-like features of a cell surface protein, termed prion protein (PrP), is thought to be the essential component of the infectious particle, though accessory co-factor molecules such as lipids and nucleotides may be involved. The cellular co-factors and environmental conditions implicated in PrP misfolding are not completely understood. To address this issue, several studies have been done inducing misfolding of recombinant PrP (recPrP) into classical amyloid structures using partially denaturing conditions. In this work, we report that misfolding of recPrP into PrP(Sc)-like aggregates can be induced by simply incubating the protein in the presence of kosmotropic salts at concentrations that are known to retain or increase the stability of the protein. We used a simple experimental reaction (protein, buffer and salts) submitted to agitation/incubation cycles at physiological temperature and pH. The formation of protease resistant-recPrP was time and salt-concentration dependent and required the presence of kosmotropic anions such as F(-) or SO(4)(-2). The molecular weights of the protease resistant recPrP fragments are reminiscent of those found in degradation assays of bona fide PrP(Sc). The aggregates also exhibited PrP(Sc)-like ultrastructural features including rod-shape morphology under electron microscope, high beta-sheet content and thioflavin-T positive signal. The formation of recPrP aggregates with PrP(Sc) biochemical features under conditions closer to physiological in the absence of organic co-factor molecules provides a simple setup that may prove helpful to understand the molecular mechanism of PrP misfolding.

  9. Lifecycle management for recombinant protein production using mammalian cell culture technology.

    Science.gov (United States)

    Moran, Enda; Gammell, Patrick

    2014-01-01

    Product lifecycle management refers to the oversight process and activities carried out to fully realize the commercial potential and value of a product in the marketplace. It is typical for many changes to be introduced to the production processes and testing methods for biopharmaceutical drugs over their lifetime in the commercial marketplace. Technology lifecycle management, as discussed here, refers to the management of the different phases or generations of processes and methods used to make and test the active biopharmaceutical ingredient or drug product, and the adoption of different devices used to present the drug product to patients. The factors to consider when making changes to a commercial biopharmaceutical manufacturing process as part of a technology lifecycle management program are discussed. A case study outlines one approach taken in bringing forward a major process change to a cell culture process for the production of a therapeutic recombinant protein.

  10. Simple control of fed-batch processes for recombinant protein production with E. coli.

    Science.gov (United States)

    Schaepe, Sebastian; Kuprijanov, Artur; Aehle, Mathias; Simutis, Rimvydas; Lübbert, Andreas

    2011-09-01

    A very simple but effective process control technique is proposed that leads to a high batch-to-batch reproducibility with respect to biomass concentration as well as the specific biomass growth rate profiles in E. coli fermentations performed during recombinant protein production. It makes use of the well-established temperature controllers in currently used fermenters, but takes its information from the difference between the controlled culture temperature T (cult) and the temperature T (coolin) of the coolant fed to the fermenter's cooling jacket as adjusted by the fermenter temperature controller. For process control purposes this measured difference is corrected regarding stirrer influences and cumulated before it is used as a new process control variable. As a spin-off of this control, it becomes possible to estimate online the oxygen mass transfer rates and the corresponding k(L)a values during the real cultivation process. © Springer Science+Business Media B.V. 2011

  11. Advances in animal cell recombinant protein production: GS-NS0 expression system.

    Science.gov (United States)

    Barnes, L M; Bentley, C M; Dickson, A J

    2000-02-01

    The production of recombinant proteins using mammalian cell expression systems is of growing importance within biotechnology, largely due to the ability of specific mammalian cells to carry out post-translational modifications of the correct fidelity. The Glutamine Synthetase-NS0 system is now one such industrially important expression system.Glutamine synthetase catalyses the formation ofglutamine from glutamate and ammonia. NS0 cellscontain extremely low levels of endogenous glutaminesynthetase activity, therefore exogenous glutaminesynthetase can be used efficiently as a selectablemarker to identify successful transfectants in theabsence of glutamine in the media. In addition, theinclusion of methionine sulphoximine, an inhibitor ofglutamine synthetase activity, enables furtherselection of those clones producing relatively highlevels of transfected glutamine synthetase and henceany heterologous gene which is coupled to it. Theglutamine synthetase system technology has been usedfor research and development purposes during thisdecade and its importance is clearly demonstrated nowthat two therapeutic products produced using thissystem have reached the market place.

  12. Induction of Mucosal and Systemic Immunity to a Recombinant Simian Immunodeficiency Viral Protein

    Science.gov (United States)

    Lehner, T.; Bergmeier, L. A.; Panagiotidi, C.; Tao, L.; Brookes, R.; Klavinskis, L. S.; Walker, P.; Walker, J.; Ward, R. G.; Hussain, L.; Gearing, A. J. H.; Adams, S. E.

    1992-11-01

    Heterosexual transmission through the cervico-vaginal mucosa is the principal route of human immunodeficiency virus (HIV) infection in Africa and is increasing in the United States and Europe. Vaginal immunization with simian immunodeficiency virus (SIV) had not yet been studied in nonhuman primates. Immune responses in macaques were investigated by stimulation of the genital and gut-associated lymphoid tissue with a recombinant, particulate SIV antigen. Vaginal, followed by oral, administration of the vaccine elicited three types of immunity: (i) gag protein p27-specific, secretory immunoglobulin A (IgA) and immunoglobulin G (IgG) in the vaginal fluid, (ii) specific CD4^+ T cell proliferation and helper function in B cell p27-specific IgA synthesis in the genital lymph nodes, and (iii) specific serum IgA and IgG, with CD4^+ T cell proliferative and helper functions in the circulating blood.

  13. Assessment of the antiviral properties of recombinant surfactant protein D against influenza B virus in vitro.

    Science.gov (United States)

    Hillaire, Marine L B; van Eijk, Martin; Vogelzang-van Trierum, Stella E; Nieuwkoop, Nella J; van Riel, Debby; Fouchier, Ron A M; Kuiken, Thijs; Osterhaus, Albert D M E; Haagsman, Henk P; Rimmelzwaan, Guus F

    2015-01-02

    The armamentarium of antiviral drugs against influenza viruses is limited. Furthermore, influenza viruses emerge that are resistant to existing antiviral drugs like the M2 and NA inhibitors. Therefore, there is an urgent need for the development of novel classes of antiviral drugs. Here we investigated the antiviral properties of recombinant porcine surfactant protein D (RpSP-D), an innate defense molecule with lectin properties, against influenza B viruses. We have previously shown that porcine SP-D has more potent neutralizing activity against influenza A viruses than human SP-D. Here we show that RpSP-D neutralizes influenza B viruses efficiently and inhibited the binding of these viruses to epithelial cells of the human trachea. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Blocking ELISA using recombinant NcSRS2 protein for diagnosing bovine neosporosis.

    Science.gov (United States)

    Sinnott, Francine A; Monte, Leonardo G; Collares, Thais F; De Matos, Bruno M; Pacheco, Diene B; Borsuk, Sibele; Andreotti, Renato; Hartleben, Cláudia P

    2015-03-01

    Neospora caninum is the etiologic agent of neosporosis, which leads to economic impacts on cattle industry. The reference method for serodiagnosis of neosporosis is the indirect fluorescent antibody test (IFAT). However, IFAT is laborious, expensive, and is not practicable in high throughput screening. In order to facilitate the serological diagnosis of neosporosis, we developed a blocking enzyme-linked immunosorbent assay (b-ELISA) based on NcSRS2 recombinant protein (rNcSRS2) and polyclonal antibodies against rNcSRS2 (b-ELISA/rNcSRS2). Compared to IFAT, b-ELISA/rNcSRS2 showed 93.7 % accuracy (98.7 % sensitivity and 88.7 % specificity), suggesting its potential as diagnostic assay to detect N. caninum antibodies in cattle sera.

  15. 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.

  16. A neutralizing recombinant single chain antibody, scFv, against BaP1, A P-I hemorrhagic metalloproteinase from Bothrops asper snake venom.

    Science.gov (United States)

    Castro, J M A; Oliveira, T S; Silveira, C R F; Caporrino, M C; Rodriguez, D; Moura-da-Silva, A M; Ramos, O H P; Rucavado, A; Gutiérrez, J M; Magalhães, G S; Faquim-Mauro, E L; Fernandes, I

    2014-09-01

    BaP1 is a P-I class snake venom metalloproteinase (SVMP) relevant in the local tissue damage associated with envenomings by Bothrops asper, a medically important snake species in Central America and parts of South and North America. The main treatment for these accidents is the passive immunotherapy using antibodies raised in horses. In order to obtain more specific and batch-to-batch consistent antivenons, recombinant antibodies are considered a good option compared to animal immunization. We constructed a recombinant single chain variable fragment (scFv) from a monoclonal antibody against BaP1 (MABaP1) formerly secreted by a hybridoma clone. This recombinant antibody was cloned into pMST3 vector in fusion with SUMO protein and contains VH and VL domains linked by a flexible (G4S)3 polypeptide (scFvBaP1). The aim of this work was to produce scFvBaP1 and to evaluate its potential concerning the neutralization of biologically important activities of BaP1. The cytoplasmic expression of this construct was successfully achieved in C43 (DE3) bacteria. Our results showed that scFvBaP1-SUMO fusion protein presented an electrophoretic band of around 43 kDa from which SUMO alone corresponded to 13.6 kDa, and only the scFv was able to recognize BaP1 as well as the whole venom by ELISA. In contrast, neither an irrelevant scFv anti-LDL nor its MoAb partner recognized it. BaP1-induced fibrinolysis was significantly neutralized by scFvBaP1, but not by SUMO, in a concentration-dependent manner. In addition, scFvBaP1, as well as MaBaP1, completely neutralized in vivo hemorrhage, muscle necrosis, and inflammation induced by the toxin. Docking analyses revealed possible modes of interaction of the recombinant antibody with BaP1. Our data showed that scFv recognized BaP1 and whole B. asper venom, and neutralized biological effects of this SVMP. This scFv antibody can be used for understanding the molecular mechanisms of neutralization of SVMPs, and for exploring the potential of

  17. Gamete Therapeutics: Recombinant Protein Adsorption by Sperm for Increasing Fertility via Artificial Insemination

    Science.gov (United States)

    Alvarez-Gallardo, Horacio; Kjelland, Michael E.; Moreno, Juan F.; Welsh, Thomas H.; Randel, Ronald D.; Lammoglia, Miguel A.; Pérez-Martínez, Mario; Lara-Sagahón, Alma V.; Esperón-Sumano, A. Enrique; Romo, Salvador

    2013-01-01

    A decrease in fertility can have a negative economic impact, both locally and over a broader geographical scope, and this is especially the case with regard to the cattle industry. Therefore, much interest exists in evaluating proteins that might be able to increase the fertility of sperm. Heparin binding proteins (HBPs), specifically the fertility associated antigen (FAA) and the Type-2 tissue inhibitor of metalloproteinase (TIMP-2), act to favor the capacitation and acrosome reaction and perhaps even modulate the immune system’s response toward the sperm. The objective of this research was to determine the effect on fertility of adding recombinant FAA (rFAA) and recombinant TIMP-2 (rTIMP-2) to bovine semen before cryopreservation for use in an artificial insemination (AI) program in a tropical environment. For this experiment, 100 crossbred (Bos taurus x Bos indicus) heifers were selected based on their estrus cycle, body condition score (BCS), of 4 to 6 on a scale of 1 to 9, and adequate anatomical conformation evaluated by pelvic and genital (normal) measurements. Heifers were synchronized using estradiol benzoate (EB), Celosil® (PGF2α) (Shering-Plough) and a controlled internal drug release (CIDR) device was inserted that contained progesterone. Inseminations were performed in two groups at random, 50 animals per group. The control group was inseminated with conventional semen. The treatment group was inseminated with semen containing rFAA (25 µg/mL) and rTIMP-2 (25 µg/mL). In the control group a 16% pregnancy rate was obtained versus a 40% pregnancy rate for the HBP treatment group, resulting in a significant difference (P = 0.0037). Given the results herein, one may conclude that the HBPs can increase fertility and could be an option for cattle in tropical conditions; however, one needs to consider the environment, nutrition, and the genetic interaction affecting the final result in whatever reproductive program that is implemented. PMID:23762288

  18. Gamete therapeutics: recombinant protein adsorption by sperm for increasing fertility via artificial insemination.

    Directory of Open Access Journals (Sweden)

    Horacio Alvarez-Gallardo

    Full Text Available A decrease in fertility can have a negative economic impact, both locally and over a broader geographical scope, and this is especially the case with regard to the cattle industry. Therefore, much interest exists in evaluating proteins that might be able to increase the fertility of sperm. Heparin binding proteins (HBPs, specifically the fertility associated antigen (FAA and the Type-2 tissue inhibitor of metalloproteinase (TIMP-2, act to favor the capacitation and acrosome reaction and perhaps even modulate the immune system's response toward the sperm. The objective of this research was to determine the effect on fertility of adding recombinant FAA (rFAA and recombinant TIMP-2 (rTIMP-2 to bovine semen before cryopreservation for use in an artificial insemination (AI program in a tropical environment. For this experiment, 100 crossbred (Bos taurus x Bos indicus heifers were selected based on their estrus cycle, body condition score (BCS, of 4 to 6 on a scale of 1 to 9, and adequate anatomical conformation evaluated by pelvic and genital (normal measurements. Heifers were synchronized using estradiol benzoate (EB, Celosil® (PGF2α (Shering-Plough and a controlled internal drug release (CIDR device was inserted that contained progesterone. Inseminations were performed in two groups at random, 50 animals per group. The control group was inseminated with conventional semen. The treatment group was inseminated with semen containing rFAA (25 µg/mL and rTIMP-2 (25 µg/mL. In the control group a 16% pregnancy rate was obtained versus a 40% pregnancy rate for the HBP treatment group, resulting in a significant difference (P = 0.0037. Given the results herein, one may conclude that the HBPs can increase fertility and could be an option for cattle in tropical conditions; however, one needs to consider the environment, nutrition, and the genetic interaction affecting the final result in whatever reproductive program that is implemented.

  19. Identification, Characterization and Down-Regulation of Cysteine Protease Genes in Tobacco for Use in Recombinant Protein Production.

    Directory of Open Access Journals (Sweden)

    Kishor Duwadi

    Full Text Available Plants are an attractive host system for pharmaceutical protein production. Many therapeutic proteins have been produced and scaled up in plants at a low cost compared to the conventional microbial and animal-based systems. The main technical challenge during this process is to produce sufficient levels of recombinant proteins in plants. Low yield is generally caused by proteolytic degradation during expression and downstream processing of recombinant proteins. The yield of human therapeutic interleukin (IL-10 produced in transgenic tobacco leaves was found to be below the critical level, and may be due to degradation by tobacco proteases. Here, we identified a total of 60 putative cysteine protease genes (CysP in tobacco. Based on their predicted expression in leaf tissue, 10 candidate CysPs (CysP1-CysP10 were selected for further characterization. The effect of CysP gene silencing on IL-10 accumulation was examined in tobacco. It was found that the recombinant protein yield in tobacco could be increased by silencing CysP6. Transient expression of CysP6 silencing construct also showed an increase in IL-10 accumulation in comparison to the control. Moreover, CysP6 localizes to the endoplasmic reticulum (ER, suggesting that ER may be the site of IL-10 degradation. Overall results suggest that CysP6 is important in determining the yield of recombinant IL-10 in tobacco leaves.

  20. Purification and Refolding to Amyloid Fibrils of (His)6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli.

    Science.gov (United States)

    Li, Qiaojing; Richard, Charles-Adrien; Moudjou, Mohammed; Vidic, Jasmina

    2015-12-19

    The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein belonging to the prion family. A chromatographic method for the purification of (His)6-tagged recombinant Shadoo expressed as inclusion bodies is described. The inclusion bodies are solubilized in 8 M urea and bound to a Ni(2+)-charged column to perform ion affinity chromatography. Bound proteins are eluted by a gradient of imidazole. Fractions containing Shadoo protein are subjected to size exclusion chromatography to obtain a highly purified protein. In the final step purified Shadoo is desalted to remove salts, urea and imidazole. Recombinant Shadoo protein is an important reagent for biophysical and biochemical studies of protein conformation disorders occurring in prion diseases. Many reports demonstrated that prion neurodegenerative diseases originate from the deposition of stable, ordered amyloid fibrils. Sample protocols describing how to fibrillate Shadoo into amyloid fibrils at acidic and neutral/basic pHs are presented. The methods on how to produce and fibrillate Shadoo can facilitate research in laboratories working on prion diseases, since it allows for production of large amounts of protein in a rapid and low cost manner.

  1. Expression and Purification of Recombinant Proteins in Escherichia coli with a His6or Dual His6-MBP Tag.

    Science.gov (United States)

    Raran-Kurussi, Sreejith; Waugh, David S

    2017-01-01

    Rapid advances in bioengineering and biotechnology over the past three decades have greatly facilitated the production of recombinant proteins in Escherichia coli. Affinity-based methods that employ protein or peptide based tags for protein purification have been instrumental in this progress. Yet insolubility of recombinant proteins in E. coli remains a persistent problem. One way around this problem is to fuse an aggregation-prone protein to a highly soluble partner. E. coli maltose-binding protein (MBP) is widely acknowledged as a highly effective solubilizing agent. In this chapter, we describe how to construct either a His 6 - or a dual His 6 -MBP tagged fusion protein by Gateway ® recombinational cloning and how to evaluate their yield and solubility. We also describe a simple and rapid procedure to test the solubility of proteins after removing their N-terminal fusion tags by tobacco etch virus (TEV) protease digestion. The choice of whether to use a His 6 tag or a His 6 -MBP tag can be made on the basis of this solubility test.

  2. Avian embryos and related cell lines: A convenient platform for recombinant proteins and vaccine production.

    Science.gov (United States)

    Farzaneh, Maryam; Hassani, Seyedeh-Nafiseh; Mozdziak, Paul; Baharvand, Hossein

    2017-05-01

    Chick embryos are a significant historical research model in basic and applied sciences. The embryonated eggs have been used for virus inoculation in order to vaccine production for nearly a century. Recently, avian eggs and cell lines derived from embryonated eggs have found wide application in biotechnology. This review will discuss about the unique characteristics of avian eggs in terms of safety, large scale and economical production of recombinant proteins. This system also provides the human-like glycosylation on target proteins and therefore can be considered as a suitable host for biomanufacturing of humanized monoclonal antibodies and therapeutic proteins. Avian derived cell lines are an alternative for rapid vaccine manufacturing during a pandemic. Based on the latest knowledge in cell and animal transgenesis, the currently available germ cell-mediated gene transfer system provides a more efficient strategy in gene targeting and creation of transgenic birds that lead to advancements in industrial, biotechnology, and biological research applications. This review covers the recent development of avian fertilized eggs and related cell lines in a variety of human biopharmaceuticals and viral vaccine manufacturing. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Surface Plasmon Resonance Investigations of Bioselective Element Based on the Recombinant Protein A for Immunoglobulin Detection

    Science.gov (United States)

    Bakhmachuk, A.; Gorbatiuk, O.; Rachkov, A.; Dons'koi, B.; Khristosenko, R.; Ushenin, I.; Peshkova, V.; Soldatkin, A.

    2017-02-01

    The developed surface plasmon resonance (SPR) biosensor based on the recombinant Staphylococcal protein A with an additional cysteine residue (SPA-Cys) used as a biorecognition component showed a good selectivity and sensitivity for the immunoglobulin detection. The developed biosensor with SPA-Cys-based bioselective element can also be used as a first step of immunosensor creation. The successful immobilization of SPA-Cys on the nanolayer gold sensor surface of the SPR spectrometer was performed. The efficiency of blocking nonspecific sorption sites on the sensor surface with milk proteins, gelatin, BSA, and HSA was studied, and a rather high efficiency of using gelatin was confirmed. The SPR biosensor selectively interacted with IgG and did not interact with the control proteins. The linear dependence of the sensor response on the IgG concentration in the range from 2 to 10 μg/ml was shown. Using the calibration curve, the IgG concentration was measured in the model samples. The determined concentrations are in good agreement ( r 2 = 0.97) with the given concentration of IgG.

  4. Production and characterization of a monoclonal antibody against recombinant fatty acid binding protein of Fasciola gigantica.

    Science.gov (United States)

    Sirisriro, A; Grams, R; Vichasri-Grams, S; Ardseungneon, P; Pankao, V; Meepool, A; Chaithirayanon, K; Viyanant, V; Tan-Ariya, P; Upatham, E S; Sobhon, P

    2002-04-30

    In Fasciola parasites fatty acid binding proteins (FABPs) are the carrier proteins that help in the uptake of fatty acids from the hosts' fluids. Attempts have been made to utilize both native and recombinant FABP (rFABP) for immunodiagnosis and vaccine development for fasciolosis. In this study, we have produced a number of monoclonal antibodies (MoAbs) against rFABP of Fasciola gigantica. These MoAbs were initially screened against rFABP by ELISA and then tested for their specificities by immunoblotting. Five stable clones were selected and characterized further: four of them were of the isotype IgG(1) while one clone was IgG(2a). All the MoAbs reacted with rFABP which has a molecular weight (MW) of 20 kD and with at least two isoforms of native proteins at MW 14.5 kD that were present in the tegumental antigen (TA) and crude worm extracts, and the excretion-secretion materials. Immunoperoxidase staining of frozen sections of adult parasites by using these MoAbs as primary antibodies indicated that FABP were present in high concentration in the parenchymal cells and reproductive tissues, in low concentration in the tegument and caecal epithelium. All MoAbs cross-reacted with a 14.5 kD antigen present in the whole body (WB) extract of Schistosoma mansoni, while no cross-reactivities were detected with antigens from Eurytrema pancreaticum and Paramphistomum spp.

  5. [Evaluation of the Recombinant Protein Tp0965 of Treponema Pallidum as Perspective Antigen for the Improved Serological Diagnosis of Syphilis].

    Science.gov (United States)

    Runina, A V; Starovoitova, A S; Deryabin, D G; Kubanov, A A

    2016-01-01

    BACKGRAUND. Treponemal tests based on the detection of antibodies against the Treponema pallidum antigens are the most specific methods for serological diagnosis of syphilis. Due to the inability to cultivate this bacterium in vitro, the most promising sources of antigens for diagnostics are recombinant proteins of T. pallidum. Evaluation of the analytical value of certain T. pallidum proteins is the approach to improve sensitivity, specificity, and reproducibility of syphilis serological tests, including possibilities of differential diagnosis of various forms of the disease. The aim of the research was to evaluate the analytical values (sensitivity and specificity) of recombinant protein Tp0965 of T. pallidum as a candidate antigen for serological diagnosis of syphilis. tp0965 gene was cloned into the expression vector pET28a and the construct was used for the transformation of E. coli BL-21 (DE3) cells and further expression and purification of the recombinant protein. The collected protein was used as T. pallidum antigen for serum analysis (ELISA) of groups of patients with various forms of syphilis (n=84) and the group of healthy donors (n = 25). High frequency of positive ELISA results was shown with serum of patients with syphilis, compared to the group of healthy donors. The sensitivity of serological reactions using recombinant protein Tp0965 was 98.8%, specificity--87.5%. The highest sensitivity (100%) was detected in the groups of patients with primary, secondary and early latent syphilis while in the group of patients with late latent syphilis it decreased to 95.2%. We concluded that due to its specificity T. pallidum recombinant protein Tp0965 can be used as a novel perspective antigen for development of syphilis serological diagnostic assays (for primary and early latent forms).

  6. A strategy for targeting recombinant proteins to protein storage vacuoles by fusion to Brassica napus napin in napin-depleted seeds.

    Science.gov (United States)

    Hegedus, Dwayne D; Baron, Marcus; Labbe, Natalie; Coutu, Cathy; Lydiate, Derek; Lui, Helen; Rozwadowski, Kevin

    2014-03-01

    Seeds are capable of accumulating high levels of seed storage proteins (SSP), as well as heterologous proteins under certain conditions. Arabidopsis thaliana was used to develop a strategy to deplete seeds of an endogenous SSP and then replenish them with the same protein fused to a heterologous protein. In several other studies, competition with endogenous SSP for space and metabolic resources was shown to affect the accumulation of recombinant proteins in seeds. We used RNAi to reduce the expression of the five napin genes and deplete the seeds of this SSP. Targeting a recombinant protein to a vacuole or structure within the seed where it can be protected from cytosolic proteases can also promote its accumulation. To achieve this, a synthetic Brassica napus napin gene (Bn napin) was designed that was both impervious to the A. thaliana napin (At napin) RNAi construct and permitted fusion to a heterologous protein, in this case green fluorescent protein (GFP). GFP was placed in several strategic locations within Bn napin with consideration to maintaining structure, processing sites and possible vacuolar targeting signals. In transgenic A. thaliana plants, GFP was strongly localized to the seed protein storage vacuole in all Bn napin fusion configurations tested, but not when expressed alone. This SSP depletion-replenishment strategy outlined here would be applicable to expression of recombinant proteins in industrial crops that generally have large repertoires of endogenous SSP genes. Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.

  7. Hexahistidine-tagged maltose-binding protein as a fusion partner for the production of soluble recombinant proteins in Escherichia coli.

    Science.gov (United States)

    Austin, Brian P; Nallamsetty, Sreedevi; Waugh, David S

    2009-01-01

    Insolubility of recombinant proteins in Escherichia coli is a major impediment to their production for structural and functional studies. One way to circumvent this problem is to fuse an aggregation-prone protein to a highly soluble partner. E. coli maltose-binding protein (MBP) has emerged as one of the most effective solubilizing agents. In this chapter, we describe how to construct combinatorially-tagged His(6)MBP fusion proteins by recombinational cloning and how to evaluate their yield and solubility. We also describe a procedure to determine how efficiently a His(6)MBP fusion protein is cleaved by tobacco etch virus (TEV) protease in E. coli and a method to assess the solubility of the target protein after it has been separated from His(6)MBP.

  8. Processing of recombinant Listeria monocytogenes proteins for MHC class I presentation follows a dedicated, high-efficiency pathway

    Science.gov (United States)

    Wolf, Benjamin J.; Princiotta, Michael F.

    2013-01-01

    CD8+ T lymphocytes recognize short peptides of ~8–10 amino acids bound to MHC class I molecules (pMHC) on the surface of antigen presenting cells. These peptides can be generated from either endogenous proteins synthesized by the biosynthetic machinery of the presenting cell or from exogenously sourced proteins. Because much of the research characterizing the MHC class I processing pathway has focused on endogenously synthesized proteins, it is not known whether differences exist in the processing pathway followed by endogenously synthesized versus exogenously sourced proteins. To highlight potential differences in the processing of endogenous versus exogenous proteins, we developed a model system to measure the efficiency of pMHC generation from nearly identical recombinant proteins expressed from vaccinia virus and Listeria monocytogenes. In these experiments, we uncovered a striking difference in the way recombinant Listeria antigens are processed and presented when compared to endogenously synthesized viral proteins. Specifically, we find that pMHC production from secreted Listeria proteins occurs at the same rate, independent of the cellular half-life of the protein from which it is derived, whereas the rate of pMHC production from endogenously synthesized viral proteins is absolutely dependent on its protein half-life. Accordingly, our data demonstrate the existence of a distinct and highly efficient MHC class I presentation pathway used for the processing of at least some exogenously synthesized proteins. PMID:23396941

  9. Single channel analysis of membrane proteins in artificial bilayer membranes.

    Science.gov (United States)

    Bartsch, Philipp; Harsman, Anke; Wagner, Richard

    2013-01-01

    The planar lipid bilayer technique is a powerful experimental approach for electrical single channel recordings of pore-forming membrane proteins in a chemically well-defined and easily modifiable environment. Here we provide a general survey of the basic materials and procedures required to set up a robust bilayer system and perform electrophysiological single channel recordings of reconstituted proteins suitable for the in-depth characterization of their functional properties.

  10. Recombinant Salivary Proteins of Phlebotomus orientalis are Suitable Antigens to Measure Exposure of Domestic Animals to Sand Fly Bites.

    Directory of Open Access Journals (Sweden)

    Michal Sima

    2016-03-01

    Full Text Available Certain salivary proteins of phlebotomine sand flies injected into the host skin during blood-feeding are highly antigenic and elicit strong antibody-mediated immune responses in repeatedly-exposed hosts. These antibodies can be measured by enzyme-linked immuno sorbent assays (ELISAs using salivary gland homogenates (SGHs as the source of antigens and serve as a markers for exposure to biting sand flies. Large-scale screening for anti-sand fly saliva antibodies requires replacement of SGH with recombinant salivary proteins. In East Africa, Phlebotomus orientalis is the main vector of Leishmania donovani, a trypanosomatid parasite causing visceral leishmaniasis. We tested recombinant salivary proteins derived from Ph. orientalis saliva to study exposure of domestic animals to this sand fly species.Antigenic salivary proteins from Ph. orientalis were identified by immunoblot and mass spectrometry. Recombinant apyrase rPorSP15, yellow-related protein rPorSP24, ParSP25-like protein rPorSP65, D7-related protein rPorSP67, and antigen 5-related protein rPorSP76 were tested using ELISA with sera of domestic animals from L. donovani foci in Ethiopia where Ph. orientalis is present. Our results highlighted recombinant yellow-related protein rPorSP24 as the most promising antigen, displaying a high positive correlation coefficient as well as good sensitivity and specificity when compared to SGH. This recombinant protein was the most suitable one for testing sera of dogs, sheep, and goats. In addition, a different antigen, rPorSP65 was found efficacious for testing canine sera.Recombinant salivary proteins of Ph. orientalis, specifically rPorSP24, were shown to successfully substitute SGH in serological experiments to measure exposure of domestic animals to Ph. orientalis, the vector of L. donovani. The results suggest that rPorSP24 might be a suitable antigen for detecting anti-Ph. orientalis antibody-mediated reactions also in other host species.

  11. Recombinant Salivary Proteins of Phlebotomus orientalis are Suitable Antigens to Measure Exposure of Domestic Animals to Sand Fly Bites.

    Science.gov (United States)

    Sima, Michal; Ferencova, Blanka; Warburg, Alon; Rohousova, Iva; Volf, Petr

    2016-03-01

    Certain salivary proteins of phlebotomine sand flies injected into the host skin during blood-feeding are highly antigenic and elicit strong antibody-mediated immune responses in repeatedly-exposed hosts. These antibodies can be measured by enzyme-linked immuno sorbent assays (ELISAs) using salivary gland homogenates (SGHs) as the source of antigens and serve as a markers for exposure to biting sand flies. Large-scale screening for anti-sand fly saliva antibodies requires replacement of SGH with recombinant salivary proteins. In East Africa, Phlebotomus orientalis is the main vector of Leishmania donovani, a trypanosomatid parasite causing visceral leishmaniasis. We tested recombinant salivary proteins derived from Ph. orientalis saliva to study exposure of domestic animals to this sand fly species. Antigenic salivary proteins from Ph. orientalis were identified by immunoblot and mass spectrometry. Recombinant apyrase rPorSP15, yellow-related protein rPorSP24, ParSP25-like protein rPorSP65, D7-related protein rPorSP67, and antigen 5-related protein rPorSP76 were tested using ELISA with sera of domestic animals from L. donovani foci in Ethiopia where Ph. orientalis is present. Our results highlighted recombinant yellow-related protein rPorSP24 as the most promising antigen, displaying a high positive correlation coefficient as well as good sensitivity and specificity when compared to SGH. This recombinant protein was the most suitable one for testing sera of dogs, sheep, and goats. In addition, a different antigen, rPorSP65 was found efficacious for testing canine sera. Recombinant salivary proteins of Ph. orientalis, specifically rPorSP24, were shown to successfully substitute SGH in serological experiments to measure exposure of domestic animals to Ph. orientalis, the vector of L. donovani. The results suggest that rPorSP24 might be a suitable antigen for detecting anti-Ph. orientalis antibody-mediated reactions also in other host species.

  12. Comparative assessment of ELISAs using recombinant saposin-like protein 2 and recombinant cathepsin L-1 from Fasciola hepatica for the serodiagnosis of human Fasciolosis.

    Directory of Open Access Journals (Sweden)

    Bruno Gottstein

    2014-06-01

    Full Text Available Two recombinant Fasciola hepatica antigens, saposin-like protein-2 (recSAP2 and cathepsin L-1 (recCL1, were assessed individually and in combination in enzyme-linked immunosorbent assays (ELISA for the specific serodiagnosis of human fasciolosis in areas of low endemicity as encountered in Central Europe. Antibody detection was conducted using ProteinA/ProteinG (PAG conjugated to alkaline phosphatase. Test characteristics as well as agreement with results from an ELISA using excretory-secretory products (FhES from adult stage liver flukes was assessed by receiver operator characteristic (ROC analysis, specificity, sensitivity, Youdens J and overall accuracy. Cross-reactivity was assessed using three different groups of serum samples from healthy individuals (n=20, patients with other parasitic infections (n=87 and patients with malignancies (n=121. The best combined diagnostic results for recombinant antigens were obtained using the recSAP2-ELISA (87% sensitivity, 99% specificity and 97% overall accuracy employing the threshold (cut-off to discriminate between positive and negative reactions that maximized Youdens J. The findings showed that recSAP2-ELISA can be used for the routine serodiagnosis of chronic fasciolosis in clinical laboratories; the use of the PAG-conjugate offers the opportunity to employ, for example, rabbit hyperimmune serum for the standardization of positive controls.

  13. Comparative Assessment of ELISAs Using Recombinant Saposin-Like Protein 2 and recombinant Cathepsin L-1 from Fasciola hepatica for the Serodiagnosis of Human Fasciolosis

    Science.gov (United States)

    Gottstein, Bruno; Schneeberger, Marianne; Boubaker, Ghalia; Merkle, Bernadette; Huber, Cristina; Spiliotis, Markus; Müller, Norbert; Garate, Teresa; Doherr, Marcus G.

    2014-01-01

    Two recombinant Fasciola hepatica antigens, saposin-like protein-2 (recSAP2) and cathepsin L-1 (recCL1), were assessed individually and in combination in enzyme-linked immunosorbent assays (ELISA) for the specific serodiagnosis of human fasciolosis in areas of low endemicity as encountered in Central Europe. Antibody detection was conducted using ProteinA/ProteinG (PAG) conjugated to alkaline phosphatase. Test characteristics as well as agreement with results from an ELISA using excretory–secretory products (FhES) from adult stage liver flukes was assessed by receiver operator characteristic (ROC) analysis, specificity, sensitivity, Youdens J and overall accuracy. Cross-reactivity was assessed using three different groups of serum samples from healthy individuals (n = 20), patients with other parasitic infections (n = 87) and patients with malignancies (n = 121). The best combined diagnostic results for recombinant antigens were obtained using the recSAP2-ELISA (87% sensitivity, 99% specificity and 97% overall accuracy) employing the threshold (cut-off) to discriminate between positive and negative reactions that maximized Youdens J. The findings showed that recSAP2-ELISA can be used for the routine serodiagnosis of chronic fasciolosis in clinical laboratories; the use of the PAG-conjugate offers the opportunity to employ, for example, rabbit hyperimmune serum for the standardization of positive controls. PMID:24922050

  14. Recombinant FAdV-4 fiber-2 protein protects chickens against hepatitis-hydropericardium syndrome (HHS).

    Science.gov (United States)

    Schachner, Anna; Marek, Ana; Jaskulska, Barbara; Bilic, Ivana; Hess, Michael

    2014-02-19

    Virulent fowl adenovirus (FAdV) serotype 4 strains are the etiological agents of hepatitis-hydropericardium syndrome (HHS), a highly infectious disease in chickens with severe economic impact. In the present study, three different FAdV-4 derived capsid proteins, fiber-1, fiber-2, and hexon loop-1, were expressed in a baculovirus system and tested for their capacity to induce protection in chickens. Purified recombinant proteins were administered to day-old specific pathogen-free (SPF) chickens allocated in three separate groups and challenged with virulent FAdV-4 at 21 days of life. Two additional groups served as controls, a challenge control group with mock-vaccinated but infected birds and a negative control group with PBS injection substituting both vaccination and challenge. The fiber-2 vaccinated group displayed high resistance against the adverse effects of the challenge with only one dead bird out of 28, as compared to the challenge control group where the infection caused 78% mortality. A moderate protective effect resulting in 38% mortality was observed for fiber-1, whereas the hexon loop-1 vaccinated group was not effectively protected as manifested by 73% mortality. While a fiber-2 specific ELISA showed a gradual antibody increase after immunization of birds with the homologous protein, a commercial ELISA did not detect vaccination-induced antibodies in any of the groups but displayed a difference in challenge virus-directed response in protected and non-protected birds. Although immunoblotting confirmed the presence of specific antibodies in all vaccinated groups, the anti-protein sera did not exhibit neutralizing activity. Fecal excretion of challenge virus DNA was detected with a real-time PCR in the majority of tested birds until termination of the study independent of protection, indicating the prevention of clinical symptoms, but not infection, by vaccination. In conclusion, recombinant fiber-2 was identified as a protective immunogen and is

  15. Protein Expression Analyses at the Single Cell Level

    Directory of Open Access Journals (Sweden)

    Masae Ohno

    2014-09-01

    Full Text Available The central dogma of molecular biology explains how genetic information is converted into its end product, proteins, which are responsible for the phenotypic state of the cell. Along with the protein type, the phenotypic state depends on the protein copy number. Therefore, quantification of the protein expression in a single cell is critical for quantitative characterization of the phenotypic states. Protein expression is typically a dynamic and stochastic phenomenon that cannot be well described by standard experimental methods. As an alternative, fluorescence imaging is being explored for the study of protein expression, because of its high sensitivity and high throughput. Here we review key recent progresses in fluorescence imaging-based methods and discuss their application to proteome analysis at the single cell level.

  16. Development of Escherichia coli Strains That Withstand Membrane Protein-Induced Toxicity and Achieve High-Level Recombinant Membrane Protein Production.

    Science.gov (United States)

    Gialama, Dimitra; Kostelidou, Kalliopi; Michou, Myrsini; Delivoria, Dafni Chrysanthi; Kolisis, Fragiskos N; Skretas, Georgios

    2017-02-17

    Membrane proteins perform critical cellular functions in all living organisms and constitute major targets for drug discovery. Escherichia coli has been the most popular overexpression host for membrane protein biochemical/structural studies. Bacterial production of recombinant membrane proteins, however, is typically hampered by poor cellular accumulation and severe toxicity for the host, which leads to low final biomass and minute volumetric yields. In this work, we aimed to rewire the E. coli protein-producing machinery to withstand the toxicity caused by membrane protein overexpression in order to generate engineered bacterial strains with the ability to achieve high-level membrane protein production. To achieve this, we searched for bacterial genes whose coexpression can suppress membrane protein-induced toxicity and identified two highly potent effectors: the membrane-bound DnaK cochaperone DjlA, and the inhibitor of the mRNA-degrading activity of the E. coli RNase E, RraA. E. coli strains coexpressing either djlA or rraA, termed SuptoxD and SuptoxR, respectively, accumulated markedly higher levels of final biomass and produced dramatically enhanced yields for a variety of prokaryotic and eukaryotic recombinant membrane proteins. In all tested cases, either SuptoxD, or SuptoxR, or both, outperformed the capabilities of commercial strains frequently utilized for recombinant membrane protein production purposes.

  17. Construction of Recombinant Single Chain Variable Fragment (ScFv) Antibody Against Superantigen for Immunodetection Using Antibody Phage Display Technology.

    Science.gov (United States)

    Singh, Pawan Kumar; Agrawal, Ranu; Kamboj, D V; Singh, Lokendra

    2016-01-01

    Superantigens are a class of antigens that bind to the major histocompatibility complex class (MHC) II and T-cell receptor (TCR) and cause the nonspecific activation of T cells, resulting in a massive release of pro-inflammatory mediators. They are produced by the gram-positive organisms Staphylococcus aureus and Streptococcus pyogenes, and by a variety of other microbes such as viruses and mycoplasma, and cause toxic shock syndrome (TSS) and even death in some cases. The immunodetection of superantigens is difficult due to the polyclonal activation of T-cells leading to nonspecific antibody production. The production of recombinant monoclonal antibodies against superantigens can solve this problem and are far better than polyclonal antibodies in terms of detection. Here, we describe the construction of recombinant single chain variable fragments (ScFv) antibodies against superantigens with specific reference to SEB (staphylococcal enterotoxin B) using antibody phage display technology.

  18. Evaluation of yeast single cell protein (SCP) diets on growth ...

    African Journals Online (AJOL)

    An investigation was carried out on the possibility of replacing fishmeal with graded levels of yeast single cell protein (SCP; 10, 20, 30, 40 and 50%) in ... that the 50% yeast SCP fed fish had the highest percentage of body protein (55.35%), but with a lower amount of fat at the end of the feeding trial compared to the control.

  19. PRODt;CTION OF SINGLE CELL PROTEIN FROM BREWERY ...

    African Journals Online (AJOL)

    BSN

    customary food and feed sources of protein (agriculnrre and fishery) to ocher sources like single cell protein (SCP); whose production from hydrocarbons is one ... origin is unicellular or simple multicellular organism such as bacteria, yeasts, fungi, algae. protozoa, mid even bacterinphagcs generally cultivated on substrates ...

  20. Conversion of Food waste to Single Cell Protein using Aspergillus ...

    African Journals Online (AJOL)

    ADOWIE PERE

    2018-03-13

    Mar 13, 2018 ... ABSTRACT: The utilization of food waste into products like single cell protein is an alternative solution to global protein shortage ... as orange, pineapple, banana, watermelon and cucumber waste as growth media for A. niger using standard techniques. ..... Waste to Wealth- Value Recovery from. Agrofood.

  1. Conversion of Food waste to Single Cell Protein using Aspergillus ...

    African Journals Online (AJOL)

    The utilization of food waste into products like single cell protein is an alternative solution to global protein shortage and to alleviate pollution problems. This investigation was carried out with food wastes such as orange, pineapple, banana, watermelon and cucumber waste as growth media for A. niger using standard ...

  2. Recombinant expression and purification of the RNA-binding LARP6 proteins from fish genetic model organisms.

    Science.gov (United States)

    Castro, José M; Horn, Daniel A; Pu, Xinzhu; Lewis, Karen A

    2017-06-01

    The RNA-binding proteins that comprise the La-related protein (LARP) superfamily have been implicated in a wide range of cellular functions, from tRNA maturation to regulation of protein synthesis. To more expansively characterize the biological function of the LARP6 subfamily, we have recombinantly expressed the full-length LARP6 proteins from two teleost fish, platyfish (Xiphophorus maculatus) and zebrafish (Danio rerio). The yields of the recombinant proteins were enhanced to >2 mg/L using a tandem approach of an N-terminal His 6 -SUMO tag and an iterative solubility screening assay to identify structurally stabilizing buffer components. The domain topologies of the purified fish proteins were probed with limited proteolysis. The fish proteins contain an internal, protease-resistant 40 kDa domain, which is considerably more stable than the comparable domain from the human LARP6 protein. The fish proteins are therefore a lucrative model system in which to study both the evolutionary divergence of this family of La-related proteins and the structure and conformational dynamics of the domains that comprise the LARP6 protein. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. PRODt;CTION OF SINGLE CELL PROTEIN FROM BREWERY ...

    African Journals Online (AJOL)

    BSN

    The production of single cell protein (SCP) by the propagation of the yeast, Saccharomyces cerevisae ... animal feed but little or no information has been documented as per its explication for the production of single cell .... use of yeasts produced from vatious carbohydrate sources, molasses, sulphite liquors and vegetable.

  4. A novel synthetic medium and expression system for subzero growth and recombinant protein production in Pseudoalteromonas haloplanktis TAC125.

    Science.gov (United States)

    Sannino, F; Giuliani, M; Salvatore, U; Apuzzo, G A; de Pascale, D; Fani, R; Fondi, M; Marino, G; Tutino, M L; Parrilli, E

    2017-01-01

    The Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 is a model organism of cold-adapted bacteria. The interest in the study of this psychrophilic bacterium stems from its capability either as a non-conventional system for production of recombinant protein and as a rich source of bioactive compounds. To further explore the biotechnological ability of P. haloplanktis TAC125, we have developed a synthetic medium, containing D-gluconate and L-glutamate (GG), which allows the bacterium to grow even at subzero temperatures. P. haloplanktis TAC125 growing in GG medium at low temperature displays growth kinetic parameters which confirm its spectacular adaptation to cold environment and subzero lifestyle, paving the way to the definition of the underlying molecular strategies. Moreover, in this paper, we report the setup of a finely regulated gene expression system inducible by D-galactose to produce recombinant protein in GG synthetic medium at temperatures as low as -2.5 °C. Thanks to the combination of the novel medium and the new expression system, we obtained for the first time the production of a recombinant protein at subzero temperature, thus providing an innovative strategy for the recombinant production of "difficult" proteins.

  5. Production of Polyclonal Antiobies to a Recombinant Potato Mop-top Virus Non-structural Triple Gene Block Protein l

    Czech Academy of Sciences Publication Activity Database

    Čeřovská, Noemi; Filigarová, Marie; Pečenková, Tamara

    2006-01-01

    Roč. 154, - (2006), s. 422-427 ISSN 0931-1785 R&D Projects: GA ČR GA522/04/1329 Institutional research plan: CEZ:AV0Z50380511 Keywords : Potato mop-top vir