WorldWideScience

Sample records for recombinant a27 protein

  1. Co-administration of recombinant major envelope proteins (rA27L and rH3L) of buffalopox virus provides enhanced immunogenicity and protective efficacy in animal models.

    Science.gov (United States)

    Kumar, Amit; Yogisharadhya, Revanaiah; Venkatesan, Gnanavel; Bhanuprakash, Veerakyathappa; Pandey, Awadh Bihari; Shivachandra, Sathish Bhadravati

    2017-05-01

    Buffalopox virus (BPXV) and other vaccinia-like viruses (VLVs) are causing an emerging/re-emerging zoonosis affecting buffaloes, cattle and humans in India and other countries. A27L and H3L are immuno-dominant major envelope proteins of intracellular mature virion (IMV) of orthopoxviruses (OPVs) and are highly conserved with an ability to elicit neutralizing antibodies. In the present study, two recombinant proteins namely; rA27L ( 21 S to E 110 ; ∼30 kDa) and rH3L( 1 M to I 280 ; ∼50 kDa) of BPXV-Vij/96 produced from Escherichia coli were used in vaccine formulation. A combined recombinant subunit vaccine comprising rA27L and rH3L antigens (10 μg of each) was used for active immunization of adult mice (20μg/dose/mice) with or without adjuvant (FCA/FIA) by intramuscular route. Immune responses revealed a gradual increase in antigen specific serum IgG as well as neutralizing antibody titers measured by using indirect-ELISA and serum neutralization test (SNT) respectively, which were higher as compared to that elicited by individual antigens. Suckling mice passively administered with combined anti-A27L and anti-H3L sera showed a complete (100%) pre-exposure protection upon challenge with virulent BPXV. Conclusively, this study highlights the potential utility of rA27L and rH3L proteins as safer candidate prophylactic antigens in combined recombinant subunit vaccine for buffalopox as well as passive protective efficacy of combined sera in employing better pre-exposure protection against virulent BPXV. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. 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.

  3. 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...

  4. Recombinant DNA production of spider silk proteins.

    Science.gov (United States)

    Tokareva, Olena; Michalczechen-Lacerda, Valquíria A; Rech, Elíbio L; Kaplan, David L

    2013-11-01

    Spider dragline silk is considered to be the toughest biopolymer on Earth due to an extraordinary combination of strength and elasticity. Moreover, silks are biocompatible and biodegradable protein-based materials. Recent advances in genetic engineering make it possible to produce recombinant silks in heterologous hosts, opening up opportunities for large-scale production of recombinant silks for various biomedical and material science applications. We review the current strategies to produce recombinant spider silks. © 2013 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  5. L1R, A27L, A33R and B5R vaccinia virus genes expressed by fowlpox recombinants as putative novel orthopoxvirus vaccines.

    Science.gov (United States)

    Pacchioni, Sole Maria; Bissa, Massimiliano; Zanotto, Carlo; Morghen, Carlo De Giuli; Illiano, Elena; Radaelli, Antonia

    2013-04-11

    The traditional smallpox vaccine, administered by scarification, was discontinued in the general population from 1980, because of the absence of new smallpox cases. However, the development of an effective prophylactic vaccine against smallpox is still necessary, to protect from the threat of deliberate release of the variola virus for bioterrorism and from new zoonotic infections, and to improve the safety of the traditional vaccine. Preventive vaccination still remains the most effective control and new vectors have been developed to generate recombinant vaccines against smallpox that induce the same immunogenicity as the traditional one. As protective antibodies are mainly directed against the surface proteins of the two infectious forms of vaccinia, the intracellular mature virions and the extracellular virions, combined proteins from these viral forms can be used to better elicit a complete and protective immunity. Four novel viral recombinants were constructed based on the fowlpox genetic background, which independently express the vaccinia virus L1 and A27 proteins present on the mature virions, and the A33 and B5 proteins present on the extracellular virions. The correct expression of the transgenes was determined by RT-PCR, Western blotting, and immunofluorescence. Using immunoprecipitation and Western blotting, the ability of the proteins expressed by the four novel FPL1R, FPA27L, FPA33R and FPB5R recombinants to be recognized by VV-specific hyperimmune mouse sera was demonstrated. By neutralisation assays, recombinant virus particles released by infected chick embryo fibroblasts were shown not be recognised by hyperimmune sera. This thus demonstrates that the L1R, A27L, A33R and B5R gene products are not inserted into the new viral progeny. Fowlpox virus replicates only in avian species, but it is permissive for entry and transgene expression in mammalian cells, while being immunologically non-cross-reactive with vaccinia virus. These recombinants might

  6. Recombinant protein blends: silk beyond natural design.

    Science.gov (United States)

    Dinjaski, Nina; Kaplan, David L

    2016-06-01

    Recombinant DNA technology and new material concepts are shaping future directions in biomaterial science for the design and production of the next-generation biomaterial platforms. Aside from conventionally used synthetic polymers, numerous natural biopolymers (e.g., silk, elastin, collagen, gelatin, alginate, cellulose, keratin, chitin, polyhydroxyalkanoates) have been investigated for properties and manipulation via bioengineering. Genetic engineering provides a path to increase structural and functional complexity of these biopolymers, and thereby expand the catalog of available biomaterials beyond that which exists in nature. In addition, the integration of experimental approaches with computational modeling to analyze sequence-structure-function relationships is starting to have an impact in the field by establishing predictive frameworks for determining material properties. Herein, we review advances in recombinant DNA-mediated protein production and functionalization approaches, with a focus on hybrids or combinations of proteins; recombinant protein blends or 'recombinamers'. We highlight the potential biomedical applications of fibrous protein recombinamers, such as Silk-Elastin Like Polypeptides (SELPs) and Silk-Bacterial Collagens (SBCs). We also discuss the possibility for the rationale design of fibrous proteins to build smart, stimuli-responsive biomaterials for diverse applications. We underline current limitations with production systems for these proteins and discuss the main trends in systems/synthetic biology that may improve recombinant fibrous protein design and production. Copyright © 2016. Published by Elsevier Ltd.

  7. Antibodies to the A27 protein of vaccinia virus neutralize and protect against infection but represent a minor component of Dryvax vaccine--induced immunity.

    Science.gov (United States)

    He, Yong; Manischewitz, Jody; Meseda, Clement A; Merchlinsky, Michael; Vassell, Russell A; Sirota, Lev; Berkower, Ira; Golding, Hana; Weiss, Carol D

    2007-10-01

    The smallpox vaccine Dryvax, which consists of replication-competent vaccinia virus, elicits antibodies that play a major role in protection. Several vaccinia proteins generate neutralizing antibodies, but their importance for protection is unknown. We investigated the potency of antibodies to the A27 protein of the mature virion in neutralization and protection experiments and the contributions of A27 antibodies to Dryvax-induced immunity. Using a recombinant A27 protein (rA27), we confirmed that A27 contains neutralizing determinants and that vaccinia immune globulin (VIG) derived from Dryvax recipients contains reactivity to A27. However, VIG neutralization was not significantly reduced when A27 antibodies were removed, and antibodies elicited by an rA27 enhanced the protection conferred by VIG in passive transfer experiments. These findings demonstrate that A27 antibodies do not represent the major fraction of neutralizing activity in VIG and suggest that immunity may be augmented by vaccines and immune globulins that include strong antibody responses to A27.

  8. 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).

  9. 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.

  10. 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.

  11. Functions and structures of eukaryotic recombination proteins

    International Nuclear Information System (INIS)

    Ogawa, Tomoko

    1994-01-01

    We have found that Rad51 and RecA Proteins form strikingly similar structures together with dsDNA and ATP. Their right handed helical nucleoprotein filaments extend the B-form DNA double helixes to 1.5 times in length and wind the helix. The similarity and uniqueness of their structures must reflect functional homologies between these proteins. Therefore, it is highly probable that similar recombination proteins are present in various organisms of different evolutional states. We have succeeded to clone RAD51 genes from human, mouse, chicken and fission yeast genes, and found that the homologues are widely distributed in eukaryotes. The HsRad51 and MmRad51 or ChRad51 proteins consist of 339 amino acids differing only by 4 or 12 amino acids, respectively, and highly homologous to both yeast proteins, but less so to Dmcl. All of these proteins are homologous to the region from residues 33 to 240 of RecA which was named ''homologous core. The homologous core is likely to be responsible for functions common for all of them, such as the formation of helical nucleoprotein filament that is considered to be involved in homologous pairing in the recombination reaction. The mouse gene is transcribed at a high level in thymus, spleen, testis, and ovary, at lower level in brain and at a further lower level in some other tissues. It is transcribed efficiently in recombination active tissues. A clear functional difference of Rad51 homologues from RecA was suggested by the failure of heterologous genes to complement the deficiency of Scrad51 mutants. This failure seems to reflect the absence of a compatible partner, such as ScRad52 protein in the case of ScRad51 protein, between different species. Thus, these discoveries play a role of the starting point to understand the fundamental gene targeting in mammalian cells and in gene therapy. (J.P.N.)

  12. 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)

  13. Recombinant Amphiphilic Protein Micelles for Drug Delivery

    OpenAIRE

    Kim, Wookhyun; Xiao, Jiantao; Chaikof, Elliot L.

    2011-01-01

    Amphiphilic block polypeptides can self-assemble into a range of nanostructures in solution, including micelles and vesicles. Our group has recently described the capacity of recombinant amphiphilic diblock copolypeptides to form highly stable micelles. In this report, we demonstrate the utility of protein nanoparticles to serve as a vehicle for controlled drug delivery. Drug-loaded micelles were produced by encapsulating dipyridamole as a model hydrophobic drug with anti-inflammatory activit...

  14. 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.

  15. 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.

  16. 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 ...

  17. 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....

  18. Insect Larvae: A New Platform to Produce Commercial Recombinant Proteins.

    Science.gov (United States)

    Targovnik, Alexandra M; Arregui, Mariana B; Bracco, Lautaro F; Urtasun, Nicolas; Baieli, Maria F; Segura, Maria M; Simonella, Maria A; Fogar, Mariela; Wolman, Federico J; Cascone, Osvaldo; Miranda, Maria V

    2016-01-01

    In Biotechnology, the expression of recombinant proteins is a constantly growing field and different hosts are used for this purpose. Some valuable proteins cannot be produced using traditional systems. Insects from the order Lepidoptera infected with recombinant baculovirus have appeared as a good choice to express high levels of proteins, especially those with post-translational modifications. Lepidopteran insects, which are extensively distributed in the world, can be used as small protein factories, the new biofactories. Species like Bombyx mori (silkworm) have been analyzed in Asian countries to produce a great number of recombinant proteins for use in basic and applied science and industry. Many proteins expressed in this larva have been commercialized. Several recombinant proteins produced in silkworms have already been commercialized. On the other hand, species like Spodoptera frugiperda, Heliothis virescens, Rachiplusia nu, Helicoverpa zea and Trichoplusia ni are widely distributed in both the occidental world and Europe. The expression of recombinant proteins in larvae has the advantage of its low cost in comparison with insect cell cultures. A wide variety of recombinant proteins, including enzymes, hormones and vaccines, have been efficiently expressed with intact biological activity. The expression of pharmaceutically proteins, using insect larvae or cocoons, has become very attractive. This review describes the use of insect larvae as an alternative to produce commercial recombinant proteins.

  19. 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

  20. Protective Effect of Surfactant Protein D in Pulmonary Vaccinia Virus Infection: Implication of A27 Viral Protein

    Directory of Open Access Journals (Sweden)

    Julien Perino

    2013-03-01

    Full Text Available Vaccinia virus (VACV was used as a surrogate of variola virus (VARV (genus Orthopoxvirus, the causative agent of smallpox, to study Orthopoxvirus infection. VARV is principally transmitted between humans by aerosol droplets. Once inhaled, VARV first infects the respiratory tract where it could encounter surfactant components, such as soluble pattern recognition receptors. Surfactant protein D (SP-D, constitutively present in the lining fluids of the respiratory tract, plays important roles in innate host defense against virus infection. We investigated the role of SP-D in VACV infection and studied the A27 viral protein involvement in the interaction with SP-D. Interaction between SP-D and VACV caused viral inhibition in a lung cell model. Interaction of SP-D with VACV was mediated by the A27 viral protein. Binding required Ca2+ and interactions were blocked in the presence of excess of SP-D saccharide ligands. A27, which lacks glycosylation, directly interacted with SP-D. The interaction between SP-D and the viral particle was also observed using electron microscopy. Infection of mice lacking SP-D (SP-D-/- resulted in increased mortality compared to SP-D+/+ mice. Altogether, our data show that SP-D participates in host defense against the vaccinia virus infection and that the interaction occurs with the viral surface protein A27.

  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. 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

  5. Recombinant human bone morphogenetic protein induces bone formation

    International Nuclear Information System (INIS)

    Wang, E.A.; Rosen, V.; D'Alessandro, J.S.; Bauduy, M.; Cordes, P.; Harada, T.; Israel, D.I.; Hewick, R.M.; Kerns, K.M.; LaPan, P.; Luxenberg, D.P.; McQuaid, D.; Moutsatsos, I.K.; Nove, J.; Wozney, J.M.

    1990-01-01

    The authors have purified and characterized active recombinant human bone morphogenetic protein (BMP) 2A. Implantation of the recombinant protein in rats showed that a single BMP can induce bone formation in vivo. A dose-response and time-course study using the rat ectopic bone formation assay revealed that implantation of 0.5-115 μg of partially purified recombinant human BMP-2A resulted in cartilage by day 7 and bone formation by day 14. The time at which bone formation occurred was dependent on the amount of BMP-2A implanted; at high doses bone formation could be observed at 5 days. The cartilage- and bone-inductive activity of the recombinant BMP-2A is histologically indistinguishable from that of bone extracts. Thus, recombinant BMP-2A has therapeutic potential to promote de novo bone formation in humans

  6. 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; ...

  7. 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.

  8. Reactions of chicken sera to recombinant Campylobacter jejuni flagellar proteins.

    Science.gov (United States)

    Yeh, Hung-Yueh; Hiett, Kelli L; Line, John E

    2015-03-01

    Campylobacter jejuni is a Gram-negative spiral rod bacterium and is the leading but underreported bacterial food-borne pathogen that causes human campylobacteriosis worldwide. Raw or undercooked poultry products are regarded as a major source for human infection. C. jejuni flagella have been implicated in colonization and adhesion to the mucosal surface of chicken gastrointestinal tracts. Therefore, flagellar proteins would be the excellent targets for further investigation. In this report, we used the recombinant technology to generate a battery of C. jejuni flagellar proteins, which were purified by His tag affinity chromatography and determined antigenic profiles of these recombinant flagellar proteins using sera from chickens older than 6 weeks of age. The immunoblot results demonstrate that each chicken serum reacted to various numbers of recombinant flagellar proteins. Among these recombinant proteins, chicken sera reacted predominantly to the FlgE1, FlgK, FlhF, FliG and FliY proteins. These antibody screening results provide a rationale for further evaluation of these recombinant flagellar proteins as potential vaccines for chickens to improve food safety as well as investigation of host immune response to C. jejuni.

  9. Viral vectors for production of recombinant proteins in plants.

    Science.gov (United States)

    Lico, Chiara; Chen, Qiang; Santi, Luca

    2008-08-01

    Global demand for recombinant proteins has steadily accelerated for the last 20 years. These recombinant proteins have a wide range of important applications, including vaccines and therapeutics for human and animal health, industrial enzymes, new materials and components of novel nano-particles for various applications. The majority of recombinant proteins are produced by traditional biological "factories," that is, predominantly mammalian and microbial cell cultures along with yeast and insect cells. However, these traditional technologies cannot satisfy the increasing market demand due to prohibitive capital investment requirements. During the last two decades, plants have been under intensive investigation to provide an alternative system for cost-effective, highly scalable, and safe production of recombinant proteins. Although the genetic engineering of plant viral vectors for heterologous gene expression can be dated back to the early 1980s, recent understanding of plant virology and technical progress in molecular biology have allowed for significant improvements and fine tuning of these vectors. These breakthroughs enable the flourishing of a variety of new viral-based expression systems and their wide application by academic and industry groups. In this review, we describe the principal plant viral-based production strategies and the latest plant viral expression systems, with a particular focus on the variety of proteins produced and their applications. We will summarize the recent progress in the downstream processing of plant materials for efficient extraction and purification of recombinant proteins. (c) 2008 Wiley-Liss, Inc.

  10. 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.

  11. Recombinant protein expression in microbial systems

    OpenAIRE

    Rosano, Germán L.; Ceccarelli, Eduardo A.

    2014-01-01

    The emergence of recombinant DNA technology during the early 70's set a revolution in molecular biology. This set of techniques was strengthened even further later on with the introduction of the polymerase chain reaction and allowed scientists to explore and understand essential life processes in an easy and straightforward way. It also marked the birth of the modern biotech industry. At that time, it was shown that eukaryotic DNA could be propagated in Escherichia coli (Morrow et al., 1974)...

  12. N-Glycosylation of Plant-produced Recombinant Proteins

    NARCIS (Netherlands)

    Bosch, H.J.; Castilho, A.; Loos, A.; Schots, A.; Steinkeller, H.

    2013-01-01

    Plants are gaining increasingly acceptance as a production platform for recombinant proteins. One reason for this is their ability to carry out posttranslational protein modifications in a similar if not identical way as mammalian cells. The capability of plants to carry out human-like complex

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

    African Journals Online (AJOL)

    user

    2010-11-22

    Nov 22, 2010 ... protein (Baneyx and Mujacic, 2004). Although ... both time-consuming and expensive (Tsumoto et al.,. 2003). Thus ... proteins (Schlieker et al., 2002). ..... Alibolandi M, Mirzahoseini H, Abedi Khalil Abad M, Azami Movahed M.

  14. 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.

  15. Manufacturing of recombinant therapeutic proteins in microbial systems.

    Science.gov (United States)

    Graumann, Klaus; Premstaller, Andreas

    2006-02-01

    Recombinant therapeutic proteins have gained enormous importance for clinical applications. The first recombinant products have been produced in E. coli more than 20 years ago. Although with the advent of antibody-based therapeutics mammalian expression systems have experienced a major boost, microbial expression systems continue to be widely used in industry. Their intrinsic advantages, such as rapid growth, high yields and ease of manipulation, make them the premier choice for expression of non-glycosylated peptides and proteins. Innovative product classes such as antibody fragments or alternative binding molecules will further expand the use of microbial systems. Even more, novel, engineered production hosts and integrated technology platforms hold enormous potential for future applications. This review summarizes current applications and trends for development, production and analytical characterization of recombinant therapeutic proteins in microbial systems.

  16. 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)

  17. Genome engineering for improved recombinant protein expression in Escherichia coli.

    Science.gov (United States)

    Mahalik, Shubhashree; Sharma, Ashish K; Mukherjee, Krishna J

    2014-12-19

    A metabolic engineering perspective which views recombinant protein expression as a multistep pathway allows us to move beyond vector design and identify the downstream rate limiting steps in expression. In E.coli these are typically at the translational level and the supply of precursors in the form of energy, amino acids and nucleotides. Further recombinant protein production triggers a global cellular stress response which feedback inhibits both growth and product formation. Countering this requires a system level analysis followed by a rational host cell engineering to sustain expression for longer time periods. Another strategy to increase protein yields could be to divert the metabolic flux away from biomass formation and towards recombinant protein production. This would require a growth stoppage mechanism which does not affect the metabolic activity of the cell or the transcriptional or translational efficiencies. Finally cells have to be designed for efficient export to prevent buildup of proteins inside the cytoplasm and also simplify downstream processing. The rational and the high throughput strategies that can be used for the construction of such improved host cell platforms for recombinant protein expression is the focus of this review.

  18. 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.

  19. Gene Delivery into Plant Cells for Recombinant Protein Production

    Directory of Open Access Journals (Sweden)

    Qiang Chen

    2015-01-01

    Full Text Available Recombinant proteins are primarily produced from cultures of mammalian, insect, and bacteria cells. In recent years, the development of deconstructed virus-based vectors has allowed plants to become a viable platform for recombinant protein production, with advantages in versatility, speed, cost, scalability, and safety over the current production paradigms. In this paper, we review the recent progress in the methodology of agroinfiltration, a solution to overcome the challenge of transgene delivery into plant cells for large-scale manufacturing of recombinant proteins. General gene delivery methodologies in plants are first summarized, followed by extensive discussion on the application and scalability of each agroinfiltration method. New development of a spray-based agroinfiltration and its application on field-grown plants is highlighted. The discussion of agroinfiltration vectors focuses on their applications for producing complex and heteromultimeric proteins and is updated with the development of bridge vectors. Progress on agroinfiltration in Nicotiana and non-Nicotiana plant hosts is subsequently showcased in context of their applications for producing high-value human biologics and low-cost and high-volume industrial enzymes. These new advancements in agroinfiltration greatly enhance the robustness and scalability of transgene delivery in plants, facilitating the adoption of plant transient expression systems for manufacturing recombinant proteins with a broad range of applications.

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

    African Journals Online (AJOL)

    USER

    2010-04-12

    Apr 12, 2010 ... protein expression with the influence of the carbon source feeding ... in the culture media, increasing the peroxisomes numbers ...... source, temperature, pH, O2, methanol feeding strategy) ..... Catabolite Inactivation in Yeast.

  1. 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.

  2. Expression and purification of recombinant polyomavirus VP2 protein and its interactions with polyomavirus proteins

    Science.gov (United States)

    Cai, X.; Chang, D.; Rottinghaus, S.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    Recombinant polyomavirus VP2 protein was expressed in Escherichia coli (RK1448), using the recombinant expression system pFPYV2. Recombinant VP2 was purified to near homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroelution, and Extracti-Gel chromatography. Polyclonal serum to this protein which reacted specifically with recombinant VP2 as well as polyomavirus virion VP2 and VP3 on Western blots (immunoblots) was produced. Purified VP2 was used to establish an in vitro protein-protein interaction assay with polyomavirus structural proteins and purified recombinant VP1. Recombinant VP2 interacted with recombinant VP1, virion VP1, and the four virion histones. Recombinant VP1 coimmunoprecipitated with recombinant VP2 or truncated VP2 (delta C12VP2), which lacked the carboxy-terminal 12 amino acids. These experiments confirmed the interaction between VP1 and VP2 and revealed that the carboxyterminal 12 amino acids of VP2 and VP3 were not necessary for formation of this interaction. In vivo VP1-VP2 interaction study accomplished by cotransfection of COS-7 cells with VP2 and truncated VP1 (delta N11VP1) lacking the nuclear localization signal demonstrated that VP2 was capable of translocating delta N11VP1 into the nucleus. These studies suggest that complexes of VP1 and VP2 may be formed in the cytoplasm and cotransported to the nucleus for virion assembly to occur.

  3. 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

  4. Step-wise refolding of recombinant proteins.

    Science.gov (United States)

    Tsumoto, Kouhei; Arakawa, Tsutomu; Chen, Linda

    2010-04-01

    Protein refolding is still on trial-and-error basis. Here we describe step-wise dialysis refolding, in which denaturant concentration is altered in step-wise fashion. This technology controls the folding pathway by adjusting the concentrations of the denaturant and other solvent additives to induce sequential folding or disulfide formation.

  5. Divergence, recombination and retention of functionality during protein evolution

    Directory of Open Access Journals (Sweden)

    Xu Yanlong O

    2005-09-01

    Full Text Available Abstract We have only a vague idea of precisely how protein sequences evolve in the context of protein structure and function. This is primarily because structural and functional contexts are not easily predictable from the primary sequence, and evaluating patterns of evolution at individual residue positions is also difficult. As a result of increasing biodiversity in genomics studies, progress is being made in detecting context-dependent variation in substitution processes, but it remains unclear exactly what context-dependent patterns we should be looking for. To address this, we have been simulating protein evolution in the context of structure and function using lattice models of proteins and ligands (or substrates. These simulations include thermodynamic features of protein stability and population dynamics. We refer to this approach as 'ab initio evolution' to emphasise the fact that the equilibrium details of fitness distributions arise from the physical principles of the system and not from any preconceived notions or arbitrary mathematical distributions. Here, we present results on the retention of functionality in homologous recombinants following population divergence. A central result is that protein structure characteristics can strongly influence recombinant functionality. Exceptional structures with many sequence options evolve quickly and tend to retain functionality -- even in highly diverged recombinants. By contrast, the more common structures with fewer sequence options evolve more slowly, but the fitness of recombinants drops off rapidly as homologous proteins diverge. These results have implications for understanding viral evolution, speciation and directed evolutionary experiments. Our analysis of the divergence process can also guide improved methods for accurately approximating folding probabilities in more complex but realistic systems.

  6. Influenza vaccines: from whole virus preparations to recombinant protein technology.

    Science.gov (United States)

    Huber, Victor C

    2014-01-01

    Vaccination against influenza represents our most effective form of prevention. Historical approaches toward vaccine creation and production have yielded highly effective vaccines that are safe and immunogenic. Despite their effectiveness, these historical approaches do not allow for the incorporation of changes into the vaccine in a timely manner. In 2013, a recombinant protein-based vaccine that induces immunity toward the influenza virus hemagglutinin was approved for use in the USA. This vaccine represents the first approved vaccine formulation that does not require an influenza virus intermediate for production. This review presents a brief history of influenza vaccines, with insight into the potential future application of vaccines generated using recombinant technology.

  7. 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...... between the anti-GLURP489-1271 and anti-(EENV)6 antibody responses. The data provide indirect evidence for a protective role of antibodies reacting with recombinant GLURP489-1271 as well as with the synthetic peptide (EENV)6 from the Pf155/RESA....

  8. Recombinant protein production from stable mammalian cell lines and pools.

    Science.gov (United States)

    Hacker, David L; Balasubramanian, Sowmya

    2016-06-01

    We highlight recent developments for the production of recombinant proteins from suspension-adapted mammalian cell lines. We discuss the generation of stable cell lines using transposons and lentivirus vectors (non-targeted transgene integration) and site-specific recombinases (targeted transgene integration). Each of these methods results in the generation of cell lines with protein yields that are generally superior to those achievable through classical plasmid transfection that depends on the integration of the transfected DNA by non-homologous DNA end-joining. This is the main reason why these techniques can also be used for the generation of stable cell pools, heterogenous populations of recombinant cells generated by gene delivery and genetic selection without resorting to single cell cloning. This allows the time line from gene transfer to protein production to be reduced. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. 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.

  10. Recombination Proteins Mediate Meiotic Spatial Chromosome Organization and Pairing

    Science.gov (United States)

    Storlazzi, Aurora; Gargano, Silvana; Ruprich-Robert, Gwenael; Falque, Matthieu; David, Michelle; Kleckner, Nancy; Zickler, Denise

    2010-01-01

    SUMMARY Meiotic chromosome pairing involves not only recognition of homology but also juxtaposition of entire chromosomes in a topologically regular way. Analysis of filamentous fungus Sordaria macrospora reveals that recombination proteins Mer3, Msh4 and Mlh1 play direct roles in all of these aspects, in advance of their known roles in recombination. Absence of Mer3 helicase results in interwoven chromosomes, thereby revealing the existence of features that specifically ensure “entanglement avoidance”. Entanglements that remain at zygotene, i.e. “interlockings”, require Mlh1 for resolution, likely to eliminate constraining recombinational connections. Patterns of Mer3 and Msh4 foci along aligned chromosomes show that the double-strand breaks mediating homologous alignment have spatially separated ends, one localized to each partner axis, and that pairing involves interference among developing interhomolog interactions. We propose that Mer3, Msh4 and Mlh1 execute all of these roles during pairing by modulating the state of nascent double-strand break/partner DNA contacts within axis-associated recombination complexes. PMID:20371348

  11. Targeted in vivo inhibition of specific protein-protein interactions using recombinant antibodies.

    Directory of Open Access Journals (Sweden)

    Matej Zábrady

    Full Text Available With the growing availability of genomic sequence information, there is an increasing need for gene function analysis. Antibody-mediated "silencing" represents an intriguing alternative for the precise inhibition of a particular function of biomolecules. Here, we describe a method for selecting recombinant antibodies with a specific purpose in mind, which is to inhibit intrinsic protein-protein interactions in the cytosol of plant cells. Experimental procedures were designed for conveniently evaluating desired properties of recombinant antibodies in consecutive steps. Our selection method was successfully used to develop a recombinant antibody inhibiting the interaction of ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 3 with such of its upstream interaction partners as the receiver domain of CYTOKININ INDEPENDENT HISTIDINE KINASE 1. The specific down-regulation of the cytokinin signaling pathway in vivo demonstrates the validity of our approach. This selection method can serve as a prototype for developing unique recombinant antibodies able to interfere with virtually any biomolecule in the living cell.

  12. Potency assay design for adjuvanted recombinant proteins as malaria vaccines.

    Science.gov (United States)

    Giersing, Birgitte K; Dubovsky, Filip; Saul, Allan; Denamur, Francoise; Minor, Philip; Meade, Bruce

    2006-05-15

    Many licensed vaccines are composed of live, attenuated or inactivated whole-cell microorganisms, or they comprise purified components from whole-cell extracts or culture supernatants. For some diseases, pathology is fairly well understood, and there may be known correlates of protection that provide obvious parameters for assessment of vaccine potency. However, this is not always the case, and some effective vaccines are routinely used even though the mechanisms or correlates of protection are unknown. Some more modern vaccine approaches employ purified recombinant proteins, based on molecules that appear on the surface of the pathogen. This is one of the strategies that has been adopted in the quest to develop a malaria vaccine. Use of these parasite antigens as vaccine candidates is supported by substantial epidemiological data, and some have demonstrated the ability to elicit protective responses in animal models of malaria infection. However, there is as yet no immunological correlate of protection and no functional assays or animal models that have demonstrated the ability to predict efficacy in humans. There is little precedence for the most appropriate and practical method for assessing potency of vaccines based on these recombinant molecules for malaria vaccines. This is likely because the majority of malaria vaccine candidates have only recently entered clinical evaluation. The PATH Malaria Vaccine Initiative (MVI) convened a panel with expertise in potency assay design from industry, governmental institutions, and regulatory bodies to discuss and review the rationale, available methods, and best approaches for assessing the potency of recombinant proteins, specifically for their use as malarial vaccines. The aim of this meeting was to produce a discussion document on the practical potency assessment of recombinant protein malaria vaccines, focusing on early phase potency assay development.

  13. 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 transcriptomics, we identify metabolic and regulatory markers that are consistent with uptake of whole proteins by endocytosis, followed by intracellular degradation and catabolism of substituent amino acids. Uptake and degradation of recombinant protein products may be common in S. cerevisiae protein secretion...... and simplifying downstream purification, compared to other systems that require complex media. As such, engineering S. cerevisiae to improve titers has been then the subject of significant attention, but the majority of previous efforts have been focused on improving protein synthesis. Here, we characterize...

  14. High-level transient expression of recombinant protein in lettuce.

    Science.gov (United States)

    Joh, Lawrence D; Wroblewski, Tadeusz; Ewing, Nicholas N; VanderGheynst, Jean S

    2005-09-30

    Transient expression following agroinfiltration of plant tissue was investigated as a system for producing recombinant protein. As a model system, Agrobacterium tumefaciens containing the beta-glucuronidase (GUS) gene was vacuum infiltrated into lettuce leaf disks. Infiltration with a suspension of 10(9) colony forming units/mL followed by incubation for 72 h at 22 degrees C in continuous darkness produced a maximum of 0.16% GUS protein based on dry tissue or 1.1% GUS protein based on total soluble protein. This compares favorably to expression levels for commercially manufactured GUS protein from transgenic corn seeds. A. tumefaciens culture medium pH between 5.6 and 7.0 and surfactant concentrations lettuce to produce GUS protein more rapidly, but final levels did not exceed the GUS production in leaves incubated in continuous darkness after 72 h at 22 degrees C. The kinetics of GUS expression during incubation in continuous light and dark were represented well using a logistic model, with rate constants of 0.30 and 0.29/h, respectively. To semi-quantitatively measure the GUS expression in large numbers of leaf disks, a photometric enhancement of the standard histochemical staining method was developed. A linear relationship with an R2 value of 0.90 was determined between log10 (% leaf darkness) versus log10 (GUS activity). Although variability in expression level was observed, agroinfiltration appears to be a promising technology that could potentially be scaled up to produce high-value recombinant proteins in planta. Copyright 2005 Wiley Periodicals, Inc

  15. Dynamic organization of genetic recombination proteins and chromosomes

    International Nuclear Information System (INIS)

    Essers, J.; Van Cappellen, G.; Van Drunen, E.; Theil, A.; Jaspers, N.N.G.J.; Houtsmuller, A.B.; Vermeulen, W.; Kanaar, R.

    2003-01-01

    Homologous recombination requires the co-ordinated action of the RAD52 group proteins, including Rad51, Rad52 and Rad54. Upon treatment of mammalian cells with ionizing radiation, these proteins accumulate into foci at sites of DSB induction. We probed the nature of the DNA damage-induced foci in living cells with the use of photobleaching techniques. These foci are not static assemblies of DNA repair proteins. Instead, they are dynamic structures of which Rad51 is a stable core component, while Rad52 and Rad54 reversibly interact with the structure. Furthermore, even though the RAD52 group proteins colocalize in the DNA damage-induced foci, the majority of the proteins are not part of the same multi-protein complex in the absence of DNA damage. Executing DNA transactions through dynamic multi-protein complexes, rather than stable holo-complexes, allows greater flexibility during the transaction. In case of DNA repair, for example, it allows cross talk between different DNA repair pathways and coupling to other DNA transactions, such as replication. In addition to the behavior of proteins in living cells, we have tracked chromosomes during cell division. Our results suggest that the relative position of chromosomes in the mother cell is conserved in its daughter cells

  16. Recombinant Sheep Pox Virus Proteins Elicit Neutralizing Antibodies.

    Science.gov (United States)

    Chervyakova, Olga V; Zaitsev, Valentin L; Iskakov, Bulat K; Tailakova, Elmira T; Strochkov, Vitaliy M; Sultankulova, Kulyaisan T; Sandybayev, Nurlan T; Stanbekova, Gulshan E; Beisenov, Daniyar K; Abduraimov, Yergali O; Mambetaliyev, Muratbay; Sansyzbay, Abylay R; Kovalskaya, Natalia Y; Nemchinov, Lev G; Hammond, Rosemarie W

    2016-06-07

    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.

  17. 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.

  18. Osteoinductive recombinant silk fusion proteins for bone regeneration.

    Science.gov (United States)

    Dinjaski, Nina; Plowright, Robyn; Zhou, Shun; Belton, David J; Perry, Carole C; Kaplan, David L

    2017-02-01

    Protein polymers provide a unique opportunity for tunable designs of material systems due to the genetic basis of sequence control. To address the challenge of biomineralization interfaces with protein based materials, we genetically engineered spider silks to design organic-inorganic hybrid systems. The spider silk inspired domain (SGRGGLGGQG AGAAAAAGGA GQGGYGGLGSQGT) 15 served as an organic scaffold to control material stability and to allow multiple modes of processing, whereas the hydroxyapatite binding domain VTKHLNQISQSY (VTK), provided control over osteogenesis. The VTK domain was fused either to the N-, C- or both terminals of the spider silk domain to understand the effect of position on material properties and mineralization. The addition of the VTK domain to silk did not affect the physical properties of the silk recombinant constructs, but it had a critical role in the induction of biomineralization. When the VTK domain was placed on both the C- and N-termini the formation of crystalline hydroxyapatite was significantly increased. In addition, all of the recombinant proteins in film format supported the growth and proliferation of human mesenchymal stem cells (hMSCs). Importantly, the presence of the VTK domain enhanced osteoinductive properties up to 3-fold compared to the control (silk alone without VTK). Therefore, silk-VTK fusion proteins have been shown suitable for mineralization and functionalization for specific biomedical applications. Organic-inorganic interfaces are integral to biomaterial functions in many areas of repair and regeneration. Several protein polymers have been investigated for this purpose. Despite their success the limited options to fine-tune their material properties, degradation patterns and functionalize them for each specific biomedical application limits their application. Various studies have shown that the biological performance of such proteins can be improved by genetic engineering. The present study provides data

  19. Recombinant proteins in therapeutics: haemophilia treatment as an example

    Directory of Open Access Journals (Sweden)

    Liras Antonio

    2008-04-01

    Full Text Available Abstract One of the most spectacular advances in the history of scientific knowledge was the discovery of deoxyribonucleic acid (DNA by Watson and Crick in 1953. This enabled certain proteins to be prepared in this way for their therapeutic use in clinical practice. Today, in the first decade of the 21st century, hundreds of therapeutic proteins have been produced recombinantly and about 50 of them have been approved for clinical use. Because of the specific procedure used for obtaining these products, which is based on expressing a atherapeutica gene from a fragment of DNA in a cell to produce a functional protein that is free from any human or animal component, they are especially acleana and thus the therapy of choice for many current diseases. The immediate question is: why are recombinant products not used more extensively given their high efficacy and maximum safety? In short, we are faced with an interesting but also unfortunate paradox of pharmacology that greater progress in therapeutic procedures is not always associated with greater introduction of those resources that are safest, for the simple reason that they are more costly.

  20. Isolation and initial structural characterization of a 27 kDa protein from Zingiber officinale

    Science.gov (United States)

    Rasheed, Saima; Malik, Shoaib Ahmad; Falke, Sven; Arslan, Ali; Fazel, Ramin; Schlüter, Hartmut; Betzel, Christian; Choudhary, M. Iqbal

    2018-03-01

    Zingiber officinale Roscoe (Ginger) is a widely used traditional medicinal plant (for different ailments such as arthritis, constipation, and hypertension). This article describes the isolation and characterization of a so far unknown protein from ginger rhizomes applying ion exchange, affinity, size-exclusion chromatography, small angle X-ray scattering (SAXS), and mass spectrometry techniques. One-dimensional Coomassie-stained SDS-PAGE was performed under non-reducing conditions, showing one band corresponding to approx. 27 kDa. Dynamic light scattering (DLS) analysis of the protein solution revealed monodispersity and a monomeric state of the purified protein. Circular dichroism (CD) spectroscopy strongly indicated a β-sheet-rich protein, and disordered regions. MALDI-TOF-MS, and LC-MS/MS analysis resulted in the identification of 27.29 kDa protein, having 32.13% and 25.34% sequence coverage with Zingipain-1 and 2, respectively. The monomeric state and molecular weight were verified by small angle X-ray scattering (SAXS) studies. An elongated ab-initio model was calculated based on the scattering intensity distribution.

  1. 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.

  2. [Eukaryotic Expression and Immunogenic Research of Recombination Ebola Virus Membrane Protein Gp-Fc].

    Science.gov (United States)

    Zhang, Xiaoguang; Yang, Ren; Wang, Jiao; Wang, Xuan; Hou, Mieling; An, Lina; Zhu, Ying; Cao, Yuxi; Zeng, Yi

    2016-01-01

    We used 293 cells to express the recombinant membrane protein of the Ebola virus. Then, the immunogenicity of the recombinant protein was studied by immunized BALB/c mice. According to the codon use frequency of humans, the gene encoding the extracellular domain of the Ebola virus membrane protein was optimized, synthesized, and inserted into the eukaryotic expression plasmid pXG-Fc to construct the human IgG Fc and Ebola GP fusion protein expression plasmid pXG-modGP-Fc. To achieve expression, the fusion protein expression vector was transfected into high-density 293 cells using transient transfection technology. The recombinant protein was purified by protein A affinity chromatography. BALB/c mice were immunized with the purified fusion protein, and serum antibody titers evaluated by an indirect enzyme-linked immunosorbent assay (ELISA). Purification and analyses of the protein revealed that the eukaryotic expression vector could express the recombinant protein GP-Fc effectively, and that the recombinant protein in the supernatant of the cell culture was present as a dimer. After immunization with the purified recombinant protein, a high titer of antigen-specific IgG could be detected in the serum of immunized mice by indirect ELISA, showing that the recombinant protein had good immunogenicity. These data suggest that we obtained a recombinant protein with good immunogenicity. Our study is the basis for development of a vaccine against the Ebola virus and for screening of monoclonal antibodies.

  3. Recombinant human prion protein inhibits prion propagation in vitro.

    Science.gov (United States)

    Yuan, Jue; Zhan, Yi-An; Abskharon, Romany; Xiao, Xiangzhu; Martinez, Manuel Camacho; Zhou, Xiaochen; Kneale, Geoff; Mikol, Jacqueline; Lehmann, Sylvain; Surewicz, Witold K; Castilla, Joaquín; Steyaert, Jan; Zhang, Shulin; Kong, Qingzhong; Petersen, Robert B; Wohlkonig, Alexandre; Zou, Wen-Quan

    2013-10-09

    Prion diseases are associated with the conformational conversion of the cellular prion protein (PrP(C)) into the pathological scrapie isoform (PrP(Sc)) in the brain. Both the in vivo and in vitro conversion of PrP(C) into PrP(Sc) is significantly inhibited by differences in amino acid sequence between the two molecules. Using protein misfolding cyclic amplification (PMCA), we now report that the recombinant full-length human PrP (rHuPrP23-231) (that is unglycosylated and lacks the glycophosphatidylinositol anchor) is a strong inhibitor of human prion propagation. Furthermore, rHuPrP23-231 also inhibits mouse prion propagation in a scrapie-infected mouse cell line. Notably, it binds to PrP(Sc), but not PrP(C), suggesting that the inhibitory effect of recombinant PrP results from blocking the interaction of brain PrP(C) with PrP(Sc). Our findings suggest a new avenue for treating prion diseases, in which a patient's own unglycosylated and anchorless PrP is used to inhibit PrP(Sc) propagation without inducing immune response side effects.

  4. Purification and Characterization of Recombinant Vaccinia L1R Protein from Escherichia coli

    Science.gov (United States)

    2016-08-01

    RECOMBINANT VACCINIA L1R PROTEIN FROM ESCHERICHIA COLI 1. INTRODUCTION 1.1 Background Vaccinia virus (VACV) is the active component of the...the preparation of the recombinant VACV L1R protein fragment by denaturing , refolding, and purifying material expressed into inclusion bodies in...PURIFICATION AND CHARACTERIZATION OF RECOMBINANT VACCINIA L1R PROTEIN FROM ESCHERICHIA COLI ECBC-TR-1370

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

    African Journals Online (AJOL)

    Jane

    2011-10-24

    Oct 24, 2011 ... protein from tomato fruit and study of its antimicrobial ... the recombinant NP24, as well as to prove the activity of native protein on the bacterial as well as fungal .... The antifungal effect of the recombinant NP24I protein was.

  6. 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.

  7. Recombiner

    International Nuclear Information System (INIS)

    Kikuchi, Nobuo.

    1983-01-01

    Purpose: To shorten the pre-heating time for a recombiner and obtain a uniform temperature distribution for the charged catalyst layer in a BWR type reactor. Constitution: A pre-heating heater is disposed to the outer periphery of a vessel for a recombiner packed with catalysts for recombining hydrogen and oxygen in gases flowing through a radioactive gaseous wastes processing system. Heat pipes for transmitting the heat applied to said container to the catalyst are disposed vertically and horizontally within the container. Different length of the heat pipes are combined. In this way, pre-heating time for the recombiner before the operation start and before the system switching can be shortened and the uniform pre-heating for the inside of the recombiner is also made possible. Further, heater control in the pre-heating can be carried out effectively and with ease. (Moriyama, K.)

  8. Recombiner

    International Nuclear Information System (INIS)

    Osumi, Morimichi.

    1979-01-01

    Purpose: To provide a recombiner which is capable of converting hydrogen gas into water by use of high-frequency heating at comparatively low temperatures and is safe and cheap in cost. Constitution: Hydrogen gas is introduced from an outer pipeline to the main structure of a recombiner, and when it passes through the vicinity of the central part of the recombiner, it is reacted with copper oxide (CuO 2 ) heated to a temperature more than 300 0 C by a high-frequency heater, and converted gently into water by reduction operation (2H 2 + CuO 2 → Cu + 2H 2 O). The thus prepared water is exhausted through the outer pipeline to a suppression pool. A part of hydrogen gas which has not been converted completely into water by the reaction and is remaining as hydrogen is recovered through exhaust nozzles and again introduced into the main structure of the recombiner. (Yoshino, Y.)

  9. 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.

  10. 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 "...

  11. Analysis of recombinant proteins by isoelectric focusing in immobilized pH gradients

    NARCIS (Netherlands)

    Bischoff, Rainer; Roecklin, D.; Roitsch, C.

    1992-01-01

    Isoelectric focusing in immobilized pH gradients (IEF-IPG) was used to analyze three different recombinant proteins. Recombinant leech hirudin (65 amino acids, three disulfide bonds) expressed in Saccharomyces cerevisiae as a secreted protein and purified by anion-exchange and reversed-phase

  12. 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...

  13. Immunogenicity of recombinant feline infectious peritonitis virus spike protein in mice and kittens

    NARCIS (Netherlands)

    Horzinek, M.C.; Vennema, H.; Groot, R. de; Harbour, D.A.; Dalderup, M.; Gruffydd-Jones, T.; Spaan, W.J.M.

    1990-01-01

    The gene encoding the fusogenic spike protein of the coronavirus causing feline infectious peritonitis (FIVP) was recombined into the genome of vaccinia virus, strain WR. The recombinant induced spike protein specific, in vitro neutralizing antibodies in mkice. When kittens were immunized with the

  14. 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

  15. Mutations in the RNA-binding domains of tombusvirus replicase proteins affect RNA recombination in vivo

    International Nuclear Information System (INIS)

    Panaviene, Zivile; Nagy, Peter D.

    2003-01-01

    RNA recombination, which is thought to occur due to replicase errors during viral replication, is one of the major driving forces of virus evolution. In this article, we show evidence that the replicase proteins of Cucumber necrosis virus, a tombusvirus, are directly involved in RNA recombination in vivo. Mutations within the RNA-binding domains of the replicase proteins affected the frequency of recombination observed with a prototypical defective-interfering (DI) RNA, a model template for recombination studies. Five of the 17 replicase mutants tested showed delay in the formation of recombinants when compared to the wild-type helper virus. Interestingly, two replicase mutants accelerated recombinant formation and, in addition, these mutants also increased the level of subgenomic RNA synthesis (Virology 308 (2003), 191-205). A trans-complementation system was used to demonstrate that mutation in the p33 replicase protein resulted in altered recombination rate. Isolated recombinants were mostly imprecise (nonhomologous), with the recombination sites clustered around a replication enhancer region and a putative cis-acting element, respectively. These RNA elements might facilitate the proposed template switching events by the tombusvirus replicase. Together with data in the article cited above, results presented here firmly establish that the conserved RNA-binding motif of the replicase proteins is involved in RNA replication, subgenomic RNA synthesis, and RNA recombination

  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 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...... 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....

  17. Cloning an artificial gene encoding angiostatic anginex: From designed peptide to functional recombinant protein

    International Nuclear Information System (INIS)

    Brandwijk, Ricardo J.M.G.E.; Nesmelova, Irina; Dings, Ruud P.M.; Mayo, Kevin H.; Thijssen, Victor L.J.L.; Griffioen, Arjan W.

    2005-01-01

    Anginex, a designed peptide 33-mer, is a potent angiogenesis inhibitor and anti-tumor agent in vivo. Anginex functions by inhibiting endothelial cell (EC) proliferation and migration leading to detachment and apoptosis of activated EC's. To better understand tumor endothelium targeting properties of anginex and enable its use in gene therapy, we constructed an artificial gene encoding the biologically exogenous peptide and produced the protein recombinantly in Pichia pastoris. Mass spectrometry shows recombinant anginex to be a dimer and circular dichroism shows the recombinant protein folds with β-strand structure like the synthetic peptide. Moreover, like parent anginex, the recombinant protein is active at inhibiting EC growth and migration, as well as inhibiting angiogenesis in vivo in the chorioallantoic membrane of the chick embryo. This study demonstrated that it is possible to produce a functionally active protein version of a rationally designed peptide, using an artificial gene and the recombinant protein approach

  18. Selective Advantage of Recombination in Evolving Protein Populations:. a Lattice Model Study

    Science.gov (United States)

    Williams, Paul D.; Pollock, David D.; Goldstein, Richard A.

    Recent research has attempted to clarify the contributions of several mutational processes, such as substitutions or homologous recombination. Simplistic, tractable protein models, which determine the compact native structure phenotype from the sequence genotype, are well-suited to such studies. In this paper, we use a lattice-protein model to examine the effects of point mutation and homologous recombination on evolving populations of proteins. We find that while the majority of mutation and recombination events are neutral or deleterious, recombination is far more likely to be beneficial. This results in a faster increase in fitness during evolution, although the final fitness level is not significantly changed. This transient advantage provides an evolutionary advantage to subpopulations that undergo recombination, allowing fixation of recombination to occur in the population.

  19. 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.

  20. Serological diagnosis of Strongylus vulgaris infection: use of a recombinant protein

    DEFF Research Database (Denmark)

    Andersen, Ulla Vestergaard; Howe, Daniel K.; Olsen, Susanne Nautrup

    , an immunoreactive cDNA clone was subcloned into E. coli and the plasmid sequenced, the open reading frame encoding the mature protein was cloned into a pET22b expression vector and expressed as a His-tagged recombinant protein in BL21 expression cells. The recombinant protein was used in an indirect enzyme....... vulgaris (n=9) reacted against the recombinant protein, expressed as optic density (OD) readings of >24 % of a positive control, while sera from negative horses had OD readings

  1. Application of native signal sequences for recombinant proteins secretion in Pichia pastoris

    DEFF Research Database (Denmark)

    Borodina, Irina; Do, Duy Duc; Eriksen, Jens C.

    Background Methylotrophic yeast Pichia pastoris is widely used for recombinant protein production, largely due to its ability to secrete correctly folded heterologous proteins to the fermentation medium. Secretion is usually achieved by cloning the recombinant gene after a leader sequence, where...... alpha‐mating factor (MF) prepropeptide from Saccharomyces cerevisiae is most commonly used. Our aim was to test whether signal peptides from P. pastoris native secreted proteins could be used to direct secretion of recombinant proteins. Results Eleven native signal peptides from P. pastoris were tested...... by optimization of expression of three different proteins in P. pastoris. Conclusions Native signal peptides from P. pastoris can be used to direct secretion of recombinant proteins. A novel USER‐based P. pastoris system allows easy cloning of protein‐coding gene with the promoter and leader sequence of choice....

  2. Recombiner

    International Nuclear Information System (INIS)

    Saalfrank, H.

    1985-01-01

    Air containing hydrogen can be oxidized by heating in a container called a recombiner, in order to avoid the collection of hydrogen. The container is long and a large number of straight heating bars are arranged in parallel in it and they are flanged to a lid. The heating bars are surrounded by tubes, in order to obtain good heat transfer by a narrow annular gap. (orig.) [de

  3. Production of Recombinant and Tagged Proteins in the Hyperthermophilic Archaeon Sulfolobus solfataricus

    NARCIS (Netherlands)

    Albers, S.-V.; Jonuscheit, M.; Dinkelaker, S.; Urich, T.; Kletzin, A.; Tampé, R.; Driessen, A.J.M.; Schleper, C.

    Many systems are available for the production of recombinant proteins in bacterial and eukaryotic model organisms, which allow us to study proteins in their native hosts and to identify protein-protein interaction partners. In contrast, only a few transformation systems have been developed for

  4. 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 ...

  5. 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.

  6. 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

    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...... therefore purified rpL23-GFP-His, rpL23-His and GFP from E. coli recombinants using affinity, ion exchange and hydrophobic interaction chromatography. These proteins could be purified with yields of 150, 150 and 1500 microg per gram cellular wet weight, respectively. However, rpL23-GFP-His could only...... proteolytic cleavage sites. We conclude that the generated antibodies can be used to evaluate ribosomal coupling of recombinant target proteins as well as the efficiency of their separation from the ribosome....

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. Recominant Pinoresino-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.

    2003-10-21

    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 from source species Forsythia intermedia, Thuja plicata, Tsuga heterophylla, Eucommia ulmoides, Linum usitatissimum, and Schisandra chinensis, 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.

  13. 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.

  14. 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.

  15. [Study on the anti-NTHi infection of Hap recombinant protein in vivo].

    Science.gov (United States)

    Li, Wan-yi; Wang, Bao-ning; Zuo, Feng-qiong; Zeng, Wei; Feng, Feng; Kuang, Yu; Jiang, Zhong-hua; Li, Ming-yuan

    2010-07-01

    To observe the immune effect of Hap recombinant protein on murine model of bronchopneumonia infected with NTHi, and explore the mechanism about the anti-NTHi infection. The C57BL/6 mice intranasally immunized with purified Hap recombinant protein and CT-B were challenged by NTHi encased in agar beads. The immunifaction of anti-infection was observed through encocyte counting of BALF, bacteria detection of lung and the pathologyical change of lung tissue. In the challenge with NTHi experiment, the inflammatory exudation of the infected murine and pathological change of lung tissue was relieved by combined immunization of Hap recombinant protein and CT-B, and quantity of NTHi in lung of the infected murine was reduced obviously. The Hap recombinant protein also had good ability of anti-NTHi infection in the murine model of NTHi bronchopneumonia. This study could offer the oretical and experimental basis for development of new vaccine against NTHi.

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

    Science.gov (United States)

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

    2016-08-09

    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.

  17. 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...

  18. 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.

  19. 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.

  20. An inducible expression system for high-level expression of recombinant proteins in slow growing mycobacteria.

    Science.gov (United States)

    Leotta, Lisa; Spratt, Joanne M; Kong, Carlyn U; Triccas, James A

    2015-09-01

    A novel protein expression vector utilising the inducible hspX promoter of Mycobacterium tuberculosis was constructed and evaluated in this study. High-level induction of three mycobacterial antigens, comprising up to 9% of bacterial sonicate, was demonstrated in recombinant Mycobacterium bovis BCG when grown under low-oxygen tension, which serves to enhance hspX promoter activity. Recombinant proteins were efficiently purified from bacterial lysates in a soluble form by virtue of a C-terminal 6-histidine tag. Purification of the immunodominant M. tuberculosis Ag85B antigen using this system resulted in a recombinant protein that stimulated significant IFN-γ release from Ag85B-reactive T cells generated after vaccination of mice with an Ag85B-expressing vaccine. Further, the M. tuberculosis L-alanine dehydrogenase (Ald) protein purified from recombinant BCG displayed strong enzymatic activity in recombinant form. This study demonstrated that high levels of native-like recombinant mycobacterial proteins can be produced in mycobacterial hosts, and this may aid the analysis of mycobacterial protein function and the development of new treatments. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. 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

  2. Regulation of homologous recombination repair protein Rad51 by Ku70

    International Nuclear Information System (INIS)

    Du Liqing; Liu Qiang; Wang Yan; Xu Chang; Cao Jia; Fu Yue; Chen Fenghua; Fan Feiyue

    2013-01-01

    Objective: To explore the regulative effect of non-homologous end joining (NHEJ)protein Ku70 on homologous recombination repair protein Rad51, and to investigate the synergistic mechanism of homologous recombination repair in combination with NHEJ. Methods: Observed Rad51 protein expression after transfect Ku70 small interfering RNA or Ku70 plasmid DNA into tumor cells using Western blot. Results: Expression of Rad51 was obviously reduced after pretreated with Ku70 small interfering RNA. And with the increasing expression of Ku70 protein after transfection of Ku70 plasmid DNA PGCsi3.0-hKu70 into tumor cell lines, the Rad51 protein expression was increased. Conclusion: Ku70 protein has regulating effect on gene expression of Rad51, and it might participate in the collaboration between homologous recombination repair and NHEJ. (authors)

  3. 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

    Surface plasmon resonance and mass spectrometry (SPR-MS) has been combined for quality check of recombinant 6xHis-tagged 14-3-3 proteins expressed in Escherichia coli. Lysates were injected over an SPR sensorchip with immobilized Ni2+ for SPR analysis of the specific Ni2+ binding response...... and stability. To validate the identity, intactness and homogeneity of the captured proteins were eluted for mass spectrometric analysis of intact molecular weight and peptide mass mapping. Additionally, the captured recombinant proteins were investigated for specific binding to known phosphorylated ligands...... 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...

  4. Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli

    DEFF Research Database (Denmark)

    Sørensen, Hans; Mortensen, Kim

    2005-01-01

    Pure, soluble and functional proteins are of high demand in modern biotechnology. Natural protein sources rarely meet the requirements for quantity, ease of isolation or price and hence recombinant technology is often the method of choice. Recombinant cell factories are constantly employed...... molecular tools available. In spite of all these qualities, expression of recombinant proteins with E. coli as the host often results in insoluble and/or nonfunctional proteins. Here we review new approaches to overcome these obstacles by strategies that focus on either controlled expression of target...... for the production of protein preparations bound for downstream purification and processing. Eschericia coli is a frequently used host, since it facilitates protein expression by its relative simplicity, its inexpensive and fast high density cultivation, the well known genetics and the large number of compatible...

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. Binding of recombinant apolipoprotein(a) to extracellular matrix proteins

    NARCIS (Netherlands)

    van der Hoek, Y. Y.; Sangrar, W.; Côté, G. P.; Kastelein, J. J.; Koschinsky, M. L.

    1994-01-01

    Elevated levels of lipoprotein(a), which consists of apolipoprotein(a) [apo(a)] covalently linked to a low-density lipoprotein-like moiety, is an independent risk factor for the development of atherosclerosis. We show that a recombinant form of apo(a) [r-apo(a)] binds strongly to fibronectin and

  10. Protection against myxomatosis and rabbit viral hemorrhagic disease with recombinant myxoma viruses expressing rabbit hemorrhagic disease virus capsid protein

    OpenAIRE

    Bertagnoli, Stéphane; Gelfi, Jacqueline; Le Gall, Ghislaine; Boilletot, Eric; Vautherot, Jean-François; Rasschaert, Denis; Laurent, Sylvie; Petit, Frédérique; Boucraut-Baralon, Corine; Milon, Alain

    1996-01-01

    Two myxoma virus-rabbit hemorrhagic disease virus (RHDV) recombinant viruses were constructed with the SG33 strain of myxoma virus to protect rabbits against myxomatosis and rabbit viral hemorrhagic disease. These recombinant viruses expressed the RHDV capsid protein (VP60). The recombinant protein, which is 60 kDa in size, was antigenic, as revealed by its reaction in immunoprecipitation with antibodies raised against RHDV. Both recombinant viruses induced high levels of RHDV- and myxoma vir...

  11. 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.

  12. 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...

  13. Isolation of recombinant antibodies directed against surface proteins of Clostridium difficile.

    Science.gov (United States)

    Shirvan, Ali Nazari; Aitken, Robert

    2016-01-01

    Clostridium difficile has emerged as an increasingly important nosocomial pathogen and the prime causative agent of antibiotic-associated diarrhoea and pseudomembranous colitis in humans. In addition to toxins A and B, immunological studies using antisera from patients infected with C. difficile have shown that a number of other bacterial factors contribute to the pathogenesis, including surface proteins, which are responsible for adhesion, motility and other interactions with the human host. In this study, various clostridial targets, including FliC, FliD and cell wall protein 66, were expressed and purified. Phage antibody display yielded a large panel of specific recombinant antibodies, which were expressed, purified and characterised. Reactions of the recombinant antibodies with their targets were detected by enzyme-linked immunosorbent assay; and Western blotting suggested that linear rather than conformational epitopes were recognised. Binding of the recombinant antibodies to surface-layer proteins and their components showed strain specificity, with good recognition of proteins from C. difficile 630. However, no reaction was observed for strain R20291-a representative of the 027 ribotype. Binding of the recombinant antibodies to C. difficile M120 extracts indicated that a component of a surface-layer protein of this strain might possess immunoglobulin-binding activities. The recombinant antibodies against FliC and FliD proteins were able to inhibit bacterial motility. Copyright © 2016. Published by Elsevier Editora Ltda.

  14. 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.

  15. Expression and the antigenicity of recombinant coat proteins of tungro viruses expressed in Escherichia coli.

    Science.gov (United States)

    Yee, Siew Fung; Chu, Chia Huay; Poili, Evenni; Sum, Magdline Sia Henry

    2017-02-01

    Rice tungro disease (RTD) is a recurring disease affecting rice farming especially in the South and Southeast Asia. The disease is commonly diagnosed by visual observation of the symptoms on diseased plants in paddy fields and by polymerase chain reaction (PCR). However, visual observation is unreliable and PCR can be costly. High-throughput as well as relatively cheap detection methods are important for RTD management for screening large number of samples. Due to this, detection by serological assays such as immunoblotting assays and enzyme-linked immunosorbent assay are preferred. However, these serological assays are limited by lack of continuous supply of antibodies as reagents due to the difficulty in preparing sufficient purified virions as antigens. This study aimed to generate and evaluate the reactivity of the recombinant coat proteins of Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV) as alternative antigens to generate antibodies. The genes encoding the coat proteins of both viruses, RTBV (CP), and RTSV (CP1, CP2 and CP3) were cloned and expressed as recombinant fusion proteins in Escherichia coli. All of the recombinant fusion proteins, with the exception of the recombinant fusion protein of the CP2 of RTSV, were reactive against our in-house anti-tungro rabbit serum. In conclusion, our study showed the potential use of the recombinant fusion coat proteins of the tungro viruses as alternative antigens for production of antibodies for diagnostic purposes. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. 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.

  17. Recombinant fusion protein of albumin-retinol binding protein inactivates stellate cells

    International Nuclear Information System (INIS)

    Choi, Soyoung; Park, Sangeun; Kim, Suhyun; Lim, Chaeseung; Kim, Jungho; Cha, Dae Ryong; Oh, Junseo

    2012-01-01

    Highlights: ► We designed novel recombinant albumin-RBP fusion proteins. ► Expression of fusion proteins inactivates pancreatic stellate cells (PSCs). ► Fusion proteins are successfully internalized into and inactivate PSCs. ► RBP moiety mediates cell specific uptake of fusion protein. -- Abstract: Quiescent pancreatic- (PSCs) and hepatic- (HSCs) stellate cells store vitamin A (retinol) in lipid droplets via retinol binding protein (RBP) receptor and, when activated by profibrogenic stimuli, they transform into myofibroblast-like cells which play a key role in the fibrogenesis. Despite extensive investigations, there is, however, currently no appropriate therapy available for tissue fibrosis. We previously showed that the expression of albumin, composed of three homologous domains (I–III), inhibits stellate cell activation, which requires its high-affinity fatty acid-binding sites asymmetrically distributed in domain I and III. To attain stellate cell-specific uptake, albumin (domain I/III) was coupled to RBP; RBP-albumin domain III (R-III) and albumin domain I -RBP-albumin III (I-R-III). To assess the biological activity of fusion proteins, cultured PSCs were used. Like wild type albumin, expression of R-III or I-R-III in PSCs after passage 2 (activated PSCs) induced phenotypic reversal from activated to fat-storing cells. On the other hand, R-III and I-R-III, but not albumin, secreted from transfected 293 cells were successfully internalized into and inactivated PSCs. FPLC-purified R-III was found to be internalized into PSCs via caveolae-mediated endocytosis, and its efficient cellular uptake was also observed in HSCs and podocytes among several cell lines tested. Moreover, tissue distribution of intravenously injected R-III was closely similar to that of RBP. Therefore, our data suggest that albumin-RBP fusion protein comprises of stellate cell inactivation-inducing moiety and targeting moiety, which may lead to the development of effective anti

  18. Rapid production of functionalized recombinant proteins: marrying ligation independent cloning and in vitro protein ligation.

    Science.gov (United States)

    Kushnir, Susanna; Marsac, Yoann; Breitling, Reinhard; Granovsky, Igor; Brok-Volchanskaya, Vera; Goody, Roger S; Becker, Christian F W; Alexandrov, Kirill

    2006-01-01

    Functional genomics and proteomics have been very active fields since the sequencing of several genomes was completed. To assign a physiological role to the newly discovered coding genes with unknown function, new generic methods for protein production, purification, and targeted functionalization are needed. This work presents a new vector, pCYSLIC, that allows rapid generation of Escherichia coli expression constructs via ligation-independent cloning (LIC). The vector is designed to facilitate protein purification by either Ni-NTA or GSH affinity chromatography. Subsequent proteolytic removal of affinity tags liberates an N-terminal cysteine residue that is then used for covalent modification of the target protein with different biophysical probes via protein ligation. The described system has been tested on 36 mammalian Rab GTPases, and it was demonstrated that recombinant GTPases produced with pCYSLIC could be efficiently modified with fluorescein or biotin in vitro. Finally, LIC was compared with the recently developed In-Fusion cloning method, and it was demonstrated that In-Fusion provides superior flexibility in choice of expression vector. By the application of In-Fusion cloning Cys-Rab6A GTPase with an N-terminal cysteine residue was generated employing unmodified pET30a vector and TVMV protease.

  19. 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...... research. Here we review the cell biological response to DSBs in mitotically growing cells with an emphasis on homologous recombination pathways in yeast Saccharomyces cerevisiae and in mammalian cells....

  20. 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...

  1. 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...

  2. The recombinant 120-kilodalton protein of Ehrlichia chaffeensis, a potential diagnostic tool.

    OpenAIRE

    Yu, X J; Crocquet-Valdes, P; Cullman, L C; Walker, D H

    1996-01-01

    DNA encoding two repeat units of 120-kDa protein of Ehrlichia chaffeensis was cloned into the expression vector pGEX and expressed in Escherichia coli. The sensitivity and specificity of a dot blot assay for detection of human antibodies with the recombinant protein were 86 and 100%, respectively, compared with an indirect immunofluorescence assay.

  3. 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...

  4. 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

  5. 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.

  6. Recombinant Expression Screening of P. aeruginosa Bacterial Inner Membrane Proteins

    Directory of Open Access Journals (Sweden)

    Jeffery Constance J

    2010-11-01

    Full Text Available Abstract Background Transmembrane proteins (TM proteins make up 25% of all proteins and play key roles in many diseases and normal physiological processes. However, much less is known about their structures and molecular mechanisms than for soluble proteins. Problems in expression, solubilization, purification, and crystallization cause bottlenecks in the characterization of TM proteins. This project addressed the need for improved methods for obtaining sufficient amounts of TM proteins for determining their structures and molecular mechanisms. Results Plasmid clones were obtained that encode eighty-seven transmembrane proteins with varying physical characteristics, for example, the number of predicted transmembrane helices, molecular weight, and grand average hydrophobicity (GRAVY. All the target proteins were from P. aeruginosa, a gram negative bacterial opportunistic pathogen that causes serious lung infections in people with cystic fibrosis. The relative expression levels of the transmembrane proteins were measured under several culture growth conditions. The use of E. coli strains, a T7 promoter, and a 6-histidine C-terminal affinity tag resulted in the expression of 61 out of 87 test proteins (70%. In this study, proteins with a higher grand average hydrophobicity and more transmembrane helices were expressed less well than less hydrophobic proteins with fewer transmembrane helices. Conclusions In this study, factors related to overall hydrophobicity and the number of predicted transmembrane helices correlated with the relative expression levels of the target proteins. Identifying physical characteristics that correlate with protein expression might aid in selecting the "low hanging fruit", or proteins that can be expressed to sufficient levels using an E. coli expression system. The use of other expression strategies or host species might be needed for sufficient levels of expression of transmembrane proteins with other physical

  7. 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.

  8. Hydroxyapatite coating on the titanium substrate modulated by a recombinant collagen-like protein

    International Nuclear Information System (INIS)

    Pan Mingli; Kong Xiangdong; Cai Yurong; Yao Juming

    2011-01-01

    Research highlights: → Hydroxyapatite was deposited on alkali-heat treated Ti substrate by immersing in 1.5 x SBF solution containing the recombinant collagen-like protein. → The recombinant collagen-like protein accelerated the preferential nucleation and growth of hydroxyapatite along c axis on the Ti substrate. → Hydroxyapatite-collagen composite on the Ti substrate promoted the attachment, subsequently proliferation and differentiation of MG-63 cells. - Abstract: Plenty of techniques have been developed to modify the surface character of titanium (Ti) and its alloys in order to realize their biological bond to natural bone. In this work, a biomimetic process was employed to form a hydroxyapatite (HAp) coating on the alkali-heat treated Ti substrate in 1.5 times simulated body fluid (1.5 x SBF) with the addition of a recombinant collagen-like protein. The coating was characterized using SEM-EDX, FESEM, and XRD. Results showed that the recombinant collagen-like protein could accelerate the preferential nucleation and directional growth along c axis of HAp on the pretreated Ti substrates. The investigation of in vitro cell cultivation showed that the existence of recombinant collagen-like protein in coating could improve the initial cell adhesion, proliferation and differentiation of MG-63 cells, which implied the materials possessed excellent biocompatibility and had a wide potential in biomedical application.

  9. Hydroxyapatite coating on the titanium substrate modulated by a recombinant collagen-like protein

    Energy Technology Data Exchange (ETDEWEB)

    Pan Mingli [Key Laboratory of Advanced Textile Materials and Manufacturing Technology of Ministry of Education, College of Materials and Textile, Zhejiang Sci-Tech University, Hangzhou 310018 (China); Kong Xiangdong [College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018 (China); Cai Yurong [Key Laboratory of Advanced Textile Materials and Manufacturing Technology of Ministry of Education, College of Materials and Textile, Zhejiang Sci-Tech University, Hangzhou 310018 (China); Yao Juming, E-mail: yaoj@zstu.edu.cn [Key Laboratory of Advanced Textile Materials and Manufacturing Technology of Ministry of Education, College of Materials and Textile, Zhejiang Sci-Tech University, Hangzhou 310018 (China)

    2011-04-15

    Research highlights: {yields} Hydroxyapatite was deposited on alkali-heat treated Ti substrate by immersing in 1.5 x SBF solution containing the recombinant collagen-like protein. {yields} The recombinant collagen-like protein accelerated the preferential nucleation and growth of hydroxyapatite along c axis on the Ti substrate. {yields} Hydroxyapatite-collagen composite on the Ti substrate promoted the attachment, subsequently proliferation and differentiation of MG-63 cells. - Abstract: Plenty of techniques have been developed to modify the surface character of titanium (Ti) and its alloys in order to realize their biological bond to natural bone. In this work, a biomimetic process was employed to form a hydroxyapatite (HAp) coating on the alkali-heat treated Ti substrate in 1.5 times simulated body fluid (1.5 x SBF) with the addition of a recombinant collagen-like protein. The coating was characterized using SEM-EDX, FESEM, and XRD. Results showed that the recombinant collagen-like protein could accelerate the preferential nucleation and directional growth along c axis of HAp on the pretreated Ti substrates. The investigation of in vitro cell cultivation showed that the existence of recombinant collagen-like protein in coating could improve the initial cell adhesion, proliferation and differentiation of MG-63 cells, which implied the materials possessed excellent biocompatibility and had a wide potential in biomedical application.

  10. Indirect Enzyme-Linked Immunosorbent Assay for Detection of Immunoglobulin G Reactive with a Recombinant Protein Expressed from the Gene Encoding the 116-Kilodalton Protein of Mycoplasma pneumoniae

    OpenAIRE

    Duffy, Michael F.; Whithear, Kevin G.; Noormohammadi, Amir H.; Markham, Philip F.; Catton, Michael; Leydon, Jennie; Browning, Glenn F.

    1999-01-01

    Serology remains the method of choice for laboratory diagnosis of Mycoplasma pneumoniae infection. Currently available serological tests employ complex cellular fractions of M. pneumoniae as antigen. To improve the specificity of M. pneumoniae diagnosis, a recombinant protein was assessed as a serodiagnostic reagent. A panel of recombinant proteins were expressed from a cloned M. pneumoniae gene that encodes a 116-kDa surface protein antigen. The recombinant proteins were assessed for reactiv...

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

    International Nuclear Information System (INIS)

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

    2011-01-01

    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.

  12. An overview on molecular chaperones enhancing solubility of expressed recombinant proteins with correct folding.

    Science.gov (United States)

    Mamipour, Mina; Yousefi, Mohammadreza; Hasanzadeh, Mohammad

    2017-09-01

    The majority of research topics declared that most of the recombinant proteins have been expressed by Escherichia coli in basic investigations. But the majority of high expressed proteins formed as inactive recombinant proteins that are called inclusion body. To overcome this problem, several methods have been used including suitable promoter, environmental factors, ladder tag to secretion of proteins into the periplasm, gene protein optimization, chemical chaperones and molecular chaperones sets. Co-expression of the interest protein with molecular chaperones is one of the common methods The chaperones are a group of proteins, which are involved in making correct folding of recombinant proteins. Chaperones are divided two groups including; cytoplasmic and periplasmic chaperones. Moreover, periplasmic chaperones and proteases can be manipulated to increase the yields of secreted proteins. In this article, we attempted to review cytoplasmic chaperones such as Hsp families and periplasmic chaperones including; generic chaperones, specialized chaperones, PPIases, and proteins involved in disulfide bond formation. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Trafficking of endoplasmic reticulum-retained recombinant proteins is unpredictable in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Thomas eDe Meyer

    2014-09-01

    Full Text Available A wide variety of recombinant proteins has been produced in the dicot model plant, Arabidopsis thaliana. Many of these proteins are targeted for secretion by means of an N terminal endoplasmic reticulum (ER signal peptide. In addition, they can also be designed for ER retention by adding a C terminal H/KDEL-tag. Despite extensive knowledge of the protein trafficking pathways, the final protein destination, especially of such H/KDEL-tagged recombinant proteins, is unpredictable. In this respect, glycoproteins are ideal study objects. Microscopy experiments reveal their deposition pattern and characterization of their N-glycans aids in elucidating the trafficking. Here, we combine microscopy and N glycosylation data generated in Arabidopsis leaves and seeds, and highlight the lack of a decent understanding of heterologous protein trafficking.

  14. 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.

    2015-01-01

    suggesting that the proteins 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. Conclusions/Significance 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. Trial Registration ClinicalTrials.gov NCT00663546 PMID:26701602

  15. Bicistronic expression plasmid for the rapid production of recombinant fused proteins in Escherichia coli.

    Science.gov (United States)

    Yero, Daniel; Pajón, Rolando; Niebla, Olivia; Sardiñas, Gretel; Vivar, Isbel; Perera, Yasser; García, Darien; Delgado, Maité; Cobas, Karem

    2006-04-01

    In the post-genomic era, every aspect of the production of proteins must be accelerated. In this way, several vectors are currently exploited for rapid production of recombinant proteins in Escherichia coli. N-terminal fusions to the first 47 amino acids of the LpdA (dihydrolipoamide dehydrogenase A) protein of Neisseria meningitidis have been shown to increase the expression of recombinant proteins. Consequently, we have constructed a modified N-terminal LpdA fusion vector, introducing the blue/white colony selection by exploiting a bicistronic gene organization. In the new vector, the sequence encoding the first 47 amino acids of meningococcal LpdA and the alpha-peptide sequence of beta-galactosidase were connected via a ribosome-binding site, and two MCSs (multiple cloning sites) were located surrounding the latter, allowing efficient cloning by colour selection of recombinants. The vector was also improved with the addition of a C-terminal polyhistidine tag, and an EKS (enterokinase recognition sequence) immediately after the LpdA fusion sequence. The new plasmid was employed in the expression and purification of six different bacterial polypeptides. One of these recombinant proteins, P6 protein from Haemophilus influenzae, was used as a model and its N-terminal fusion sequence was totally removed from the recombinant version after incubation with the enterokinase protease, while the polyhistidine tail successfully allowed the purification of the unfused protein from the protease reaction. Two completely new neisserial vaccine candidates, NMB0088 and NMB1126 proteins, were cloned, expressed and purified using this system. To our knowledge, this constitutes the first report of the cloning and expression of these proteins in E. coli.

  16. 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.

  17. Application of Recombinant Proteins for Serodiagnosis of Visceral Leishmaniasis in Humans and Dogs.

    Science.gov (United States)

    Farahmand, Mahin; Nahrevanian, Hossein

    2016-07-01

    Visceral leishmaniasis (VL) is a zoonotic disease caused by leishmania species. Dogs are considered to be the main reservoir of VL. A number of methods and antigen-based assays are used for the diagnosis of leishmaniasis. However, currently available methods are mainly based on direct examination of tissues for the presence of parasites, which is highly invasive. A variety of serological tests are commonly applied for VL diagnosis, including indirect fluorescence antibody test, enzyme-linked immunosorbent assay (ELISA), dot-ELISA, direct agglutination test, Western-blotting, and immunochromatographic test. However, when soluble antigens are used, serological tests are less specific due to cross-reactivity with other parasitic diseases. Several studies have attempted to replace soluble antigens with recombinant proteins to improve the sensitivity and the specificity of the immunodiagnostic tests. Major technological advances in recombinant antigens as reagents for the serological diagnosis of VL have led to high sensitivity and specificity of these serological tests. A great number of recombinant proteins have been shown to be effective for the diagnosis of leishmania infection in dogs, the major reservoir of L. infantum. Although few recombinant proteins with high efficacy provide reasonable results for the diagnosis of human and canine VL, more optimization is still needed for the appropriate antigens to provide high-throughput performance. This review aims to explore the application of different recombinant proteins for the serodiagnosis of VL in humans and dogs.

  18. 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.

  19. Study of recombinant proteins derived from Ser-2 gene of Bombyx mori

    OpenAIRE

    STAŠKOVÁ, Tereza

    2012-01-01

    Four different variants of recombinant proteins derived from Bombyx mori Ser-2 gene were expressed in bacteria. The ability of these proteins to coat hydrofobic surfaces and to support growth of various types of adherent cells in vitro were examined. It was shown that these proteins support cell adhesion and proliferation, and could be used as coating agents to realize surfaces suitable for growth of vertebrate and insect cells.

  20. 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.

  1. 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.

  2. 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

  3. Serum from Nipah Virus Patients Recognises Recombinant Viral Proteins Produced in Escherichia coli.

    Science.gov (United States)

    Tiong, Vunjia; Lam, Chui-Wan; Phoon, Wai-Hong; AbuBakar, Sazaly; Chang, Li-Yen

    2017-01-24

    The genes for Nipah virus (NiV) proteins were amplified from viral RNA, cloned into the plasmid pTriEx-3 Hygro, expressed, and purified using immobilized metal affinity chromatography. The recombinant N, F, and G NiV proteins (rNiV-N, rNiV-F, and rNiV-G), were successfully expressed in Escherichia coli and purified with a yield of 4, 16, and 4 mg/L, respectively. All 3 recombinant viral proteins reacted with all 19 samples of NiV-positive human sera. The rNiV-N and rNiV-G proteins were the most immunogenic. The recombinant viral proteins did not react with any of the 12 NiV-negative sera. However, serum from a patient with a late-onset relapsing NiV infection complication was found to be primarily reactive to rNiV-G only. Additionally, there is a distinctive variation in the profile of antigen-reactive bands between the sample from a case of relapsing NiV encephalitis and that of acute NiV infection. The overall findings of this study suggest that the recombinant viral proteins have the potential to be developed further for use in the detection of NiV infection, and continuous biosurveillance of NiV infection in resource-limited settings.

  4. N-terminal processing of affinity-tagged recombinant proteins purified by IMAC procedures.

    Science.gov (United States)

    Mooney, Jane T; Fredericks, Dale P; Christensen, Thorkild; Bruun Schiødt, Christine; Hearn, Milton T W

    2015-07-01

    The ability of a new class of metal binding tags to facilitate the purification of recombinant proteins, exemplified by the tagged glutathione S-transferase and human growth hormone, from Escherichia coli fermentation broths and lysates has been further investigated. These histidine-containing tags exhibit high affinity for borderline metal ions chelated to the immobilised ligand, 1,4,7-triazacyclononane (tacn). The use of this tag-tacn immobilised metal ion affinity chromatography (IMAC) system engenders high selectivity with regard to host cell protein removal and permits facile tag removal from the E. coli-expressed recombinant protein. In particular, these tags were specifically designed to enable their efficient removal by the dipeptidyl aminopeptidase 1 (DAP-1), thus capturing the advantages of high substrate specificity and rates of cleavage. MALDI-TOF MS analysis of the cleaved products from the DAP-1 digestion of the recombinant N-terminally tagged proteins confirmed the complete removal of the tag within 4-12 h under mild experimental conditions. Overall, this study demonstrates that the use of tags specifically designed to target tacn-based IMAC resins offers a comprehensive and flexible approach for the purification of E. coli-expressed recombinant proteins, where complete removal of the tag is an essential prerequisite for subsequent application of the purified native proteins in studies aimed at delineating the molecular and cellular basis of specific biological processes. Copyright © 2015 John Wiley & Sons, Ltd.

  5. 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.

  6. 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.

  7. 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

  8. Recombinant dioscorins of the yam storage protein expressed in Escherichia coli exhibit antioxidant and immunomodulatory activities.

    Science.gov (United States)

    Jheng, Yi-Jyun; Tsai, Wei-Yi; Chen, Kuo-Hsuan; Lin, Kuo-Wei; Chyan, Chia-Lin; Yang, Ching-Chi; Lin, Kuo-Chih

    2012-09-01

    Dioscorins, the major storage proteins in yam tubers, exhibit biochemical and immunomodulatroy activities. To investigate the potential application of dioscorins in biomedical research, we expressed the dioscorin genes Dj-dioA3 and Dp-dioA2 from Dioscorea japonica and Dioscorea pseudojaponica, respectively, in E. coli and routinely obtained approximately 15 mg proteins per liter Escherichia coli culture (mg/L) to 30 mg/L of rDj-dioscorinA3 and 4 to 8 mg/L of rDp-dioscorinA2. Western blot analyses revealed that both recombinant dioscorins contained epitopes with similar antigenicities to those of the native dioscorins. Results from dithiothreitol (DTT) treatment followed by monobromobimane (mBBr) staining showed that both recombinant dioscorins, like the native dioscorins, contain an intramolecular disulfide bond between Cys(28) and Cys(187) residues. Circular dichroism spectroscopy findings indicated that the secondary structural contents of the recombinant dioscorins showed high similarity to those of their corresponding native dioscorins. Both recombinant dioscorins, like the native dioscorins, exhibited 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and Toll-like receptor 4 signaling activities, and stimulated the phagocytosis of E. coli by macrophage. Overall, our results indicated that substantial amounts of recombinant dioscorins can be purified easily from E. coli and that these recombinant dioscorins are appropriate for application in future investigations of the biomedical functions of dioscorins. Copyright © 2012 Elsevier Inc. All rights reserved.

  9. 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.

  10. Recombinant fusion protein of albumin-retinol binding protein inactivates stellate cells

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Soyoung; Park, Sangeun; Kim, Suhyun [Laboratory of Cellular Oncology, Korea University Graduate School of Medicine, Ansan, Gyeonggi do 425-707 (Korea, Republic of); Lim, Chaeseung [Department of Laboratory Medicine, Korea University Guro Hospital, Seoul 152-703 (Korea, Republic of); Kim, Jungho [Department of Life Science, Sogang University, Seoul 121-742 (Korea, Republic of); Cha, Dae Ryong [Department of Internal Medicine, Korea University Ansan Hospital, Ansan, Gyeonggi do 425-020 (Korea, Republic of); Oh, Junseo, E-mail: ohjs@korea.ac.kr [Laboratory of Cellular Oncology, Korea University Graduate School of Medicine, Ansan, Gyeonggi do 425-707 (Korea, Republic of)

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer We designed novel recombinant albumin-RBP fusion proteins. Black-Right-Pointing-Pointer Expression of fusion proteins inactivates pancreatic stellate cells (PSCs). Black-Right-Pointing-Pointer Fusion proteins are successfully internalized into and inactivate PSCs. Black-Right-Pointing-Pointer RBP moiety mediates cell specific uptake of fusion protein. -- Abstract: Quiescent pancreatic- (PSCs) and hepatic- (HSCs) stellate cells store vitamin A (retinol) in lipid droplets via retinol binding protein (RBP) receptor and, when activated by profibrogenic stimuli, they transform into myofibroblast-like cells which play a key role in the fibrogenesis. Despite extensive investigations, there is, however, currently no appropriate therapy available for tissue fibrosis. We previously showed that the expression of albumin, composed of three homologous domains (I-III), inhibits stellate cell activation, which requires its high-affinity fatty acid-binding sites asymmetrically distributed in domain I and III. To attain stellate cell-specific uptake, albumin (domain I/III) was coupled to RBP; RBP-albumin{sup domain} {sup III} (R-III) and albumin{sup domain} {sup I}-RBP-albumin{sup III} (I-R-III). To assess the biological activity of fusion proteins, cultured PSCs were used. Like wild type albumin, expression of R-III or I-R-III in PSCs after passage 2 (activated PSCs) induced phenotypic reversal from activated to fat-storing cells. On the other hand, R-III and I-R-III, but not albumin, secreted from transfected 293 cells were successfully internalized into and inactivated PSCs. FPLC-purified R-III was found to be internalized into PSCs via caveolae-mediated endocytosis, and its efficient cellular uptake was also observed in HSCs and podocytes among several cell lines tested. Moreover, tissue distribution of intravenously injected R-III was closely similar to that of RBP. Therefore, our data suggest that albumin-RBP fusion protein comprises

  11. Recombinant Protein Production of Earthworm Lumbrokinase for Potential Antithrombotic Application

    Directory of Open Access Journals (Sweden)

    Kevin Yueju Wang

    2013-01-01

    Full Text Available Earthworms have been used as a traditional medicine in China, Japan, and other Far East countries for thousands of years. Oral administration of dry earthworm powder is considered as a potent and effective supplement for supporting healthy blood circulation. Lumbrokinases are a group of enzymes that were isolated and purified from different species of earthworms. These enzymes are recognized as fibrinolytic agents that can be used to treat various conditions associated with thrombosis. Many lumbrokinase (LK genes have been cloned and characterized. Advances in genetic technology have provided the ability to produce recombinant LK and have made it feasible to purify a single lumbrokinase enzyme for potential antithrombotic application. In this review, we focus on expression systems that can be used for lumbrokinase production. In particular, the advantages of using a transgenic plant system to produce edible lumbrokinase are described.

  12. 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.

  13. 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).

  14. The Use of Recombinant Hemagglutinine Protein of Rinderpest Virus in Enzyme Immunoassay

    OpenAIRE

    BULUT, Hakan; BOLAT, Yusuf

    2003-01-01

    In this study, Rinderpest virus (RPV) recombinant hemagglutinine protein (rH) fused with protein A region of Staphylococcus aureus was expressed in Escherichia coli and purified by IgG affinity chromatography. rH protein was also used to establish enzyme immunoassay. Therefore, to prevent IgG binding to the protein A the wells coated with the rH proteins were blocked by human serum. Afterwards, RPV antigens were added to the wells to evaluate this assay. To this end, serum from mice immunized...

  15. Rational design of new materials using recombinant structural proteins: Current state and future challenges.

    Science.gov (United States)

    Sutherland, Tara D; Huson, Mickey G; Rapson, Trevor D

    2018-01-01

    Sequence-definable polymers are seen as a prerequisite for design of future materials, with many polymer scientists regarding such polymers as the holy grail of polymer science. Recombinant proteins are sequence-defined polymers. Proteins are dictated by DNA templates and therefore the sequence of amino acids in a protein is defined, and molecular biology provides tools that allow redesign of the DNA as required. Despite this advantage, proteins are underrepresented in materials science. In this publication we investigate the advantages and limitations of using proteins as templates for rational design of new materials. Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.

  16. 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.

  17. 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.

  18. The quaternary structure of the recombinant bovine odorant-binding protein is modulated by chemical denaturants.

    Directory of Open Access Journals (Sweden)

    Olga V Stepanenko

    Full Text Available A large group of odorant-binding proteins (OBPs has attracted great scientific interest as promising building blocks in constructing optical biosensors for dangerous substances, such as toxic and explosive molecules. Native tissue-extracted bovine OBP (bOBP has a unique dimer folding pattern that involves crossing the α-helical domain in each monomer over the other monomer's β-barrel. In contrast, recombinant bOBP maintaining the high level of stability inherent to native tissue bOBP is produced in a stable native-like state with a decreased tendency for dimerization and is a mixture of monomers and dimers in a buffered solution. This work is focused on the study of the quaternary structure and the folding-unfolding processes of the recombinant bOBP in the absence and in the presence of guanidine hydrochloride (GdnHCl. Our results show that the recombinant bOBP native dimer is only formed at elevated GdnHCl concentrations (1.5 M. This process requires re-organizing the protein structure by progressing through the formation of an intermediate state. The bOBP dimerization process appears to be irreversible and it occurs before the protein unfolds. Though the observed structural changes for recombinant bOBP at pre-denaturing GdnHCl concentrations show a local character and the overall protein structure is maintained, such changes should be considered where the protein is used as a sensitive element in a biosensor system.

  19. 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.

  20. that Bind Specifically to Recombinant Envelope Protein of Dengue

    African Journals Online (AJOL)

    Tropical Journal of Pharmaceutical Research June 2015; 14 (6): 997-1003 ... Revised accepted: 30 April 2015. Abstract ... Results: The 45 KDa, 43 KDa and 30 KDa plasma membrane proteins were identified as viral envelope targets.

  1. 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)

  2. Respiratory syncytial virus subunit vaccine based on a recombinant fusion protein expressed transiently in mammalian cells.

    Science.gov (United States)

    Nallet, Sophie; Amacker, Mario; Westerfeld, Nicole; Baldi, Lucia; König, Iwo; Hacker, David L; Zaborosch, Christiane; Zurbriggen, Rinaldo; Wurm, Florian M

    2009-10-30

    Although respiratory syncytial virus (RSV) causes severe lower respiratory tract infection in infants and adults at risk, no RSV vaccine is currently available. In this report, efforts toward the generation of an RSV subunit vaccine using recombinant RSV fusion protein (rRSV-F) are described. The recombinant protein was produced by transient gene expression (TGE) in suspension-adapted human embryonic kidney cells (HEK-293E) in 4 L orbitally shaken bioreactors. It was then purified and formulated in immunostimulating reconstituted influenza virosomes (IRIVs). The candidate vaccine induced anti-RSV-F neutralizing antibodies in mice, and challenge studies in cotton rats are ongoing. If successful in preclinical and clinical trials, this will be the first recombinant subunit vaccine produced by large-scale TGE in mammalian cells.

  3. [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.

  4. A novel genetic system for recombinant protein secretion in the Antarctic Pseudoalteromonas haloplanktis TAC125

    Directory of Open Access Journals (Sweden)

    Marino Gennaro

    2006-12-01

    Full Text Available Abstract Background The final aim of recombinant protein production is both to have a high specific production rate and a high product quality. It was already shown that using cold-adapted bacteria as host vectors, some "intractable" proteins can be efficiently produced at temperature as low as 4°C. Results A novel genetic system for the production and secretion of recombinant proteins in the Antarctic Gram-negative bacterium Pseudoalteromonas haloplanktis TAC125 was set up. This system aims at combining the low temperature recombinant product production with the advantages of extra-cellular protein targeting. The psychrophilic α-amylase from Pseudoalteromonas haloplanktis TAB23 was used as secretion carrier. Three chimerical proteins were produced by fusing intra-cellular proteins to C-terminus of the psychrophilic α-amylase and their secretion was analysed. Data reported in this paper demonstrate that all tested chimeras were translocated with a secretion yield always higher than 80%. Conclusion Data presented here demonstrate that the "cold" gene-expression system is efficient since the secretion yield of tested chimeras is always above 80%. These secretion performances place the α-amylase derived secretion system amongst the best heterologous secretion systems in Gram-negative bacteria reported so far. As for the quality of the secreted passenger proteins, data presented suggest that the system also allows the correct disulphide bond formation of chimera components, secreting a fully active passenger.

  5. 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.

  6. 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.

  7. 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.

  8. Refolding Techniques for Recovering Biologically Active Recombinant Proteins from Inclusion Bodies

    Directory of Open Access Journals (Sweden)

    Hiroshi Yamaguchi

    2014-02-01

    Full Text Available 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.

  9. Chemical Ligation of Folded Recombinant Proteins: Segmental Isotopic Labeling of Domains for NMR Studies

    Science.gov (United States)

    Xu, Rong; Ayers, Brenda; Cowburn, David; Muir, Tom W.

    1999-01-01

    A convenient in vitro chemical ligation strategy has been developed that allows folded recombinant proteins to be joined together. This strategy permits segmental, selective isotopic labeling of the product. The src homology type 3 and 2 domains (SH3 and SH2) of Abelson protein tyrosine kinase, which constitute the regulatory apparatus of the protein, were individually prepared in reactive forms that can be ligated together under normal protein-folding conditions to form a normal peptide bond at the ligation junction. This strategy was used to prepare NMR sample quantities of the Abelson protein tyrosine kinase-SH(32) domain pair, in which only one of the domains was labeled with 15N Mass spectrometry and NMR analyses were used to confirm the structure of the ligated protein, which was also shown to have appropriate ligand-binding properties. The ability to prepare recombinant proteins with selectively labeled segments having a single-site mutation, by using a combination of expression of fusion proteins and chemical ligation in vitro, will increase the size limits for protein structural determination in solution with NMR methods. In vitro chemical ligation of expressed protein domains will also provide a combinatorial approach to the synthesis of linked protein domains.

  10. 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

  11. IgM-specific serodiagnosis of acute human cytomegalovirus infection using recombinant autologous fusion proteins

    NARCIS (Netherlands)

    Vornhagen, R; Hinderer, W; Sonneborn, HH; Bein, G; Matter, L; The, T. Hauw; Enders, G; Jahn, G; Plachter, B

    Portions of three human cytomegalovirus (HCMV) polypeptides, which were shown previously to be highly reactive with patient sera, were expressed in Escherichia coli as autologous fusion proteins. Purified recombinant polypeptides were used as antigens in enzyme linked immunosorbent assay (ELISA) and

  12. 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....

  13. 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 ...

  14. 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

  15. 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

  16. 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.

  17. Molecular chaperone assisted expression systems: obtaining pure soluble and active recombinant proteins for structural and therapeutic purposes

    CSIR Research Space (South Africa)

    Makhoba, XH

    2015-09-01

    Full Text Available For many years recombinant protein production has been at the center of biosciences used for structural and therapeutic purposes. The production of recombinant proteins in foreign host system such as E. coli has been a biggest challenge. This has...

  18. 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.

  19. New sensitive and specific assay for human immunodeficiency virus antibodies using labeled recombinant fusion protein and time-resolved fluoroimmunoassay.

    OpenAIRE

    Siitari, H; Turunen, P; Schrimsher, J; Nunn, M

    1990-01-01

    A new, rapid method for the detection of human immunodeficiency virus type 1 (HIV-1) antibody by time-resolved fluoroimmunoassay (TR-FIA) was developed. In this assay format, microtitration strips were coated with a recombinant fusion protein, and the same protein was labeled with europium and added into the wells simultaneously with the test specimens. The recombinant fusion protein contained the HIV-1 p24 gag protein sequence that carried an insertion, near the carboxyl terminus, of a 23-am...

  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. Advanced technologies for improved expression of recombinant proteins in bacteria: perspectives and applications.

    Science.gov (United States)

    Gupta, Sanjeev K; Shukla, Pratyoosh

    2016-12-01

    Prokaryotic expression systems are superior in producing valuable recombinant proteins, enzymes and therapeutic products. Conventional microbial technology is evolving gradually and amalgamated with advanced technologies in order to give rise to improved processes for the production of metabolites, recombinant biopharmaceuticals and industrial enzymes. Recently, several novel approaches have been employed in a bacterial expression platform to improve recombinant protein expression. These approaches involve metabolic engineering, use of strong promoters, novel vector elements such as inducers and enhancers, protein tags, secretion signals, high-throughput devices for cloning and process screening as well as fermentation technologies. Advancement of the novel technologies in E. coli systems led to the production of "difficult to express" complex products including small peptides, antibody fragments, few proteins and full-length aglycosylated monoclonal antibodies in considerable large quantity. Wacker's secretion technologies, Pfenex system, inducers, cell-free systems, strain engineering for post-translational modification, such as disulfide bridging and bacterial N-glycosylation, are still under evaluation for the production of complex proteins and peptides in E. coli in an efficient manner. This appraisal provides an impression of expression technologies developed in recent times for enhanced production of heterologous proteins in E. coli which are of foremost importance for diverse applications in microbiology and biopharmaceutical production.

  2. 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

  3. Preparative Purification of Recombinant Proteins: Current Status and Future Trends

    Directory of Open Access Journals (Sweden)

    Mayank Saraswat

    2013-01-01

    Full Text Available Advances in fermentation technologies have resulted in the production of increased yields of proteins of economic, biopharmaceutical, and medicinal importance. Consequently, there is an absolute requirement for the development of rapid, cost-effective methodologies which facilitate the purification of such products in the absence of contaminants, such as superfluous proteins and endotoxins. Here, we provide a comprehensive overview of a selection of key purification methodologies currently being applied in both academic and industrial settings and discuss how innovative and effective protocols such as aqueous two-phase partitioning, membrane chromatography, and high-performance tangential flow filtration may be applied independently of or in conjunction with more traditional protocols for downstream processing applications.

  4. 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...

  5. Preparation of recombinant coat protein of Prunus necrotic ringspot virus.

    Science.gov (United States)

    Petrzik, K; Mráz, I; Kubelková, D

    2001-02-01

    The coat protein (CP) gene of Prunus necrotic ringspot virus (PNRSV) was cloned into pET 16b vector and expressed in Escherichia coli. CP-enriched fractions were prepared from whole cell lysate by differential centrifugation. The fraction sedimenting at 20,000 x g for 30 mins was used for preparation of a rabbit antiserum to CP. This antiserum had a titer of 1:2048 and reacted in a double-antibody sandwich ELISA (DAS-ELISA).

  6. 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.

  7. Construction of green fluorescent protein-tagged recombinant iridovirus to assess viral replication.

    Science.gov (United States)

    Huang, Youhua; Huang, Xiaohong; Cai, Jia; Ye, Fuzhou; Guan, Liya; Liu, Hong; Qin, Qiwei

    2011-09-01

    Green fluorescent protein-tagged recombinant virus has been successfully applied to observing the infective dynamics and evaluating viral replication. Here, we identified soft-shelled turtle iridovirus (STIV) ORF55 as an envelope protein (VP55), and developed a recombinant STIV expressing an enhanced green fluorescent protein (EGFP) fused to VP55 (EGFP-STIV). Recombinant EGFP-STIV shared similar single-step growth curves and ultrastructural morphology with wild type STIV (wt-STIV). The green fluorescence distribution during EGFP-STIV infection was consistent with the intracellular distribution of VP55 which was mostly co-localized with virus assembly sites. Furthermore, EGFP-STIV could be used to evaluate viral replication conveniently under drug treatment, and the result showed that STIV replication was significantly inhibited after the addition of antioxidant pyrrolidine dithiocarbamate (PDTC). Thus, the EGFP-tagged recombinant iridovirus will not only be useful for further investigations on the viral replicative dynamics, but also provide an alternative simple strategy to screen for antiviral substances. Copyright © 2011 Elsevier B.V. All rights reserved.

  8. BNGR-A25L and -A27 are two functional G protein-coupled receptors for CAPA periviscerokinin neuropeptides in the silkworm Bombyx mori.

    Science.gov (United States)

    Shen, Zhangfei; Chen, Yu; Hong, Lingjuan; Cui, Zhenteng; Yang, Huipeng; He, Xiaobai; Shi, Ying; Shi, Liangen; Han, Feng; Zhou, Naiming

    2017-10-06

    CAPA peptides, such as periviscerokinin (PVK), are insect neuropeptides involved in many signaling pathways controlling, for example, metabolism, behavior, and reproduction. They are present in a large number of insects and, together with their cognate receptors, are important for research into approaches for improving insect control. However, the CAPA receptors in the silkworm ( Bombyx mori ) insect model are unknown. Here, we cloned cDNAs of two putative CAPA peptide receptor genes, BNGR-A27 and -A25, from the brain of B. mori larvae. We found that the predicted BNGR-A27 ORF encodes 450 amino acids and that one BNGR-A25 splice variant encodes a full-length isoform (BNGR-A25L) of 418 amino acid residues and another a short isoform (BNGR-A25S) of 341 amino acids with a truncated C-terminal tail. Functional assays indicated that both BNGR-A25L and -A27 are activated by the PVK neuropeptides Bom -CAPA-PVK-1 and -PVK-2, leading to a significant increase in cAMP-response element-controlled luciferase activity and Ca 2+ mobilization in a G q inhibitor-sensitive manner. In contrast, BNGR-A25S was not significantly activated in response to the PVK peptides. Moreover, Bom -CAPA-PVK-1 directly bound to BNGR-A25L and -A27, but not BNGR-A25S. Of note, CAPA-PVK-mediated ERK1/2 phosphorylation and receptor internalization confirmed that BNGR-A25L and -A27 are two canonical receptors for Bombyx CAPA-PVKs. However, BNGR-A25S alone is a nonfunctional receptor but serves as a dominant-negative protein for BNGR-A25L. These results provide evidence that BNGR-A25L and -A27 are two functional G q -coupled receptors for Bombyx CAPA-PVKs, enabling the further elucidation of the endocrinological roles of Bom -CAPA-PVKs and their receptors in insect biology. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Multiplexed expression and screening for recombinant protein production in mammalian cells

    Directory of Open Access Journals (Sweden)

    McCafferty John

    2006-12-01

    Full Text Available Abstract Background A variety of approaches to understanding protein structure and function require production of recombinant protein. Mammalian based expression systems have advantages over bacterial systems for certain classes of protein but can be slower and more laborious. Thus the availability of a simple system for production and rapid screening of constructs or conditions for mammalian expression would be of great benefit. To this end we have coupled an efficient recombinant protein production system based on transient transfection in HEK-293 EBNA1 (HEK-293E suspension cells with a dot blot method allowing pre-screening of proteins expressed in cells in a high throughput manner. Results A nested PCR approach was used to clone 21 extracellular domains of mouse receptors as CD4 fusions within a mammalian GATEWAY expression vector system. Following transient transfection, HEK-293E cells grown in 2 ml cultures in 24-deep well blocks showed similar growth kinetics, viability and recombinant protein expression profiles, to those grown in 50 ml shake flask cultures as judged by western blotting. Following optimisation, fluorescent dot blot analysis of transfection supernatants was shown to be a rapid method for analysing protein expression yielding similar results as western blot analysis. Addition of urea enhanced the binding of glycoproteins to a nitrocellulose membrane. A good correlation was observed between the results of a plate based small scale transient transfection dot blot pre-screen and successful purification of proteins expressed at the 50 ml scale. Conclusion The combination of small scale multi-well plate culture and dot blotting described here will allow the multiplex analysis of different mammalian expression experiments enabling a faster identification of high yield expression constructs or conditions prior to large scale protein production. The methods for parallel GATEWAY cloning and expression of multiple constructs in cell

  10. Efficient sortase-mediated N-terminal labeling of TEV protease cleaved recombinant proteins.

    Science.gov (United States)

    Sarpong, Kwabena; Bose, Ron

    2017-03-15

    A major challenge in attaching fluorophores or other handles to proteins is the availability of a site-specific labeling strategy that provides stoichiometric modification without compromising protein integrity. We developed a simple approach that combines TEV protease cleavage, sortase modification and affinity purification to N-terminally label proteins. To achieve stoichiometrically-labeled protein, we included a short affinity tag in the fluorophore-containing peptide for post-labeling purification of the modified protein. This strategy can be easily applied to any recombinant protein with a TEV site and we demonstrate this on Epidermal Growth Factor Receptor (EGFR) and Membrane Scaffold Protein (MSP) constructs. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Nuclear Engineering of Microalgae for High Yield Secretion of Recombinant Proteins

    DEFF Research Database (Denmark)

    Ramos Martinez, Erick Miguel

    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...... to the glycomodules, accumulation of a fusion protein was dramatically increased by up to 12 folds, with the maximum yield of 15 mg L-1. Characterization of the secreted Venus showed the presence of glycosylations and increased resistance to proteolytic degradation. The results from this thesis demonstrate...... the potential of microalgae as a cell factory for secretion of recombinant proteins. The second research project presented in this thesis aimed to establish a new robust method to allow in vivo measurements of metabolic enzyme activities in cyanobacteria, with a hope that the method would facilitate further...

  12. 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

  13. 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.

  14. 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.

  15. Tobacco BY-2 Media Component Optimization for a Cost-Efficient Recombinant Protein Production.

    Science.gov (United States)

    Häkkinen, Suvi T; Reuter, Lauri; Nuorti, Ninni; Joensuu, Jussi J; Rischer, Heiko; Ritala, Anneli

    2018-01-01

    Plant cells constitute an attractive platform for production of recombinant proteins as more and more animal-free products and processes are desired. One of the challenges in using plant cells as production hosts has been the costs deriving from expensive culture medium components. In this work, the aim was to optimize the levels of most expensive components in the nutrient medium without compromising the accumulation of biomass and recombinant protein yields. Wild-type BY-2 culture and transgenic tobacco BY-2 expressing green fluorescent protein-Hydrophobin I (GFP-HFBI) fusion protein were used to determine the most inexpensive medium composition. One particularly high-accumulating BY-2 clone, named 'Hulk,' produced 1.1 ± 0.2 g/l GFP-HFBI in suspension and kept its high performance during prolonged subculturing. In addition, both cultures were successfully cryopreserved enabling truly industrial application of this plant cell host. With the optimized culture medium, 43-55% cost reduction with regard to biomass and up to 69% reduction with regard to recombinant protein production was achieved.

  16. 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.

  17. Improved Method for the Incorporation of Heme Cofactors into Recombinant Proteins Using Escherichia coli Nissle 1917.

    Science.gov (United States)

    Fiege, Kerstin; Querebillo, Christine Joy; Hildebrandt, Peter; Frankenberg-Dinkel, Nicole

    2018-05-15

    Recombinant production of heme proteins in Escherichia coli is often limited by the availability of heme in the host. Therefore, several methods, including the reconstitution of heme proteins after production but prior to purification or the HPEX system, conferring the ability to take up external heme have been developed and used in the past. Here we describe the use of the apathogenic E. coli strain Nissle 1917 (EcN) as a suitable host for the recombinant production of heme proteins. EcN has an advantage over commonly used lab strains in that it is able to take up heme from the environment through the heme receptor ChuA. Expression of several heme proteins from different prokaryotic sources led to high yield and quantitative incorporation of the cofactor when heme was supplied in the growth medium. Comparative UV-vis and resonance Raman measurements revealed that the method employed has significant influence on heme coordination with the EcN system representing the most native situation. Therefore, the use of EcN as a host for recombinant heme protein production represents an inexpensive and straightforward method to facilitate further investigations of structure and function.

  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. Protection against myxomatosis and rabbit viral hemorrhagic disease with recombinant myxoma viruses expressing rabbit hemorrhagic disease virus capsid protein.

    Science.gov (United States)

    Bertagnoli, S; Gelfi, J; Le Gall, G; Boilletot, E; Vautherot, J F; Rasschaert, D; Laurent, S; Petit, F; Boucraut-Baralon, C; Milon, A

    1996-08-01

    Two myxoma virus-rabbit hemorrhagic disease virus (RHDV) recombinant viruses were constructed with the SG33 strain of myxoma virus to protect rabbits against myxomatosis and rabbit viral hemorrhagic disease. These recombinant viruses expressed the RHDV capsid protein (VP60). The recombinant protein, which is 60 kDa in size, was antigenic, as revealed by its reaction in immunoprecipitation with antibodies raised against RHDV. Both recombinant viruses induced high levels of RHDV- and myxoma virus-specific antibodies in rabbits after immunization. Inoculations by the intradermal route protected animals against virulent RHDV and myxoma virus challenges.

  20. 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...

  1. 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.

  2. A systematic investigation of production of synthetic prions from recombinant prion protein.

    Science.gov (United States)

    Schmidt, Christian; Fizet, Jeremie; Properzi, Francesca; Batchelor, Mark; Sandberg, Malin K; Edgeworth, Julie A; Afran, Louise; Ho, Sammy; Badhan, Anjna; Klier, Steffi; Linehan, Jacqueline M; Brandner, Sebastian; Hosszu, Laszlo L P; Tattum, M Howard; Jat, Parmjit; Clarke, Anthony R; Klöhn, Peter C; Wadsworth, Jonathan D F; Jackson, Graham S; Collinge, John

    2015-12-01

    According to the protein-only hypothesis, infectious mammalian prions, which exist as distinct strains with discrete biological properties, consist of multichain assemblies of misfolded cellular prion protein (PrP). A critical test would be to produce prion strains synthetically from defined components. Crucially, high-titre 'synthetic' prions could then be used to determine the structural basis of infectivity and strain diversity at the atomic level. While there have been multiple reports of production of prions from bacterially expressed recombinant PrP using various methods, systematic production of high-titre material in a form suitable for structural analysis remains a key goal. Here, we report a novel high-throughput strategy for exploring a matrix of conditions, additives and potential cofactors that might generate high-titre prions from recombinant mouse PrP, with screening for infectivity using a sensitive automated cell-based bioassay. Overall, approximately 20,000 unique conditions were examined. While some resulted in apparently infected cell cultures, this was transient and not reproducible. We also adapted published methods that reported production of synthetic prions from recombinant hamster PrP, but again did not find evidence of significant infectious titre when using recombinant mouse PrP as substrate. Collectively, our findings are consistent with the formation of prion infectivity from recombinant mouse PrP being a rare stochastic event and we conclude that systematic generation of prions from recombinant PrP may only become possible once the detailed structure of authentic ex vivo prions is solved. © 2015 The Authors.

  3. 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.

  4. Nonreplicative RNA Recombination of an Animal Plus-Strand RNA Virus in the Absence of Efficient Translation of Viral Proteins.

    Science.gov (United States)

    Kleine Büning, Maximiliane; Meyer, Denise; Austermann-Busch, Sophia; Roman-Sosa, Gleyder; Rümenapf, Tillmann; Becher, Paul

    2017-04-01

    RNA recombination is a major driving force for the evolution of RNA viruses and is significantly implicated in the adaptation of viruses to new hosts, changes of virulence, as well as in the emergence of new viruses including drug-resistant and escape mutants. However, the molecular details of recombination in animal RNA viruses are only poorly understood. In order to determine whether viral RNA recombination depends on translation of viral proteins, a nonreplicative recombination system was established which is based on cotransfection of cells with synthetic bovine viral diarrhea virus (family Flaviviridae) RNA genome fragments either lacking the internal ribosome entry site required for cap-independent translation or lacking almost the complete polyprotein coding region. The emergence of a number of recombinant viruses demonstrated that IRES-mediated translation of viral proteins is dispensable for efficient recombination and suggests that RNA recombination can occur in the absence of viral proteins. Analyses of 58 independently emerged viruses led to the detection of recombinant genomes with duplications, deletions and insertions in the 5' terminal region of the open reading frame, leading to enlarged core fusion proteins detectable by Western blot analysis. This demonstrates a remarkable flexibility of the pestivirus core protein. Further experiments with capped and uncapped genome fragments containing a luciferase gene for monitoring the level of protein translation revealed that even a ∼1,000-fold enhancement of translation of viral proteins did not increase the frequency of RNA recombination. Taken together, this study highlights that nonreplicative RNA recombination does not require translation of viral proteins. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  5. Nonreplicative RNA Recombination of an Animal Plus-Strand RNA Virus in the Absence of Efficient Translation of Viral Proteins

    Science.gov (United States)

    Kleine Büning, Maximiliane; Meyer, Denise; Austermann-Busch, Sophia; Roman-Sosa, Gleyder; Rümenapf, Tillmann

    2017-01-01

    RNA recombination is a major driving force for the evolution of RNA viruses and is significantly implicated in the adaptation of viruses to new hosts, changes of virulence, as well as in the emergence of new viruses including drug-resistant and escape mutants. However, the molecular details of recombination in animal RNA viruses are only poorly understood. In order to determine whether viral RNA recombination depends on translation of viral proteins, a nonreplicative recombination system was established which is based on cotransfection of cells with synthetic bovine viral diarrhea virus (family Flaviviridae) RNA genome fragments either lacking the internal ribosome entry site required for cap-independent translation or lacking almost the complete polyprotein coding region. The emergence of a number of recombinant viruses demonstrated that IRES-mediated translation of viral proteins is dispensable for efficient recombination and suggests that RNA recombination can occur in the absence of viral proteins. Analyses of 58 independently emerged viruses led to the detection of recombinant genomes with duplications, deletions and insertions in the 5′ terminal region of the open reading frame, leading to enlarged core fusion proteins detectable by Western blot analysis. This demonstrates a remarkable flexibility of the pestivirus core protein. Further experiments with capped and uncapped genome fragments containing a luciferase gene for monitoring the level of protein translation revealed that even a ∼1,000-fold enhancement of translation of viral proteins did not increase the frequency of RNA recombination. Taken together, this study highlights that nonreplicative RNA recombination does not require translation of viral proteins. PMID:28338950

  6. Finding trans-regulatory genes and protein complexes modulating meiotic recombination hotspots of human, mouse and yeast.

    Science.gov (United States)

    Wu, Min; Kwoh, Chee-Keong; Li, Xiaoli; Zheng, Jie

    2014-09-11

    The regulatory mechanism of recombination is one of the most fundamental problems in genomics, with wide applications in genome wide association studies (GWAS), birth-defect diseases, molecular evolution, cancer research, etc. Recombination events cluster into short genomic regions called "recombination hotspots". Recently, a zinc finger protein PRDM9 was reported to regulate recombination hotspots in human and mouse genomes. In addition, a 13-mer motif contained in the binding sites of PRDM9 is found to be enriched in human hotspots. However, this 13-mer motif only covers a fraction of hotspots, indicating that PRDM9 is not the only regulator of recombination hotspots. Therefore, the challenge of discovering other regulators of recombination hotspots becomes significant. Furthermore, recombination is a complex process. Hence, multiple proteins acting as machinery, rather than individual proteins, are more likely to carry out this process in a precise and stable manner. Therefore, the extension of the prediction of individual trans-regulators to protein complexes is also highly desired. In this paper, we introduce a pipeline to identify genes and protein complexes associated with recombination hotspots. First, we prioritize proteins associated with hotspots based on their preference of binding to hotspots and coldspots. Second, using the above identified genes as seeds, we apply the Random Walk with Restart algorithm (RWR) to propagate their influences to other proteins in protein-protein interaction (PPI) networks. Hence, many proteins without DNA-binding information will also be assigned a score to implicate their roles in recombination hotspots. Third, we construct sub-PPI networks induced by top genes ranked by RWR for various species (e.g., yeast, human and mouse) and detect protein complexes in those sub-PPI networks. The GO term analysis show that our prioritizing methods and the RWR algorithm are capable of identifying novel genes associated with

  7. Bovine Pancreatic Trypsin Inhibitor-Trypsin Complex as a Detection System for Recombinant Proteins

    Science.gov (United States)

    Borjigin, Jimo; Nathans, Jeremy

    1993-01-01

    Bovine pancreatic trypsin inhibitor (BPTI) binds to trypsin and anhydrotrypsin (an enzymatically inactive derivative of trypsin) with affinities of 6 x 10-14 and 1.1 x 10-13 M, respectively. We have taken advantage of the high affinity and specificity of this binding reaction to develop a protein tagging system in which biotinylated trypsin or biotinylated anhydrotrypsin is used as the reagent to detect recombinant fusion proteins into which BPTI has been inserted. Two proteins, opsin and growth hormone, were used as targets for insertional mutagenesis with BPTI. In each case, both domains of the fusion protein appear to be correctly folded. The fusion proteins can be specifically and efficiently detected by biotinylated trypsin or biotinylated anhydrotrypsin, as demonstrated by staining of transfected cells, protein blotting, affinity purification, and a mobility shift assay in SDS/polyacrylamide gels.

  8. 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.

  9. 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.

  10. 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.

  11. Single vector system for efficient N-myristoylation of recombinant proteins in E. coli.

    Directory of Open Access Journals (Sweden)

    Julian M Glück

    Full Text Available BACKGROUND: N-myristoylation is a crucial covalent modification of numerous eukaryotic and viral proteins that is catalyzed by N-myristoyltransferase (NMT. Prokaryotes are lacking endogenous NMT activity. Recombinant production of N-myristoylated proteins in E. coli cells can be achieved by coexpression of heterologous NMT with the target protein. In the past, dual plasmid systems were used for this purpose. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe a single vector system for efficient coexpression of substrate and enzyme suitable for production of co- or posttranslationally modified proteins. The approach was validated using the HIV-1 Nef protein as an example. A simple and efficient protocol for production of highly pure and completely N-myristoylated Nef is presented. The yield is about 20 mg myristoylated Nef per liter growth medium. CONCLUSIONS/SIGNIFICANCE: The single vector strategy allows diverse modifications of target proteins recombinantly coexpressed in E. coli with heterologous enzymes. The method is generally applicable and provides large amounts of quantitatively processed target protein that are sufficient for comprehensive biophysical and structural studies.

  12. 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.

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

    International Nuclear Information System (INIS)

    Kim, Hyunhee; Kang, Da-Yeon; Goh, Hyun-Jeong; Oh, Byung-Keun; Singh, Ravindra P.; Oh, Soo-Min; Choi, Jeong-Woo

    2008-01-01

    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

  14. Pichia pastoris: a recombinant microfactory for antibodies and human membrane proteins.

    Science.gov (United States)

    Gonçalves, A M; Pedro, A Q; Maia, C; Sousa, F; Queiroz, J A; Passarinha, L A

    2013-05-01

    During the last few decades, it has become evident that the compatibility of the yeast biochemical environment with the ability to process and translate the RNA transcript, along with its capacity to modify a translated protein, are relevant requirements for selecting this host cell for protein expression in several pharmaceutical and clinical applications. In particular, Pichia pastoris is used as an industrial host for recombinant protein and metabolite production, showing a powerful capacity to meet required biomolecular target production levels in high-throughput assays for functional genomics and drug screening. In addition, there is a great advantage to using P. pastoris for protein secretion, even at high molecular weights, since the recovery and purification steps are simplified owing to relatively low levels of endogenous proteins in the extracellular medium. Clearly, no single microexpression system can provide all of the desired properties for human protein production. Moreover, chemical and physical bioprocess parameters, including culture medium formulation, temperature, pH, agitation, aeration rates, induction, and feeding strategies, can highly influence product yield and quality. In order to benefit from the currently available wide range of biosynthesis strategies using P. pastoris, this mini review focuses on the developments and technological fermentation achievements, providing both a comparative and an overall integration analysis. The main aim is to highlight the relevance and versatility of the P. pastoris biosystem to the design of more cost-effective microfactories to meet the increasing demands for recombinant membrane proteins and clinical antibodies for several therapeutic applications.

  15. 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, P<0.01; 156.32±10.42 μg/mL vs 66.52±7.35 μg/mL, 5.73±2.89 μg/mL, P<0.01). The tumor volume in mAFP/HSP70 group was significantly smaller than that in mAFP and HSP70 groups (42.44±7.14 mm3 vs 392.23±12.46 mm3, 838.63±13.84 mm3, P<0.01). Conclusions: The study further confirmed the function of heat shock protein 70's immune 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.

  16. Higher accumulation of F1-V fusion recombinant protein in plants after induction of protein body formation.

    Science.gov (United States)

    Alvarez, M Lucrecia; Topal, Emel; Martin, Federico; Cardineau, Guy A

    2010-01-01

    Improving foreign protein accumulation is crucial for enhancing the commercial success of plant-based production systems since product yields have a major influence on process economics. Cereal grain evolved to store large amounts of proteins in tightly organized aggregates. In maize, gamma-Zein is the major storage protein synthesized by the rough endoplasmic reticulum (ER) and stored in specialized organelles called protein bodies (PB). Zera (gamma-Zein ER-accumulating domain) is the N-terminal proline-rich domain of gamma-zein that is sufficient to induce the assembly of PB formation. Fusion of the Zera domain to proteins of interest results in assembly of dense PB-like, ER-derived organelles, containing high concentration of recombinant protein. Our main goal was to increase recombinant protein accumulation in plants in order to enhance the efficiency of orally-delivered plant-made vaccines. It is well known that oral vaccination requires substantially higher doses than parental formulations. As a part of a project to develop a plant-made plague vaccine, we expressed our model antigen, the Yersinia pestis F1-V antigen fusion protein, with and without a fused Zera domain. We demonstrated that Zera-F1-V protein accumulation was at least 3x higher than F1-V alone when expressed in three different host plant systems: Ncotiana benthamiana, Medicago sativa (alfalfa) and Nicotiana tabacum NT1 cells. We confirmed the feasibility of using Zera technology to induce protein body formation in non-seed tissues. Zera expression and accumulation did not affect plant development and growth. These results confirmed the potential exploitation of Zera technology to substantially increase the accumulation of value-added proteins in plants.

  17. 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

    a less leaky Cu2+-inducible promoter based on CUP1. The basal expression level of the new promoter was approx. 61% below the wild-type CUP1 promoter, thus expanding the absolute range of Cu2+-based gene control. The stability of 3vGFP towards direct-repeat recombination was assayed in S. cerevisiae......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...

  18. Advanced control of dissolved oxygen concentration in fed batch cultures during recombinant protein production.

    Science.gov (United States)

    Kuprijanov, A; Gnoth, S; Simutis, R; Lübbert, A

    2009-02-01

    Design and experimental validation of advanced pO(2) controllers for fermentation processes operated in the fed-batch mode are described. In most situations, the presented controllers are able to keep the pO(2) in fermentations for recombinant protein productions exactly on the desired value. The controllers are based on the gain-scheduling approach to parameter-adaptive proportional-integral controllers. In order to cope with the most often appearing distortions, the basic gain-scheduling feedback controller was complemented with a feedforward control component. This feedforward/feedback controller significantly improved pO(2) control. By means of numerical simulations, the controller behavior was tested and its parameters were determined. Validation runs were performed with three Escherichia coli strains producing different recombinant proteins. It is finally shown that the new controller leads to significant improvements in the signal-to-noise ratio of other key process variables and, thus, to a higher process quality.

  19. 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.

  20. 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...... 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.......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...

  1. Hijacked then lost in translation: the plight of the recombinant host cell in membrane protein structural biology projects.

    Science.gov (United States)

    Bill, Roslyn M; von der Haar, Tobias

    2015-06-01

    Membrane protein structural biology is critically dependent upon the supply of high-quality protein. Over the last few years, the value of crystallising biochemically characterised, recombinant targets that incorporate stabilising mutations has been established. Nonetheless, obtaining sufficient yields of many recombinant membrane proteins is still a major challenge. Solutions are now emerging based on an improved understanding of recombinant host cells; as a 'cell factory' each cell is tasked with managing limited resources to simultaneously balance its own growth demands with those imposed by an expression plasmid. This review examines emerging insights into the role of translation and protein folding in defining high-yielding recombinant membrane protein production in a range of host cells. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  2. 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/

  3. Studies to Prevent Degradation of Recombinant Fc-Fusion Protein Expressed in Mammalian Cell Line and Protein Characterization

    Directory of Open Access Journals (Sweden)

    Sanjukta Chakrabarti

    2016-06-01

    Full Text Available Clipping of recombinant proteins is a major issue in animal cell cultures. A recombinant Fc-fusion protein, VEGFR1(D1–D3-Fc expressed in CHOK1SV GS-KO cells was observed to be undergoing clippings in lab scale cultures. Partial cleaving of expressed protein initiated early on in cell culture and was observed to increase over time in culture and also on storage. In this study, a few parameters were explored in a bid to inhibit clipping in the fusion protein The effects of culture temperature, duration of culture, the addition of an anti-clumping agent, ferric citrate and use of protease inhibitor cocktail on inhibition of proteolysis of the Fc fusion were studied. Lowering of culture temperature from 37 to 30 °C alone appears to be the best solution for reducing protein degradation from the quality, cost and regulatory points of view. The obtained Fc protein was characterized and found to be in its stable folded state, exhibiting a high affinity for its ligand and also biological and functional activities.

  4. Robotic high-throughput purification of affinity-tagged recombinant proteins.

    Science.gov (United States)

    Wiesler, Simone C; Weinzierl, Robert O J

    2015-01-01

    Affinity purification of recombinant proteins has become the method of choice to obtain good quantities and qualities of proteins for a variety of downstream biochemical applications. While manual or FPLC-assisted purification techniques are generally time-consuming and labor-intensive, the advent of high-throughput technologies and liquid handling robotics has simplified and accelerated this process significantly. Additionally, without the human factor as a potential source of error, automated purification protocols allow for the generation of large numbers of proteins simultaneously and under directly comparable conditions. The delivered material is ideal for activity comparisons of different variants of the same protein. Here, we present our strategy for the simultaneous purification of up to 24 affinity-tagged proteins for activity measurements in biochemical assays. The protocol described is suitable for the scale typically required in individual research laboratories.

  5. Main Strategies of Plant Expression System Glycoengineering for Producing Humanized Recombinant Pharmaceutical Proteins.

    Science.gov (United States)

    Rozov, S M; Permyakova, N V; Deineko, E V

    2018-03-01

    Most the pharmaceutical proteins are derived not from their natural sources, rather their recombinant analogs are synthesized in various expression systems. Plant expression systems, unlike mammalian cell cultures, combine simplicity and low cost of procaryotic systems and the ability for posttranslational modifications inherent in eucaryotes. More than 50% of all human proteins and more than 40% of the currently used pharmaceutical proteins are glycosylated, that is, they are glycoproteins, and their biological activity, pharmacodynamics, and immunogenicity depend on the correct glycosylation pattern. This review examines in detail the similarities and differences between N- and O-glycosylation in plant and mammalian cells, as well as the effect of plant glycans on the activity, pharmacokinetics, immunity, and intensity of biosynthesis of pharmaceutical proteins. The main current strategies of glycoengineering of plant expression systems aimed at obtaining fully humanized proteins for pharmaceutical application are summarized.

  6. Structure of Mycobacterium tuberculosis RuvA, a protein involved in recombination

    International Nuclear Information System (INIS)

    Prabu, J. Rajan; Thamotharan, S.; Khanduja, Jasbeer Singh; Alipio, Emily Zabala; Kim, Chang-Yub; Waldo, Geoffrey S.; Terwilliger, Thomas C.; Segelke, Brent; Lekin, Tim; Toppani, Dominique; Hung, Li-Wei; Yu, Minmin; Bursey, Evan; Muniyappa, K.; Chandra, Nagasuma R.; Vijayan, M.

    2006-01-01

    RuvA, a protein from M. tuberculosis H37Rv involved in recombination, has been cloned, expressed, purified and analysed by X-ray crystallography. The process of recombinational repair is crucial for maintaining genomic integrity and generating biological diversity. In association with RuvB and RuvC, RuvA plays a central role in processing and resolving Holliday junctions, which are a critical intermediate in homologous recombination. Here, the cloning, purification and structure determination of the RuvA protein from Mycobacterium tuberculosis (MtRuvA) are reported. Analysis of the structure and comparison with other known RuvA proteins reveal an octameric state with conserved subunit–subunit interaction surfaces, indicating the requirement of octamer formation for biological activity. A detailed analysis of plasticity in the RuvA molecules has led to insights into the invariant and variable regions, thus providing a framework for understanding regional flexibility in various aspects of RuvA function

  7. Evaluation of Six Recombinant Proteins for Serological Diagnosis of Lyme Borreliosis in China.

    Science.gov (United States)

    Liu, Wei; Liu, Hui Xin; Zhang, Lin; Hou, Xue Xia; Wan, Kang Lin; Hao, Qin

    2016-05-01

    In this study, we evaluated the diagnostic efficiency of six recombinant proteins for the serodiagnosis of Lyme borreliosis (LB) and screened out the appropriate antigens to support the production of a Chinese clinical ELISA (enzyme-linked immunosorbent assay) kit for LB. Six recombinant antigens, Fla B.g, OspC B.a, OspC B.g, P39 B.g, P83 B.g, and VlsE B.a, were used for ELISA to detect serum antibodies in LB, syphilis, and healthy controls. The ELISA results were used to generate receiver operating characteristic (ROC) curves, and the sensitivity and specificity of each protein was evaluated. All recombinant proteins were evaluated and screened by using logistic regression models. Two IgG (VlsE and OspC B.g) and two IgM (OspC B.g and OspC B.a) antigens were left by the logistic regression model screened. VlsE had the highest specificity for syphilis samples in the IgG test (87.7%, Precombinant antigens, OspC B.g, OspC B.a, and VlsE B.a, were useful for ELISAs of LB. Additionally, the interaction between OspC B.a and Fla B.g should be examined in future research. Copyright © 2016 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

  8. 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.

  9. Tetratricopeptide-motif-mediated interaction of FANCG with recombination proteins XRCC3 and BRCA2.

    Science.gov (United States)

    Hussain, Shobbir; Wilson, James B; Blom, Eric; Thompson, Larry H; Sung, Patrick; Gordon, Susan M; Kupfer, Gary M; Joenje, Hans; Mathew, Christopher G; Jones, Nigel J

    2006-05-10

    Fanconi anaemia is an inherited chromosomal instability disorder characterised by cellular sensitivity to DNA interstrand crosslinkers, bone-marrow failure and a high risk of cancer. Eleven FA genes have been identified, one of which, FANCD1, is the breast cancer susceptibility gene BRCA2. At least eight FA proteins form a nuclear core complex required for monoubiquitination of FANCD2. The BRCA2/FANCD1 protein is connected to the FA pathway by interactions with the FANCG and FANCD2 proteins, both of which co-localise with the RAD51 recombinase, which is regulated by BRCA2. These connections raise the question of whether any of the FANC proteins of the core complex might also participate in other complexes involved in homologous recombination repair. We therefore tested known FA proteins for direct interaction with RAD51 and its paralogs XRCC2 and XRCC3. FANCG was found to interact with XRCC3, and this interaction was disrupted by the FA-G patient derived mutation L71P. FANCG was co-immunoprecipitated with both XRCC3 and BRCA2 from extracts of human and hamster cells. The FANCG-XRCC3 and FANCG-BRCA2 interactions did not require the presence of other FA proteins from the core complex, suggesting that FANCG also participates in a DNA repair complex that is downstream and independent of FANCD2 monoubiquitination. Additionally, XRCC3 and BRCA2 proteins co-precipitate in both human and hamster cells and this interaction requires FANCG. The FANCG protein contains multiple tetratricopeptide repeat motifs (TPRs), which function as scaffolds to mediate protein-protein interactions. Mutation of one or more of these motifs disrupted all of the known interactions of FANCG. We propose that FANCG, in addition to stabilising the FA core complex, may have a role in building multiprotein complexes that facilitate homologous recombination repair.

  10. Purification of Microbially Expressed Recombinant Proteins via a Dual ELP Split Intein System.

    Science.gov (United States)

    Shi, Changhua; Han, Tzu-Chiang; Wood, David W

    2017-01-01

    Fusions of elastin-like peptide (ELP) purification tags and self-cleaving inteins provide a powerful platform for purifying tagless recombinant proteins without the need for conventional packed-bed columns. A drawback to this method has been premature cleaving of the ELP tag during expression, before the purification procedure can take place. Here we demonstrate a split-intein method, where the self-cleaving intein is divided into two inactive segments during expression and purification. Spontaneous assembly of the purified intein segments then restores self-cleaving activity to deliver the tagless target protein.

  11. 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.

  12. Exaggerated inflammatory response after use of recombinant bone morphogenetic protein in recurrent unicameral bone cysts.

    Science.gov (United States)

    MacDonald, Kevin M; Swanstrom, Morgan M; McCarthy, James J; Nemeth, Blaise A; Guliani, Teresa A; Noonan, Kenneth J

    2010-03-01

    Recurrent unicameral bone cysts (UBCs) can result in significant morbidity during a child's physical and emotional development. Multiple treatment options are available and a review of the literature fails to clearly define the optimal treatment for UBCs. Recombinant bone morphogenetic protein (BMP) has been used with success in other disorders of poor bone formation. This manuscript is the first to report on the use of recombinant BMP in the treatment of UBCs. Three patients with recurrent UBCs underwent revision surgery with recombinant BMP. Radiographic and medical review was performed and is reported here. In these patients, the use of BMP failed to fully resolve their UBC; 2 patients had complete recurrence that required further surgery. In addition to poor radiographic results, all patients developed exaggerated inflammatory responses in the acute postoperative period. Each child developed clinically significant limb swelling and pain that mimicked infection. On the basis of our poor radiographic results and a paradoxical clinical result, we no longer recommend the use of recombinant BMP in the manner reported here for the treatment of recurrent UBCs. Level IV, case series.

  13. Construction and analysis of the transgenic carrot and celery plants expressing the recombinant thaumatin II protein

    Directory of Open Access Journals (Sweden)

    Luchakivska Yu. S.

    2015-08-01

    Full Text Available Aim To obtain the transgenic carrot and celery plants able to express recombinant thaumatin II in order to increase plant stress tolerance. Methods. Agrobacterium-mediated transformation of the carrot and celery seedlings was used for obtaining the transgenic plants. Presence and transcription of the transgene in plant tissues were proved by PCR and RT-PCR analysis. The plants were tested for the biotic stress tolerance by in vitro antifungal and antibacterial activity assays and for the salinity and osmotic stress tolerance by plant survival test in presence of NaCl and PEG in different concentrations. Results. Transgenic plants able to express recombinant thaumatin II gene (transcription proved for 60–100 % were obtained by agrobacterial transformation. The transgenic carrot plant extracts inhibited the growth of the studied phytopathogenic bacteria strains but exhibited no antifungal activity. Survival level of transgenic plants under the salinity and osmotic stress effect was definitely higher comparing to the untransgenic ones. The analysis of the photosynthetic pigment content in the transgenic carrot plants showed no significant difference of this parameter under salinity stress that may indicate a possible protective activity of the recombinant protein. Conclusions. The obtained in our study transgenic carrot and celery plants able to express the recombinant thaumatin II gene were characterized by antibacterial activity and increased tolerance to salinity and osmotic stress factors.

  14. 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.

  15. Overexpression of the transcription factor Yap1 modifies intracellular redox conditions and enhances recombinant protein secretion

    Directory of Open Access Journals (Sweden)

    Marizela Delic

    2014-10-01

    Full Text Available Oxidative folding of secretory proteins in the endoplasmic reticulum (ER is a redox active process, which also impacts the redox conditions in the cytosol. As the transcription factor Yap1 is involved in the transcriptional response to oxidative stress, we investigate its role upon the production of secretory proteins, using the yeast Pichia pastoris as model, and report a novel important role of Yap1 during oxidative protein folding. Yap1 is needed for the detoxification of reactive oxygen species (ROS caused by increased oxidative protein folding. Constitutive co-overexpression of PpYAP1 leads to increased levels of secreted recombinant protein, while a lowered Yap1 function leads to accumulation of ROS and strong flocculation. Transcriptional analysis revealed that more than 150 genes were affected by overexpression of YAP1, in particular genes coding for antioxidant enzymes or involved in oxidation-reduction processes. By monitoring intracellular redox conditions within the cytosol and the ER using redox-sensitive roGFP1 variants, we could show that overexpression of YAP1 restores cellular redox conditions of protein-secreting P. pastoris by reoxidizing the cytosolic redox state to the levels of the wild type. These alterations are also reflected by increased levels of oxidized intracellular glutathione (GSSG in the YAP1 co-overexpressing strain. Taken together, these data indicate a strong impact of intracellular redox balance on the secretion of (recombinant proteins without affecting protein folding per se. Re-establishing suitable redox conditions by tuning the antioxidant capacity of the cell reduces metabolic load and cell stress caused by high oxidative protein folding load, thereby increasing the secretion capacity.

  16. Scale-up of hydrophobin-assisted recombinant protein production in tobacco BY-2 suspension cells.

    Science.gov (United States)

    Reuter, Lauri J; Bailey, Michael J; Joensuu, Jussi J; Ritala, Anneli

    2014-05-01

    Plant suspension cell cultures are emerging as an alternative to mammalian cells for production of complex recombinant proteins. Plant cell cultures provide low production cost, intrinsic safety and adherence to current regulations, but low yields and costly purification technology hinder their commercialization. Fungal hydrophobins have been utilized as fusion tags to improve yields and facilitate efficient low-cost purification by surfactant-based aqueous two-phase separation (ATPS) in plant, fungal and insect cells. In this work, we report the utilization of hydrophobin fusion technology in tobacco bright yellow 2 (BY-2) suspension cell platform and the establishment of pilot-scale propagation and downstream processing including first-step purification by ATPS. Green fluorescent protein-hydrophobin fusion (GFP-HFBI) induced the formation of protein bodies in tobacco suspension cells, thus encapsulating the fusion protein into discrete compartments. Cultivation of the BY-2 suspension cells was scaled up in standard stirred tank bioreactors up to 600 L production volume, with no apparent change in growth kinetics. Subsequently, ATPS was applied to selectively capture the GFP-HFBI product from crude cell lysate, resulting in threefold concentration, good purity and up to 60% recovery. The ATPS was scaled up to 20 L volume, without loss off efficiency. This study provides the first proof of concept for large-scale hydrophobin-assisted production of recombinant proteins in tobacco BY-2 cell suspensions. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  17. Use of recombinant purified protein derivative (PPD) antigens as specific skin test for tuberculosis.

    Science.gov (United States)

    Stavri, Henriette; Bucurenci, Nadia; Ulea, Irina; Costache, Adriana; Popa, Loredana; Popa, Mircea Ioan

    2012-11-01

    Purified protein derivative (PPD) is currently the only available skin test reagent used worldwide for the diagnosis of tuberculosis (TB). The aim of this study was to develop a Mycobacterium tuberculosis specific skin test reagent, without false positive results due to Bacillus Calmette-Guerin (BCG) vaccination using recombinant antigens. Proteins in PPD IC-65 were analyzed by tandem mass spectrometry and compared to proteins in M. tuberculosis culture filtrate; 54 proteins were found in common. Top candidates MPT64, ESAT 6, and CFP 10 were overexpressed in Escherichia coli expression strains and purified as recombinant proteins. To formulate optimal immunodiagnostic PPD cocktails, the antigens were evaluated by skin testing guinea pigs sensitized with M. tuberculosis H37Rv and BCG. For single antigens and a cocktail mixture of these antigens, best results were obtained using 3 μg/0.1 ml, equivalent to 105 TU (tuberculin units). Each animal was simultaneously tested with PPD IC-65, 2 TU/0.1 ml, as reference. Reactivity of the multi-antigen cocktail was greater than that of any single antigen. The skin test results were between 34.3 and 76.6 per cent the level of reactivity compared to that of the reference when single antigens were tested and 124 per cent the level of reactivity compared to the reference for the multi-antigen cocktail. Our results showed that this specific cocktail could represent a potential candidate for a new skin diagnostic test for TB.

  18. 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......-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...

  19. 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.

  20. Methods for efficient high-throughput screening of protein expression in recombinant Pichia pastoris strains.

    Science.gov (United States)

    Camattari, Andrea; Weinhandl, Katrin; Gudiminchi, Rama K

    2014-01-01

    The methylotrophic yeast Pichia pastoris is becoming one of the favorite industrial workhorses for protein expression. Due to the widespread use of integration vectors, which generates significant clonal variability, screening methods allowing assaying hundreds of individual clones are of particular importance. Here we describe methods to detect and analyze protein expression, developed in a 96-well format for high-throughput screening of recombinant P. pastoris strains. The chapter covers essentially three common scenarios: (1) an enzymatic assay for proteins expressed in the cell cytoplasm, requiring cell lysis; (2) a whole-cell assay for a fungal cytochrome P450; and (3) a nonenzymatic assay for detection and quantification of tagged protein secreted into the supernatant.

  1. [A study of recombinant human sestrin 1 and sestrin 2 proteins produced in a prokaryotic system].

    Science.gov (United States)

    Rai, N; Kumar, R; Haque, Md A; Hassan, Md I; Dey, S

    2017-01-01

    Sestrins are highly conserved stress-inducible proteins capable of suppressing the production of ROS and signalling through mTORC1. Here we report a study of human sestrin1 (sesn1) and sestrin2 (sesn2) proteins produced in a pET28^(+) vector based prokaryotic system. Mass spectrometry analysis, western blot and surface plasmon resonance (SPR) of affinity purified sesn1 and sesn2 proteins confirmed their identity; biophysical characteristics were observed using circular dichroism (CD) showing that sesn1 and sesn2 have a predominant α-helical structure. Here we describe a simple, one step purification process to purify a large amount of sestrin proteins with significant yield. Further study of recombinant human sestrins may further facilitate the understanding of their roles in eukaryotic cells.

  2. Recombinant expression, purification, and characterization of an acyl-CoA binding protein from Aspergillus oryzae.

    Science.gov (United States)

    Hao, Qing; Liu, Xiaoguang; Zhao, Guozhong; Jiang, Lu; Li, Ming; Zeng, Bin

    2016-03-01

    To characterize biochemically the lipid metabolism-regulating acyl-CoA binding protein (ACBP) from the industrially-important fungus Aspergillus oryzae. A full-length cDNA encoding a candidate ACBP from A. oryzae (AoACBP) was cloned and expressed in Escherichia coli as a maltose-binding protein (MBP) fusion protein. The MBP-AoACBP protein was purified by an amylose resin chromatography column. SDS-PAGE showed that MBP-AoACBP has an estimated molecular weight of 82 kDa. Microscale thermophoresis binding assay showed that the recombinant AoACBP displayed much greater affinity for palmitoyl-CoA (K d = 80 nM) than for myristoyl-CoA (K d = 510 nM), thus demonstrating the preference of AoACBP for long-chain acyl-CoA. The data support the identification of AoACBP as a long-chain ACBP in A. oryzae.

  3. 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...

  4. Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins.

    Directory of Open Access Journals (Sweden)

    Nan Zhong

    Full Text Available We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in vivo biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 in vivo biotinylated antigen domains. High-affinity Fabs (<20nM were obtained for 196. We constructed and optimized a new expression vector to produce in vivo biotinylated Fabs in E. coli. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP protocols.

  5. 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.

  6. A synthetic arabinose-inducible promoter confers high levels of recombinant protein expression in hyperthermophilic archaeon Sulfolobus islandicus

    DEFF Research Database (Denmark)

    Peng, Nan; Deng, Ling; Mei, Yuxia

    2012-01-01

    Despite major progresses in genetic studies of hyperthermophilic archaea, recombinant protein production in these organisms always suffers from low yields and a robust expression system is still in great demand. Here we report a versatile vector that confers high levels of protein expression...... to remove the peptide tags from expressed recombinant proteins. While pEXA employed an araS promoter for protein expression, pSeSD utilized P(araS-SD), an araS derivative promoter carrying an engineered ribosome-binding site (RBS; a Shine-Dalgarno [SD] sequence). We found that P(araS-SD) directed high...... levels of target gene expression. More strikingly, N-terminal amino acid sequencing of recombinant proteins unraveled that the protein synthesized from pEXA-N-lacS lacked the designed 6×His tag and that translation initiation did not start at the ATG codon of the fusion gene. Instead, it started...

  7. Subdominant Outer Membrane Antigens in Anaplasma marginale: Conservation, Antigenicity, and Protective Capacity Using Recombinant Protein.

    Directory of Open Access Journals (Sweden)

    Deirdre R Ducken

    Full Text Available Anaplasma marginale is a tick-borne rickettsial pathogen of cattle with a worldwide distribution. Currently a safe and efficacious vaccine is unavailable. Outer membrane protein (OMP extracts or a defined surface protein complex reproducibly induce protective immunity. However, there are several knowledge gaps limiting progress in vaccine development. First, are these OMPs conserved among the diversity of A. marginale strains circulating in endemic regions? Second, are the most highly conserved outer membrane proteins in the immunogens recognized by immunized and protected animals? Lastly, can this subset of OMPs recognized by antibody from protected vaccinates and conserved among strains recapitulate the protection of outer membrane vaccines? To address the first goal, genes encoding OMPs AM202, AM368, AM854, AM936, AM1041, and AM1096, major subdominant components of the outer membrane, were cloned and sequenced from geographically diverse strains and isolates. AM202, AM936, AM854, and AM1096 share 99.9 to 100% amino acid identity. AM1041 has 97.1 to 100% and AM368 has 98.3 to 99.9% amino acid identity. While all four of the most highly conserved OMPs were recognized by IgG from animals immunized with outer membranes, linked surface protein complexes, or unlinked surface protein complexes and shown to be protected from challenge, the highest titers and consistent recognition among vaccinates were to AM854 and AM936. Consequently, animals were immunized with recombinant AM854 and AM936 and challenged. Recombinant vaccinates and purified outer membrane vaccinates had similar IgG and IgG2 responses to both proteins. However, the recombinant vaccinates developed higher bacteremia after challenge as compared to adjuvant-only controls and outer membrane vaccinates. These results provide the first evidence that vaccination with specific antigens may exacerbate disease. Progressing from the protective capacity of outer membrane formulations to

  8. Recombinant Vaccinia Viruses Coding Transgenes of Apoptosis-Inducing Proteins Enhance Apoptosis But Not Immunogenicity of Infected Tumor Cells

    Science.gov (United States)

    Tkachenko, Anastasiya; Richter, Vladimir

    2017-01-01

    Genetic modifications of the oncolytic vaccinia virus (VV) improve selective tumor cell infection and death, as well as activation of antitumor immunity. We have engineered a double recombinant VV, coding human GM-CSF, and apoptosis-inducing protein apoptin (VV-GMCSF-Apo) for comparing with the earlier constructed double recombinant VV-GMCSF-Lact, coding another apoptosis-inducing protein, lactaptin, which activated different cell death pathways than apoptin. We showed that both these recombinant VVs more considerably activated a set of critical apoptosis markers in infected cells than the recombinant VV coding GM-CSF alone (VV-GMCSF-dGF): these were phosphatidylserine externalization, caspase-3 and caspase-7 activation, DNA fragmentation, and upregulation of proapoptotic protein BAX. However, only VV-GMCSF-Lact efficiently decreased the mitochondrial membrane potential of infected cancer cells. Investigating immunogenic cell death markers in cancer cells infected with recombinant VVs, we demonstrated that all tested recombinant VVs were efficient in calreticulin and HSP70 externalization, decrease of cellular HMGB1, and ATP secretion. The comparison of antitumor activity against advanced MDA-MB-231 tumor revealed that both recombinants VV-GMCSF-Lact and VV-GMCSF-Apo efficiently delay tumor growth. Our results demonstrate that the composition of GM-CSF and apoptosis-inducing proteins in the VV genome is very efficient tool for specific killing of cancer cells and for activation of antitumor immunity. PMID:28951871

  9. 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

    , and surprisingly, can form in the absence of Rad52 mediation. However, these Rad51 foci do not represent repair-proficient filaments, as determined by recombination assays. Antagonistic roles for Rad52 and Srs2 in Rad51 filament formation are also observed in vitro. Furthermore, we provide evidence that Srs2......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....

  10. Residual endotoxin contaminations in recombinant proteins are sufficient to activate human CD1c+ dendritic cells.

    Directory of Open Access Journals (Sweden)

    Harald Schwarz

    Full Text Available Many commercially available recombinant proteins are produced in Escherichia coli, and most suppliers guarantee contamination levels of less than 1 endotoxin unit (EU. When we analysed commercially available proteins for their endotoxin content, we found contamination levels in the same range as generally stated in the data sheets, but also some that were higher. To analyse whether these low levels of contamination have an effect on immune cells, we stimulated the monocytic cell line THP-1, primary human monocytes, in vitro differentiated human monocyte-derived dendritic cells, and primary human CD1c+ dendritic cells (DCs with very low concentrations of lipopolysaccharide (LPS; ranging from 0.002-2 ng/ml. We show that CD1c+ DCs especially can be activated by minimal amounts of LPS, equivalent to the levels of endotoxin contamination we detected in some commercially available proteins. Notably, the enhanced endotoxin sensitivity of CD1c+ DCs was closely correlated with high CD14 expression levels observed in CD1c+ DCs that had been maintained in cell culture medium for 24 hours. When working with cells that are particularly sensitive to LPS, even low endotoxin contamination may generate erroneous data. We therefore recommend that recombinant proteins be thoroughly screened for endotoxin contamination using the limulus amebocyte lysate test, fluorescence-based assays, or a luciferase based NF-κB reporter assay involving highly LPS-sensitive cells overexpressing TLR4, MD-2 and CD14.

  11. 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.

  12. 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.

  13. Manufacturing recombinant proteins in kg-ton quantities using animal cells in bioreactors.

    Science.gov (United States)

    De Jesus, Maria; Wurm, Florian M

    2011-06-01

    Mammalian cells in bioreactors as production host are the focus of this review. We wish to briefly describe today's technical status and to highlight emerging trends in the manufacture of recombinant therapeutic proteins, focusing on Chinese hamster ovary (CHO) cells. CHO cells are the manufacturing host system of choice for more than 70% of protein pharmaceuticals on the market [21]. The current global capacity to grow mammalian cells in bioreactors stands at about 0.5 million liters, whereby the largest vessels can have a working volume of about 20,000l. We are focusing in this article on the upstream part of protein manufacturing. Over the past 25 years, volumetric yields for recombinant cell lines have increased about 20-fold mainly as the result of improvements in media and bioprocess design. Future yield increases are expected to come from improved gene delivery methods, from improved, possibly genetically modified host systems, and from further improved bioprocesses in bioreactors. Other emerging trends in protein manufacturing that are discussed include the use of disposal bioreactors and transient gene expression. We specifically highlight here current research in our own laboratories. Copyright © 2011 Elsevier B.V. All rights reserved.

  14. Protein misfolding cyclic amplification induces the conversion of recombinant prion protein to PrP oligomers causing neuronal apoptosis.

    Science.gov (United States)

    Yuan, Zhen; Yang, Lifeng; Chen, Baian; Zhu, Ting; Hassan, Mohammad Farooque; Yin, Xiaomin; Zhou, Xiangmei; Zhao, Deming

    2015-06-01

    The formation of neurotoxic prion protein (PrP) oligomers is thought to be a key step in the development of prion diseases. Recently, it was determined that the sonication and shaking of recombinant PrP can convert PrP monomers into β-state oligomers. Herein, we demonstrate that β-state oligomeric PrP can be generated through protein misfolding cyclic amplification from recombinant full-length hamster, human, rabbit, and mutated rabbit PrP, and that these oligomers can be used for subsequent research into the mechanisms of PrP-induced neurotoxicity. We have characterized protein misfolding cyclic amplification-induced monomer-to-oligomer conversion of PrP from three species using western blotting, circular dichroism, size-exclusion chromatography, and resistance to proteinase K (PK) digestion. We have further shown that all of the resulting β-oligomers are toxic to primary mouse cortical neurons independent of the presence of PrP(C) in the neurons, whereas the corresponding monomeric PrP were not toxic. In addition, we found that this toxicity is the result of oligomer-induced apoptosis via regulation of Bcl-2, Bax, and caspase-3 in both wild-type and PrP(-/-) cortical neurons. It is our hope that these results may contribute to our understanding of prion transformation within the brain. We found that β-state oligomeric PrPs can be generated through protein misfolding cyclic amplification (PMCA) from recombinant full-length hamster, human, rabbit, and mutated rabbit PrP. β-oligomers are toxic to primary mouse cortical neurons independent of the presence of PrP(C) in the neurons, while the corresponding monomeric PrPs were not toxic. This toxicity is the result of oligomers-induced apoptosis via regulation of Bcl-2, Bax, and caspase-3. These results may contribute to our understanding of prion transformation within the brain. © 2015 International Society for Neurochemistry.

  15. 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.

  16. Importance of Heat and Pressure for Solubilization of Recombinant Spider Silk Proteins in Aqueous Solution.

    Science.gov (United States)

    Jones, Justin A; Harris, Thomas I; Oliveira, Paula F; Bell, Brianne E; Alhabib, Abdulrahman; Lewis, Randolph V

    2016-11-23

    The production of recombinant spider silk proteins continues to be a key area of interest for a number of research groups. Several key obstacles exist in their production as well as in their formulation into useable products. The original reported method to solubilize recombinant spider silk proteins (rSSp) in an aqueous solution involved using microwaves to quickly generate heat and pressure inside of a sealed vial containing rSSp and water. Fibers produced from this system are remarkable in their mechanical ability and demonstrate the ability to be stretched and recover 100 times. The microwave method dissolves the rSSPs with dissolution time increasing with higher molecular weight constructs, increasing concentration of rSSPs, protein type, and salt concentration. It has proven successful in solvating a number of different rSSPs including native-like sequences (MaSp1, MaSp2, piriform, and aggregate) as well as chimeric sequences (FlAS) in varied concentrations that have been spun into fibers and formed into films, foams, sponges, gels, coatings, macro and micro spheres and adhesives. The system is effective but inherently unpredictable and difficult to control. Provided that the materials that can be generated from this method of dissolution are impressive, an alternative means of applying heat and pressure that is controllable and predictable has been developed. Results indicate that there are combinations of heat and pressure (135 °C and 140 psi) that result in maximal dissolution without degrading the recombinant MaSp2 protein tested, and that heat and pressure are the key elements to the method of dissolution.

  17. Protein-only, antimicrobial peptide-containing recombinant nanoparticles with inherent built-in antibacterial activity.

    Science.gov (United States)

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

    2017-09-15

    The emergence of bacterial antibiotic resistances is a serious concern in human and animal health. In this context, naturally occurring cationic antimicrobial peptides (AMPs) might play a main role in a next generation of drugs against bacterial infections. Taking an innovative approach to design self-organizing functional proteins, we have generated here protein-only nanoparticles with intrinsic AMP microbicide activity. Using a recombinant version of the GWH1 antimicrobial peptide as building block, these materials show a wide antibacterial activity spectrum in absence of detectable toxicity on mammalian cells. The GWH1-based nanoparticles combine clinically appealing properties of nanoscale materials with full biocompatibility, structural and functional plasticity and biological efficacy exhibited by proteins. Because of the largely implemented biological fabrication of recombinant protein drugs, the protein-based platform presented here represents a novel and scalable strategy in antimicrobial drug design, that by solving some of the limitations of AMPs offers a promising alternative to conventional antibiotics. The low molecular weight antimicrobial peptide GWH1 has been engineered to oligomerize as self-assembling protein-only nanoparticles of around 50nm. In this form, the peptide exhibits potent and broad antibacterial activities against both Gram-positive and Gram-negative bacteria, without any harmful effect over mammalian cells. As a solid proof-of-concept, this finding strongly supports the design and biofabrication of nanoscale antimicrobial materials with in-built functionalities. The protein-based homogeneous composition offer advantages over alternative materials explored as antimicrobial agents, regarding biocompatibility, biodegradability and environmental suitability. Beyond the described prototype, this transversal engineering concept has wide applicability in the design of novel nanomedicines for advanced treatments of bacterial infections

  18. 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

  19. 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

  20. 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...... was performed on ultracryosections of pig small intestine using antibodies directed against aminopeptidase N and sucrase-isomaltase. The labelling efficiency of RPGG was compared to that of protein A-gold conjugates (PAG) in different compartments of the enterocyte. Quantification showed that the labelling...... intensity was dependent on the size of the marker as well as on the kind of protein used for complex formation. The distributions for RPGG and PAG were respectively: for the 12 nm particles, 10.3 and 6.2 particles/micron of length of microvillar membrane, 3.5 and 1.0 particles/micron2 of Golgi profile and 5...

  1. Serological Analysis of Immunogenic Properties of Recombinant Meningococcus IgA1 Protease-Based Proteins.

    Science.gov (United States)

    Kotelnikova, O V; Zinchenko, A A; Vikhrov, A A; Alliluev, A P; Serova, O V; Gordeeva, E A; Zhigis, L S; Zueva, V S; Razgulyaeva, O A; Melikhova, T D; Nokel, E A; Drozhzhina, E Yu; Rumsh, L D

    2016-07-01

    Using the genome sequence of IgA1 protease of N. meningitidis of serogroup B, four recombinant proteins of different structure and molecular weight were constructed. These proteins were equal in inducing the formation of specific antibodies to IgA1 protease and had protective properties against meningococci. In the sera of immunized mice, anti-IgA1 protease antibodies were detected by whole-cell ELISA, which indicated the presence of IgA1 protease on the surface of these bacteria. We hypothesized that the protective properties of IgA1 protease-based antigens and IgA1 protease analogs could be realized not only via impairment of bacterium adhesion to the mucosa, but also via suppression of this pathogen in the organism. The presented findings seem promising for using these proteins as the basis for anti-meningococcus vaccine.

  2. 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.

  3. Immunogenicity of recombinant class 1 protein from Neisseria meningitidis refolded into phospholipid vesicles and detergent.

    Science.gov (United States)

    Niebla, O; Alvarez, A; Martín, A; Rodríguez, A; Delgado, M; Falcón, V; Guillén, G

    2001-05-14

    The possibility of eliciting bactericidal antibodies against a recombinant class 1 protein (P1) from Neisseria meningitidis, joined to the first 45 amino acids of the neisserial LpdA protein (PM82), was examined. P1 was produced in Escherichia coli as intracellular inclusion bodies, from which it was purified and reconstituted by (a) inclusion into phospholipid vesicles and detergent and (b) refolding in 0.1% SDS. When Balb/c mice were immunised, high titres of subtype-specific bactericidal antibodies against P1 were obtained in both cases. These results suggest that in spite of being a denaturing agent, it is possible to use SDS to reconstitute the P1 protein in a conformation that exposes the immunodominat regions.

  4. 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.

  5. 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.

  6. 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.

  7. A recombinant pseudorabies virus co-expressing capsid proteins precursor P1-2A of FMDV and VP2 protein of porcine parvovirus: a trivalent vaccine candidate.

    Science.gov (United States)

    Hong, Qi; Qian, Ping; Li, Xiang-Min; Yu, Xiao-Lan; Chen, Huan-Chun

    2007-11-01

    Pseudorabies (PR), foot-and-mouth disease (FMD), and porcine parvovirus disease are three important infectious diseases in swine worldwide. The gene-deleted pseudorabies virus (PRV) has been used as a live-viral vector to develop multivalent genetic engineering vaccine. In this study, a recombinant PRV, which could co-express protein precursor P1-2A of FMDV and VP2 protein of PPV, was constructed using PRV TK(-)/gE(-)/LacZ(+) mutant as the vector. After homologous recombination and plaque purification, recombinant virus PRV TK(-)/gE(-)/P1-2A-VP2 was acquired and identified. Immunogenicity, safety of the recombinant PRV and its protection against PRV were confirmed in a mouse model by indirect ELISA and serum neutralization test. The results show that the recombinant PRV is a candidate vaccine strain to develop a novel trivalent vaccine against PRV, FMDV and PPV in swine.

  8. A novel mouse synaptonemal complex protein is essential for loading of central element proteins, recombination, and fertility.

    Directory of Open Access Journals (Sweden)

    Sabine Schramm

    2011-05-01

    Full Text Available The synaptonemal complex (SC is a proteinaceous, meiosis-specific structure that is highly conserved in evolution. During meiosis, the SC mediates synapsis of homologous chromosomes. It is essential for proper recombination and segregation of homologous chromosomes, and therefore for genome haploidization. Mutations in human SC genes can cause infertility. In order to gain a better understanding of the process of SC assembly in a model system that would be relevant for humans, we are investigating meiosis in mice. Here, we report on a newly identified component of the murine SC, which we named SYCE3. SYCE3 is strongly conserved among mammals and localizes to the central element (CE of the SC. By generating a Syce3 knockout mouse, we found that SYCE3 is required for fertility in both sexes. Loss of SYCE3 blocks synapsis initiation and results in meiotic arrest. In the absence of SYCE3, initiation of meiotic recombination appears to be normal, but its progression is severely impaired resulting in complete absence of MLH1 foci, which are presumed markers of crossovers in wild-type meiocytes. In the process of SC assembly, SYCE3 is required downstream of transverse filament protein SYCP1, but upstream of the other previously described CE-specific proteins. We conclude that SYCE3 enables chromosome loading of the other CE-specific proteins, which in turn would promote synapsis between homologous chromosomes.

  9. Deleting multiple lytic genes enhances biomass yield and production of recombinant proteins by Bacillus subtilis.

    Science.gov (United States)

    Wang, Yi; Chen, Zhenmin; Zhao, Ruili; Jin, Tingting; Zhang, Xiaoming; Chen, Xiangdong

    2014-08-31

    Bacillus subtilis is widely used in agriculture and industrial biotechnology; however, cell autolysis significantly decreases its yield in liquid cultures. Numerous factors mediate the lysis of B. subtilis, such as cannibalism factors, prophages, and peptidoglycan (PG) hydrolases. The aim of this work was to use molecular genetic techniques to develop a new strategy to prevent cell lysis and enhance biomass as well as the production of recombinant proteins. Five genes or genetic elements representing three different functional categories were studied as follows: lytC encoding PG hydrolases, the prophage genes xpf and yqxG-yqxH-cwlA (yGlA), and skfA and sdpC that encode cannibalism factors. Cell lysis was reduced and biomass was enhanced by deleting individually skfA, sdpC, xpf, and lytC. We constructed the multiple deletion mutant LM2531 (skfA sdpC lytC xpf) and found that after 4 h of culture, its biomass yield was significantly increased compared with that of prototypical B. subtilis 168 (wild-type) strain and that 15% and 92% of the cells were lysed in cultures of LM2531 and wild-type, respectively. Moreover, two expression vectors were constructed for producing recombinant proteins (β-galactosidase and nattokinase) under the control of the P43 promoter. Cultures of LM2531 and wild-type transformants produced 13741 U/ml and 7991 U/ml of intracellular β-galactosidase, respectively (1.72-fold increase). Further, the level of secreted nattokinase produced by strain LM2531 increased by 2.6-fold compared with wild-type (5226 IU/ml vs. 2028 IU/ml, respectively). Our novel, systematic multigene deletion approach designed to inhibit cell lysis significantly increased the biomass yield and the production of recombinant proteins by B. subtilis. These findings show promise for guiding efforts to manipulate the genomes of other B. subtilis strains that are used for industrial purposes.

  10. 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

  11. Positive and negative regulation of V(D)J recombination by the E2A proteins.

    Science.gov (United States)

    Bain, G; Romanow, W J; Albers, K; Havran, W L; Murre, C

    1999-01-18

    A key feature of B and T lymphocyte development is the generation of antigen receptors through the rearrangement and assembly of the germline variable (V), diversity (D), and joining (J) gene segments. However, the mechanisms responsible for regulating developmentally ordered gene rearrangements are largely unknown. Here we show that the E2A gene products are essential for the proper coordinated temporal regulation of V(D)J rearrangements within the T cell receptor (TCR) gamma and delta loci. Specifically, we show that E2A is required during adult thymocyte development to inhibit rearrangements to the gamma and delta V regions that normally recombine almost exclusively during fetal thymocyte development. The continued rearrangement of the fetal Vgamma3 gene segment in E2A-deficient adult thymocytes correlates with increased levels of Vgamma3 germline transcripts and increased levels of double-stranded DNA breaks at the recombination signal sequence bordering Vgamma3. Additionally, rearrangements to a number of Vgamma and Vdelta gene segments used predominantly during adult development are significantly reduced in E2A-deficient thymocytes. Interestingly, at distinct stages of T lineage development, both the increased and decreased rearrangement of particular Vdelta gene segments is highly sensitive to the dosage of the E2A gene products, suggesting that the concentration of the E2A proteins is rate limiting for the recombination reaction involving these Vdelta regions.

  12. Diagnostic value of recombinant Tp0821 protein in serodiagnosis for syphilis.

    Science.gov (United States)

    Xie, Y; Xu, M; Wang, C; Xiao, J; Xiao, Y; Jiang, C; You, X; Zhao, F; Zeng, T; Liu, S; Kuang, X; Wu, Y

    2016-04-01

    Syphilis is a multistage sexually transmitted disease that remains a serious public health concern worldwide. The coexistence of Treponema pallidum with other closely related members of spirochaeta, such as Leptospira spp. and Borrelia burgdorferi, has complicated the serodiagnosis due to cross-reactive antigens. In this study, recombinant Tp0821 protein was expressed in Escherichia coli and purified by metal affinity chromatography. Then enzyme-linked immunosorbent assays (ELISAs) based on Tp0821 for the detection of specific antibodies were established. The relative positive rates of the IgM ELISA and the IgG ELISA were found to be 91·0 and 98·3%, respectively, when screening 578 syphilis specimens. The specificities were 94·3 and 100%, respectively, when cross-checking with serum samples obtained from 30 patients with Lyme disease, five patients with leptospirosis, and 52 uninfected controls. In addition, relative positive rates and specificities of Tp0821 for human sera were all 100% in Western blotting. When compared to the syphilis diagnostic tests commonly used in clinical settings, we found that the results of Tp0821-based ELISAs correlated well with the results of the treponemal tests, specifically the T. pallidum particle agglutination (TP-PA) test and the chemiluminescent immunoassay (CIA). Thus, these findings identify Tp0821 as a novel serodiagnostic candidate for syphilis. In this study, we expressed and purified the Treponema pallidum protein Tp0821 and developed Tp0821-based enzyme-linked immunosorbent assays (ELISAs) for the detection of specific antibodies. The serodiagnostic performance of the recombinant protein was then evaluated. When compared to the results of syphilis diagnostic tests commonly used in clinical settings, we found that the reactivities of syphilitic sera with the recombinant antigen correlated well with the results of the treponemal tests, specifically the T. pallidum particle agglutination (TP-PA) test and the

  13. Maxillary anterior ridge augmentation with recombinant human bone morphogenetic protein 2.

    Science.gov (United States)

    Edmunds, Ryan K; Mealey, Brian L; Mills, Michael P; Thoma, Daniel S; Schoolfield, John; Cochran, David L; Mellonig, Jim

    2014-01-01

    No human studies exist on the use of recombinant human bone morphogenetic protein 2 (rhBMP-2) on an absorbable collagen sponge (ACS) as a sole graft material for lateral ridge augmentation in large ridge defect sites. This series evaluates the treatment outcome of maxillary anterior lateral ridge augmentation with rhBMP-2/ACS. Twenty patients were treated with rhBMP-2/ACS and fixation screws for space maintenance. Cone beam volumetric tomography measurements were used to determine gain in ridge width, and a bone core biopsy was obtained. The mean horizontal ridge gain was 1.2 mm across sites, and every site gained width.

  14. 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.

  15. 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

  16. Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway

    Directory of Open Access Journals (Sweden)

    Krainer Florian W

    2012-02-01

    Full Text Available Abstract 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

  17. 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

  18. Recombinant production and solution structure of lipid transfer protein from lentil Lens culinaris

    Energy Technology Data Exchange (ETDEWEB)

    Gizatullina, Albina K. [Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya str., 16/10, 117997 Moscow (Russian Federation); Moscow Institute of Physics and Technology (State University), Department of Physicochemical Biology and Biotechnology, Institutskii per., 9, 141700, Dolgoprudny, Moscow Region (Russian Federation); Finkina, Ekaterina I.; Mineev, Konstantin S.; Melnikova, Daria N.; Bogdanov, Ivan V. [Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya str., 16/10, 117997 Moscow (Russian Federation); Telezhinskaya, Irina N.; Balandin, Sergey V. [Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya str., 16/10, 117997 Moscow (Russian Federation); Moscow Institute of Physics and Technology (State University), Department of Physicochemical Biology and Biotechnology, Institutskii per., 9, 141700, Dolgoprudny, Moscow Region (Russian Federation); Shenkarev, Zakhar O. [Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya str., 16/10, 117997 Moscow (Russian Federation); Arseniev, Alexander S. [Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya str., 16/10, 117997 Moscow (Russian Federation); Moscow Institute of Physics and Technology (State University), Department of Physicochemical Biology and Biotechnology, Institutskii per., 9, 141700, Dolgoprudny, Moscow Region (Russian Federation); Ovchinnikova, Tatiana V., E-mail: ovch@ibch.ru [Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya str., 16/10, 117997 Moscow (Russian Federation); Moscow Institute of Physics and Technology (State University), Department of Physicochemical Biology and Biotechnology, Institutskii per., 9, 141700, Dolgoprudny, Moscow Region (Russian Federation)

    2013-10-04

    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{sup 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.

  19. 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

  20. Factors Affecting the Binding of a Recombinant Heavy Metal-Binding Domain (CXXC motif Protein to Heavy Metals

    Directory of Open Access Journals (Sweden)

    Kamala Boonyodying

    2012-06-01

    Full Text Available A number of heavy metal-binding proteins have been used to study bioremediation. CXXC motif, a metal binding domain containing Cys-X-X-Cys motif, has been identified in various organisms. These proteins are capable of binding various types of heavy metals. In this study, heavy metal binding domain (CXXC motif recombinant protein encoded from mcsA gene of S. aureus were cloned and overexpressed in Escherichia coli. The factors involved in the metal-binding activity were determined in order to analyze the potential of recombinant protein for bioremediation. A recombinant protein can be bound to Cd2+, Co2+, Cu2+ and Zn2+. The thermal stability of a recombinant protein was tested, and the results showed that the metal binding activity to Cu2+ and Zn2+ still exist after treating the protein at 85ºC for 30 min. The temperature and pH that affected the metal binding activity was tested and the results showed that recombinant protein was still bound to Cu2+ at 65ºC, whereas a pH of 3-7 did not affect the metal binding E. coli harboring a pRset with a heavy metal-binding domain CXXC motif increased the resistance of heavy metals against CuCl2 and CdCl2. This study shows that metal binding domain (CXXC motif recombinant protein can be effectively bound to various types of heavy metals and may be used as a potential tool for studying bioremediation.

  1. Relationship between recombinant protein expression and host metabolome as determined by two-dimensional NMR spectroscopy.

    Directory of Open Access Journals (Sweden)

    Young Kee Chae

    Full Text Available Escherichia coli has been the most widely used host to produce large amounts of heterologous proteins. However, given an input plasmid DNA, E. coli may produce soluble protein, produce only inclusion bodies, or yield little or no protein at all. Many efforts have been made to surmount these problems, but most of them have involved time-consuming and labor-intensive trial-and-error. We hypothesized that different metabolomic fingerprints might be associated with different protein production outcomes. If so, then it might be possible to change the expression pattern by manipulating the metabolite environment. As a first step in testing this hypothesis, we probed a subset of the intracellular metabolites by partially labeling it with 13C-glucose. We tested 71 genes and identified 17 metabolites by employing the two-dimensional NMR spectroscopy. The statistical analysis showed that there existed the metabolite compositions favoring protein production. We hope that this work would help devise a systematic and predictive approach to the recombinant protein production.

  2. 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.

  3. 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 available...... ELISA kits. We observed that estimations of recombinant human ø1-antitrypsin (rø1AT) titer by Coomassie-stained SDS-PAGE gels did not correspond to previously obtained titers obtained by a commercially available ELISA kit. This prompted us to develop two independent quantification assays based...... on biolayer interferometry and reversed-phase high-performance liquid chromatography. We compared the rø1AT titer obtained by these assays with three different off-the-shelf ELISA kits and found that the ELISA kits led to inconsistent results. The data presented here show that recombinant protein titers...

  4. 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

  5. Application of electrospray mass spectrometry to the characterization of recombinant proteins up to 44 kDa

    NARCIS (Netherlands)

    Van Dorsselaer, A.; Bitsch, F.; Green, B.; Jarvis, S.; Lepage, P.; Bischoff, Rainer; Kolbe, V.J.; Roitsch, C.

    1990-01-01

    Mass measurement by electrospray mass spectrometry (ESMS) is used as a rapid preliminary verification of the identity of various recombinant proteins ranging from 7 to 44 kDa with an accuracy of 0.01-0.03%. ESMS not only improves the speed but also the reliability of the protein structure

  6. 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.

  7. "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,…

  8. Cloning, expression, and purification of recombinant protein MPT-64 from a virulent strain of Mycobacterium bovis in a prokaryotic system

    Directory of Open Access Journals (Sweden)

    Maryam Mohammadi Tashakkori

    2016-01-01

    Conclusion: These results indicated successful expression and purification of recombinant MPT-64 protein in a prokaryotic system. This protein can be used for serological diagnosis, improved detection of pathogenicity and non-pathogenicity between infected cattle, and for verification of suspected cases of bovine TB.

  9. Modulating secretory pathway pH by proton channel co-expression can increase recombinant protein stability in plants.

    Science.gov (United States)

    Jutras, Philippe V; D'Aoust, Marc-André; Couture, Manon M-J; Vézina, Louis-Philippe; Goulet, Marie-Claire; Michaud, Dominique; Sainsbury, Frank

    2015-09-01

    Eukaryotic expression systems are used for the production of complex secreted proteins. However, recombinant proteins face considerable biochemical challenges along the secretory pathway, including proteolysis and pH variation between organelles. As the use of synthetic biology matures into solutions for protein production, various host-cell engineering approaches are being developed to ameliorate host-cell factors that can limit recombinant protein quality and yield. We report the potential of the influenza M2 ion channel as a novel tool to neutralize the pH in acidic subcellular compartments. Using transient expression in the plant host, Nicotiana benthamiana, we show that ion channel expression can significantly raise pH in the Golgi apparatus and that this can have a strong stabilizing effect on a fusion protein separated by an acid-susceptible linker peptide. We exemplify the utility of this effect in recombinant protein production using influenza hemagglutinin subtypes differentially stable at low pH; the expression of hemagglutinins prone to conformational change in mildly acidic conditions is considerably enhanced by M2 co-expression. The co-expression of a heterologous ion channel to stabilize acid-labile proteins and peptides represents a novel approach to increasing the yield and quality of secreted recombinant proteins in plants and, possibly, in other eukaryotic expression hosts. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Structural and functional characterization of the recombinant death domain from death-associated protein kinase.

    Science.gov (United States)

    Dioletis, Evangelos; Dingley, Andrew J; Driscoll, Paul C

    2013-01-01

    Death-associated protein kinase (DAPk) is a calcium/calmodulin-regulated Ser/Thr-protein kinase that functions at an important point of integration for cell death signaling pathways. DAPk has a structurally unique multi-domain architecture, including a C-terminally positioned death domain (DD) that is a positive regulator of DAPk activity. In this study, recombinant DAPk-DD was observed to aggregate readily and could not be prepared in sufficient yield for structural analysis. However, DAPk-DD could be obtained as a soluble protein in the form of a translational fusion protein with the B1 domain of streptococcal protein G. In contrast to other DDs that adopt the canonical six amphipathic α-helices arranged in a compact fold, the DAPk-DD was found to possess surprisingly low regular secondary structure content and an absence of a stable globular fold, as determined by circular dichroism (CD), NMR spectroscopy and a temperature-dependent fluorescence assay. Furthermore, we measured the in vitro interaction between extracellular-regulated kinase-2 (ERK2) and various recombinant DAPk-DD constructs. Despite the low level of structural order, the recombinant DAPk-DD retained the ability to interact with ERK2 in a 1∶1 ratio with a K d in the low micromolar range. Only the full-length DAPk-DD could bind ERK2, indicating that the apparent 'D-motif' located in the putative sixth helix of DAPk-DD is not sufficient for ERK2 recognition. CD analysis revealed that binding of DAPk-DD to ERK2 is not accompanied by a significant change in secondary structure. Taken together our data argue that the DAPk-DD, when expressed in isolation, does not adopt a classical DD fold, yet in this state retains the capacity to interact with at least one of its binding partners. The lack of a stable globular structure for the DAPk-DD may reflect either that its folding would be supported by interactions absent in our experimental set-up, or a limitation in the structural bioinformatics

  11. 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

    2013-01-01

    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. PMID:23863502

  12. 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.

  13. De novo generation of infectious prions with bacterially expressed recombinant prion protein.

    Science.gov (United States)

    Zhang, Zhihong; Zhang, Yi; Wang, Fei; Wang, Xinhe; Xu, Yuanyuan; Yang, Huaiyi; Yu, Guohua; Yuan, Chonggang; Ma, Jiyan

    2013-12-01

    The prion hypothesis is strongly supported by the fact that prion infectivity and the pathogenic conformer of prion protein (PrP) are simultaneously propagated in vitro by the serial protein misfolding cyclic amplification (sPMCA). However, due to sPMCA's enormous amplification power, whether an infectious prion can be formed de novo with bacterially expressed recombinant PrP (rPrP) remains to be satisfactorily resolved. To address this question, we performed unseeded sPMCA with rPrP in a laboratory that has never been exposed to any native prions. Two types of proteinase K (PK)-resistant and self-perpetuating recombinant PrP conformers (rPrP-res) with PK-resistant cores of 17 or 14 kDa were generated. A bioassay revealed that rPrP-res(17kDa) was highly infectious, causing prion disease in wild-type mice with an average survival time of about 172 d. In contrast, rPrP-res(14kDa) completely failed to induce any disease. Our findings reveal that sPMCA is sufficient to initiate various self-perpetuating PK-resistant rPrP conformers, but not all of them possess in vivo infectivity. Moreover, generating an infectious prion in a prion-free environment establishes that an infectious prion can be formed de novo with bacterially expressed rPrP.

  14. Application of recombinant hemagglutinin proteins as alternative antigen standards for pandemic influenza vaccines.

    Science.gov (United States)

    Choi, Yejin; Kwon, Seong Yi; Oh, Ho Jung; Shim, Sunbo; Chang, Seokkee; Chung, Hye Joo; Kim, Do Keun; Park, Younsang; Lee, Younghee

    2017-09-01

    The single radial immunodiffusion (SRID) assay, used to quantify hemagglutinin (HA) in influenza vaccines, requires reference reagents; however, because centralized production of reference reagents may slow the emergency deployment of vaccines, alternatives are needed. We investigated the production of HA proteins using recombinant DNA technology, rather than a traditional egg-based production process. The HA proteins were then used in an SRID assay as a reference antigen. We found that HA can be quantified in both egg-based and cell-based influenza vaccines when recombinant HAs (rHAs) are used as the reference antigen. Furthermore, we confirmed that rHAs obtained from strains with pandemic potential, such as H5N1, H7N3, H7N9, and H9N2 strains, can be utilized in the SRID assay. The rHA production process takes just one month, in contrast to the traditional process that takes three to four months. The use of rHAs may reduce the time required to produce reference reagents and facilitate timely introduction of vaccines during emergencies.

  15. 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.

  16. Determination of immune status in dogs against CPV-2 by recombinant protein based latex agglutination test.

    Science.gov (United States)

    Thomas, Jobin; Singh, Mithilesh; Goswami, T K; Glora, Philma; Chakravarti, Soumendu; Chander, Vishal; Upmanyu, Vikramaditya; Verma, Suman; Sharma, Chhavi; Mahendran, K

    2017-09-01

    Canine parvoviral enteritis is a highly contagious viral illness caused by canine parvovirus-2 (CPV-2) which affects puppies of mainly 6-20 weeks of age. Vaccination is pivotal in preventing and controlling CPV-2 infection. Determination of antibody status is a critical determinant for successful vaccination. The hemagglutination inhibition (HI) test is 'gold standard' test for quantification of antibodies specific to CPV-2, although the execution of this test is not feasible under field conditions. The present study was undertaken to develop a point of care testing to determine immune status prior to CPV-2 vaccination or to detect seroconversion in immunized dogs by latex agglutination test (LAT) using recombinant antigen. Truncated portion of VP2 protein (tVP2) of CPV-2 was selected on the basis of antigenic indices, overexpressed the recombinant protein in E. coli system and was subsequently used in development of LAT. A total of 59 serum samples obtained from vaccinated (n = 54) and healthy unvaccinated (n = 5) dogs were tested. The positivity was observed in 85% (46/54) of these dogs with varying agglutination pattern. The overall sensitivity and specificity of latex agglutination test in comparison to HI test was recorded as 90% and 88% respectively with an agreement value of 90% (CI = 95%). Copyright © 2017 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

  17. 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.

  18. A simple Pichia pastoris fermentation and downstream processing strategy for making recombinant pandemic Swine Origin Influenza a virus Hemagglutinin protein.

    Science.gov (United States)

    Athmaram, T N; Singh, Anil Kumar; Saraswat, Shweta; Srivastava, Saurabh; Misra, Princi; Kameswara Rao, M; Gopalan, N; Rao, P V L

    2013-02-01

    The present Influenza vaccine manufacturing process has posed a clear impediment to initiation of rapid mass vaccination against spreading pandemic influenza. New vaccine strategies are therefore needed that can accelerate the vaccine production. Pichia offers several advantages for rapid and economical bulk production of recombinant proteins and, hence, can be attractive alternative for producing an effective influenza HA based subunit vaccine. The recombinant Pichia harboring the transgene was subjected to fed-batch fermentation at 10 L scale. A simple fermentation and downstream processing strategy is developed for high-yield secretory expression of the recombinant Hemagglutinin protein of pandemic Swine Origin Influenza A virus using Pichia pastoris via fed-batch fermentation. Expression and purification were optimized and the expressed recombinant Hemagglutinin protein was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blot and MALDI-TOF analysis. In this paper, we describe a fed-batch fermentation protocol for the secreted production of Swine Influenza A Hemagglutinin protein in the P. pastoris GS115 strain. We have shown that there is a clear relationship between product yield and specific growth rate. The fed-batch fermentation and downstream processing methods optimized in the present study have immense practical application for high-level production of the recombinant H1N1 HA protein in a cost effective way using P. pastoris.

  19. Polyclonal antibodies against the recombinantly expressed coat protein of the Citrus psorosis virus

    Directory of Open Access Journals (Sweden)

    Reda Salem

    2018-05-01

    Full Text Available Psorosis is a damaging disease of citrus that is widespread in many parts of the world. Citrus psorosis virus (CPsV, the type species of the genus Ophiovirus, is the putative causal agent of psorosis. Detection of CPsV by laboratory methods, serology in particular is a primary requirement for large-scale surveys but their production has been impaired by the difficulty of obtaining sufficient clean antigen for immunization. Specific PAbs against coat protein were produced in E. coli using recombinant DNA approach. The full length CP gene fragment was amplified by RT-PCR using total RNA extracted from CPsV infected citrus leaves and CP specific primers. The obtained product (1320bp was cloned, sequenced and sub-cloned into pET-30(+ expression vector. Expression was induced and screened in different bacterial clones by the presence of the expressed protein (48kDa and optimized in one clone. Expressed CP was purified using batch chromatography under denaturing conditions. Specificity of expressed protein was demonstrated by ELISA before used as antigen for raising PAbs in mice. Specificity of the raised PAbs to CPsV was verified by ELISA and western blotting. The raised PAbs were showed highly effectiveness in screening by ELISA comparing with the commercial antibodies purchased from Agritest, Valanzano, Italy.The expression of CPsV CP gene in E. coli, production of PAbs using recombinant protein as an antigen, the suitability of these antibodies for use in immunodiagnostics against the CPsV Egyptian isolate have been accomplished in this work. Keywords: CPsV, CP, PAbs, RT-PCR, ELISA, Western blotting

  20. Attenuation of Recombinant Yellow Fever 17D Viruses Expressing Foreign Protein Epitopes at the Surface

    Science.gov (United States)

    Bonaldo, Myrna C.; Garratt, Richard C.; Marchevsky, Renato S.; Coutinho, Evandro S. F.; Jabor, Alfredo V.; Almeida, Luís F. C.; Yamamura, Anna M. Y.; Duarte, Adriana S.; Oliveira, Prisciliana J.; Lizeu, Jackeline O. P.; Camacho, Luiz A. B.; Freire, Marcos S.; Galler, Ricardo

    2005-01-01

    The yellow fever (YF) 17D vaccine is a live attenuated virus. Three-dimensional (3D) homology modeling of the E protein structure from YF 17D virus and its comparison with that from tick-borne encephalitis virus revealed that it is possible to accommodate inserts of different sizes and amino acid compositions in the flavivirus E protein fg loop. This is consistent with the 3D structures of both the dimeric and trimeric forms in which the fg loop lies exposed to solvents. We demonstrate here that YF 17D viruses bearing foreign humoral (17D/8) and T-cell (17D/13) epitopes, which vary in sequence and length, displayed growth restriction. It is hypothesized that interference with the dimer-trimer transition and with the formation of a ring of such trimers in order to allow fusion compromises the capability of the E protein to induce fusion of viral and endosomal membranes, and a slower rate of fusion may delay the extent of virus production. This would account for the lower levels of replication in cultured cells and of viremia in monkeys, as well as for the more attenuated phenotype of the recombinant viruses in monkeys. Testing of both recombinant viruses (17D/8 and 17D/13) for monkey neurovirulence also suggests that insertion at the 17D E protein fg loop does not compromise the attenuated phenotype of YF 17D virus, further confirming the potential use of this site for the development of new live attenuated 17D virus-based vaccines. PMID:15956601

  1. 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.

  2. 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.

  3. 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

  4. Complex relationship between mismatch repair proteins and MBD4 during immunoglobulin class switch recombination.

    Science.gov (United States)

    Grigera, Fernando; Bellacosa, Alfonso; Kenter, Amy L

    2013-01-01

    Mismatch repair (MMR) safeguards against genomic instability and is required for efficient Ig class switch recombination (CSR). Methyl CpG binding domain protein 4 (MBD4) binds to MutL homologue 1 (MLH1) and controls the post-transcriptional level of several MMR proteins, including MutS homologue 2 (MSH2). We show that in WT B cells activated for CSR, MBD4 is induced and interacts with MMR proteins, thereby implying a role for MBD4 in CSR. However, CSR is in the normal range in Mbd4 deficient mice deleted for exons 2-5 despite concomitant reduction of MSH2. We show by comparison in Msh2(+/-) B cells that a two-fold reduction of MSH2 and MBD4 proteins is correlated with impaired CSR. It is therefore surprising that CSR occurs at normal frequencies in the Mbd4 deficient B cells where MSH2 is reduced. We find that a variant Mbd4 transcript spanning exons 1,6-8 is expressed in Mbd4 deficient B cells. This transcript can be ectopically expressed and produces a truncated MBD4 peptide. Thus, the 3' end of the Mbd4 locus is not silent in Mbd4 deficient B cells and may contribute to CSR. Our findings highlight a complex relationship between MBD4 and MMR proteins in B cells and a potential reconsideration of their role in CSR.

  5. Production and purification of avian antibodies (IgYs from inclusion bodies of a recombinant protein central in NAD+ metabolism

    Directory of Open Access Journals (Sweden)

    Paula A. Moreno-González

    2013-08-01

    Full Text Available The use of hens for the production of polyclonal antibodies reduces animal intervention and moreover yields a higher quantity of antibodies than other animal models.  The phylogenetic distance between bird and mammal antigens, often leads to more specific avian antibodies than their mammalian counterparts.Since a large amount of antigen is required for avian antibody production, the use of recombinant proteins for this procedure has been growing faster over the last years. Nevertheless, recombinant protein production through heterologous systems frequently prompts the protein to precipitate, forming insoluble aggregates of limited utility (inclusion bodies. A methodology for the production of avian polyclonal antibodies, using recombinant protein from inclusion bodies is presented in this article.In order to produce the antigen, a recombinant Nicotinamide mononucleotide adenylyltransferase from Giardia intestinalis (His-GiNMNAT was expressed in Escherichia coli.  The protein was purified through solubilization from inclusion bodies prior to its renaturalization.  Antibodies were purified from egg yolk of immunized hens by water dilution, followed by ammonium sulfate precipitation and thiophilic affinity chromatography.The purified antibodies were tested against His-GiNMNAT protein in Western blot essays. From one egg yolk, 14.4 mg of highly pure IgY were obtained; this antibody was able to detect 15ng of His-GiNMNAT.  IgY specificity was improved by means of antigen affinity purification, allowing its use for parasite protein recognition.

  6. 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

  7. 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.

  8. 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

  9. Biomimetic mineralization of recombinant collagen type I derived protein to obtain hybrid matrices for bone regeneration.

    Science.gov (United States)

    Ramírez-Rodríguez, Gloria Belén; Delgado-López, José Manuel; Iafisco, Michele; Montesi, Monica; Sandri, Monica; Sprio, Simone; Tampieri, Anna

    2016-11-01

    Understanding the mineralization mechanism of synthetic protein has recently aroused great interest especially in the development of advanced materials for bone regeneration. Herein, we propose the synthesis of composite materials through the mineralization of a recombinant collagen type I derived protein (RCP) enriched with RGD sequences in the presence of magnesium ions (Mg) to closer mimic bone composition. The role of both RCP and Mg ions in controlling the precipitation of the mineral phase is in depth evaluated. TEM and X-ray powder diffraction reveal the crystallization of nanocrystalline apatite (Ap) in all the evaluated conditions. However, Raman spectra point out also the precipitation of amorphous calcium phosphate (ACP). This amorphous phase is more evident when RCP and Mg are at work, indicating the synergistic role of both in stabilizing the amorphous precursor. In addition, hybrid matrices are prepared to tentatively address their effectiveness as scaffolds for bone tissue engineering. SEM and AFM imaging show an homogeneous mineral distribution on the RCP matrix mineralized in presence of Mg, which provides a surface roughness similar to that found in bone. Preliminary in vitro tests with pre-osteoblast cell line show good cell-material interaction on the matrices prepared in the presence of Mg. To the best of our knowledge this work represents the first attempt to mineralize recombinant collagen type I derived protein proving the simultaneous effect of the organic phase (RCP) and Mg on ACP stabilization. This study opens the possibility to engineer, through biomineralization process, advanced hybrid matrices for bone regeneration. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Production of a soluble recombinant prion protein fused to blue fluorescent protein without refolding or detergents in Escherichia coli cells.

    Science.gov (United States)

    Arii, Yasuhiro; Yamaguchi, Hidenori; Fukuoka, Shin-Ichi

    2007-10-01

    The physiological function of prion proteins (PrP) remains unclear. To investigate the physiological relevance of PrP, we constructed a fusion protein of PrP with enhanced blue fluorescent protein (PrP-EBFP) to quantify the interaction of PrP with other molecules. Production of soluble PrP-EBFP was achieved by lowering the expression temperature in Escherichia coli (E. coli) cells to 15 degrees C. Soluble PrP-EBFP was purified on cation exchange and heparin-affinity columns to yield high purity protein. This is the first report of the preparation of soluble recombinant PrP without refolding following solubilization using denaturants or disruption using detergents. To confirm the integrity of PrP-EBFP, anisotropy was estimated under physiological conditions in the presence of heparin, which interacts with PrP. The dissociation constant was determined to be 0.88+/-0.07 microM. PrP-EBFP should be useful in the quantification of PrP interactions with other molecules.

  11. GroEL-GroES assisted folding of multiple recombinant proteins simultaneously over-expressed in Escherichia coli.

    Science.gov (United States)

    Goyal, Megha; Chaudhuri, Tapan K

    2015-07-01

    Folding of aggregation prone recombinant proteins through co-expression of chaperonin GroEL and GroES has been a popular practice in the effort to optimize preparation of functional protein in Escherichia coli. Considering the demand for functional recombinant protein products, it is desirable to apply the chaperone assisted protein folding strategy for enhancing the yield of properly folded protein. Toward the same direction, it is also worth attempting folding of multiple recombinant proteins simultaneously over-expressed in E. coli through the assistance of co-expressed GroEL-ES. The genesis of this thinking was originated from the fact that cellular GroEL and GroES assist in the folding of several endogenous proteins expressed in the bacterial cell. Here we present the experimental findings from our study on co-expressed GroEL-GroES assisted folding of simultaneously over-expressed proteins maltodextrin glucosidase (MalZ) and yeast mitochondrial aconitase (mAco). Both proteins mentioned here are relatively larger and aggregation prone, mostly form inclusion bodies, and undergo GroEL-ES assisted folding in E. coli cells during over-expression. It has been reported that the relative yield of properly folded functional forms of MalZ and mAco with the exogenous GroEL-ES assistance were comparable with the results when these proteins were overexpressed alone. This observation is quite promising and highlights the fact that GroEL and GroES can assist in the folding of multiple substrate proteins simultaneously when over-expressed in E. coli. This method might be a potential tool for enhanced production of multiple functional recombinant proteins simultaneously in E. coli. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. 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.

  13. Construction and Antiapoptosis Activities of Recombinant Adenoviral Expression Vector Carrying EBV Latent Membrane Protein 2A

    Directory of Open Access Journals (Sweden)

    Xishuang Liu

    2011-01-01

    Full Text Available To evaluate the possible effects of LMP2A (EBV latent membrane protein 2A on human gastric cancer cell line SGC-7901, LMP2A coding gene was subcloned into shuttle plasmid pAdTrackCMV to form transfer plasmid pAdTrackCMV-2A, which was linearized with PmeI and cotransformed into E.coli BJ5183 with adenovirus genomic plasmid of pAdeasy-1. The identified recombinant adenovirus plasmid DNA was digested with PacI and transfected into 293 cells to package recombinant adenovirus particles named vAd-2A. Then the expression and antiapoptosis activities of LMP2A on SGC-7901 infected with vAd-2A were analyzed. The vAd-2A was successfully constructed and identified by PCR, restriction digestion, and sequencing. LMP2A expression in SGC was identified by strong green fluorescence expression with fluorescence microscopic photograph and Southern blotting. The growth of LMP2A expressing SGC cells was apparently improved. Both cyclin E expression and S phase ratio in LMP2A expressing SGC cells were upregulated by cell cycle analysis and confocal microscopic analysis respectively. The replication-deficient recombinant adenovirus vector can express LMP2A antigen in SGC cells and inhibit their apoptosis. The results indicate that LMP2A might play an important role in pathogenesis of EBV-associated gastric cancer (EBVaGC. This study establishes a foundation for further study on EBVaGC and its gene therapy.

  14. Recombinant probiotic expressing Listeria adhesion protein attenuates Listeria monocytogenes virulence in vitro.

    Directory of Open Access Journals (Sweden)

    Ok Kyung Koo

    Full Text Available BACKGROUND: Listeria monocytogenes, an intracellular foodborne pathogen, infects immunocompromised hosts. The primary route of transmission is through contaminated food. In the gastrointestinal tract, it traverses the epithelial barrier through intracellular or paracellular routes. Strategies to prevent L. monocytogenes entry can potentially minimize infection in high-risk populations. Listeria adhesion protein (LAP aids L. monocytogenes in crossing epithelial barriers via the paracellular route. The use of recombinant probiotic bacteria expressing LAP would aid targeted clearance of Listeria from the gut and protect high-risk populations from infection. METHODOLOGY/PRINCIPAL FINDINGS: The objective was to investigate the ability of probiotic bacteria or LAP-expressing recombinant probiotic Lactobacillus paracasei (Lbp(LAP to prevent L. monocytogenes adhesion, invasion, and transwell-based transepithelial translocation in a Caco-2 cell culture model. Several wild type probiotic bacteria showed strong adhesion to Caco-2 cells but none effectively prevented L. monocytogenes infection. Pre-exposure to Lbp(LAP for 1, 4, 15, or 24 h significantly (P<0.05 reduced adhesion, invasion, and transepithelial translocation of L. monocytogenes in Caco-2 cells, whereas pre-exposure to parental Lb. paracasei had no significant effect. Similarly, Lbp(LAP pre-exposure reduced L. monocytogenes translocation by as much as 46% after 24 h. Lbp(LAP also prevented L. monocytogenes-mediated cell damage and compromise of tight junction integrity. Furthermore, Lbp(LAP cells reduced L. monocytogenes-mediated cell cytotoxicity by 99.8% after 1 h and 79% after 24 h. CONCLUSIONS/SIGNIFICANCE: Wild type probiotic bacteria were unable to prevent L. monocytogenes infection in vitro. In contrast, Lbp(LAP blocked adhesion, invasion, and translocation of L. monocytogenes by interacting with host cell receptor Hsp60, thereby protecting cells from infection. These data show promise

  15. 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.

  16. Suppression of cell division by pKi-67 antisense-RNA and recombinant protein.

    Science.gov (United States)

    Duchrow, M; Schmidt, M H; Zingler, M; Anemüller, S; Bruch, H P; Broll, R

    2001-01-01

    The human antigen defined by the monoclonal antibody Ki-67 (pKi-67) is a human nuclear protein strongly associated with cell proliferation and found in all tissues studied. It is widely used as a marker of proliferating cells, yet its function is unknown. To investigate its function we suppressed pKi-67 expression by antisense RNA and overexpressed a partial structure of pKi-67 in HeLa cells. A BrdU-incorporation assay showed a significant decrease in DNA synthesis after antisense inhibition. Cell cycle analysis indicated a higher proportion of cells in G1 phase and a lower proportion of cells in S phase while the number of G(2)/M phase cells remained constant. Overexpression of a recombinant protein encoding three of the repetitive elements from exon 13 of pKi-67 had a similar effect to that obtained by antisense inhibition. The similarity of the effect of expressing 'Ki-67 repeats' and pKi-67 antisense RNA could be explained by a negative effect on the folding of the endogenous protein in the endoplasmatic reticulum. Furthermore excessive self-association of pKi-67 via the repeat structure could inhibit its nuclear transport, preventing it from getting to its presumptive site of action. We conclude that the Ki-67 protein has an important role in the regulation of the cell cycle, which is mediated in part by its repetitive elements. Copyright 2001 S. Karger AG, Basel

  17. Evidence of chemical exchange in recombinant Major Urinary Protein and quenching thereof upon pheromone binding

    Energy Technology Data Exchange (ETDEWEB)

    Perazzolo, Chiara, E-mail: Chiara.Perazzolo@epfl.ch; Verde, Mariachiara [Ecole Polytechnique Federale de Lausanne, Institut des Sciences et Ingenierie Chimiques (Switzerland); Homans, Steve W. [University of Leeds, Institute of Molecular and Cellular Biology (United Kingdom); Bodenhausen, Geoffrey [Ecole Polytechnique Federale de Lausanne, Institut des Sciences et Ingenierie Chimiques (Switzerland)

    2007-05-15

    The internal dynamics of recombinant Major Urinary Protein (rMUP) have been investigated by monitoring transverse nitrogen-15 relaxation using multiple-echo Carr-Purcell-Meiboom-Gill (CPMG) experiments. While the ligand-free protein (APO-rMUP) features extensive evidence of motions on the milliseconds time scale, the complex with 2-methoxy-3-isobutylpyrazine (HOLO-rMUP) appears to be much less mobile on this time scale. At 308 K, exchange rates k{sub ex} = 500-2000 s{sup -1} were typically observed in APO-rMUP for residues located adjacent to a {beta}-turn comprising residues 83-87. These residues occlude an entry to the binding pocket and have been proposed to be a portal for ligand entry in other members of the lipocalin family, such as the retinol binding protein and the human fatty-acid binding protein. Exchange rates and populations are largely uncorrelated, suggesting local 'breathing' motions rather than a concerted global conformational change.

  18. Assessment of the structure of pegylated-recombinant protein therapeutics by the NMR fingerprint assay.

    Science.gov (United States)

    Hodgson, Derek J; Aubin, Yves

    2017-05-10

    A number of recombinant protein therapeutic products, such as filgrastim (methionyl granulocyte colony stimulating factor [Met-GCSF] used to boost the immune system in chemotherapy treated cancer patients), and interferon alpha-2 (used for the treatment of various viral infections), have been chemically modified with the addition of a polyethylene glycol (PEG) chain. This modification prolongs residency of the drug in the body and reduces metabolic degradation, which allows less frequent administration of the products. Here we show how NMR spectroscopy methods can assess the higher order structure (HOS) of pegylated-filgrastim (Neulasta®), pegylated interferon-α2a (Pegasys®) pegylated interferon-α2b (PEG-Intron®) purchased from the marketplace. The addition of the PEG moiety effectively doubles the molecular weight of the three products. This presents a significant challenge for the application of NMR techniques. Nevertheless, the results showed that high-resolution spectra could be recorded for two of the three products. Comparison of the spectra of the pegylated protein and the non-pegylated protein shows that the chemical modification did not alter the HOS of these proteins. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  19. 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.

  20. 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.

  1. N-Lauroylation during the Expression of Recombinant N-Myristoylated Proteins: Implications and Solutions.

    Science.gov (United States)

    Flamm, Andrea Gabriele; Le Roux, Anabel-Lise; Mateos, Borja; Díaz-Lobo, Mireia; Storch, Barbara; Breuker, Kathrin; Konrat, Robert; Pons, Miquel; Coudevylle, Nicolas

    2016-01-01

    Incorporation of myristic acid onto the N terminus of a protein is a crucial modification that promotes membrane binding and correct localization of important components of signaling pathways. Recombinant expression of N-myristoylated proteins in Escherichia coli can be achieved by co-expressing yeast N-myristoyltransferase and supplementing the growth medium with myristic acid. However, undesired incorporation of the 12-carbon fatty acid lauric acid can also occur (leading to heterogeneous samples), especially when the available carbon sources are scarce, as it is the case in minimal medium for the expression of isotopically enriched samples. By applying this method to the brain acid soluble protein 1 and the 1-185 N-terminal region of c-Src, we show the significant, and protein-specific, differences in the membrane binding properties of lauroylated and myristoylated forms. We also present a robust strategy for obtaining lauryl-free samples of myristoylated proteins in both rich and minimal media. © 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  2. Evidence of chemical exchange in recombinant Major Urinary Protein and quenching thereof upon pheromone binding

    International Nuclear Information System (INIS)

    Perazzolo, Chiara; Verde, Mariachiara; Homans, Steve W.; Bodenhausen, Geoffrey

    2007-01-01

    The internal dynamics of recombinant Major Urinary Protein (rMUP) have been investigated by monitoring transverse nitrogen-15 relaxation using multiple-echo Carr-Purcell-Meiboom-Gill (CPMG) experiments. While the ligand-free protein (APO-rMUP) features extensive evidence of motions on the milliseconds time scale, the complex with 2-methoxy-3-isobutylpyrazine (HOLO-rMUP) appears to be much less mobile on this time scale. At 308 K, exchange rates k ex = 500-2000 s -1 were typically observed in APO-rMUP for residues located adjacent to a β-turn comprising residues 83-87. These residues occlude an entry to the binding pocket and have been proposed to be a portal for ligand entry in other members of the lipocalin family, such as the retinol binding protein and the human fatty-acid binding protein. Exchange rates and populations are largely uncorrelated, suggesting local 'breathing' motions rather than a concerted global conformational change

  3. 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

  4. 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.

  5. 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......-derived neurotropic factor (BDNF). In conclusion, non-viral gene therapy to RBECs leads to protein secretion and signifies a method for therapeutic proteins to target cells inside the CNS otherwise omitted due to the BBB....

  6. 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.

  7. Comparative evaluation of the diagnostic potential of recombinant envelope proteins and native cell culture purified viral antigens of Chikungunya virus.

    Science.gov (United States)

    Khan, Mohsin; Dhanwani, Rekha; Kumar, Jyoti S; Rao, P V Lakshmana; Parida, Manmohan

    2014-07-01

    Despite the fact that Chikungunya resurgence is associated with epidemic of unprecedented magnitude, there are challenges in the field of its clinical diagnosis. However, serological tests in an ELISA format provide a rapid tool for the diagnosis of Chikungunya infection. Indeed, ELISAs based on recombinant proteins hold a great promise as these methods are cost effective and are free from the risk of handling biohazardous material. In this study, the performance of recombinant CHIKV antigens was compared in various ELISA formats for the diagnosis of Chikungunya. Two recombinant antigens derived from the envelope proteins of Chikungunya virus were prepared and evaluated by comparing their competence for detecting circulating antibodies in serum samples of patients infected with CHIKV using MAC-ELISA and indirect IgM-ELISA. The efficacy of the recombinant antigens was also compared with the native antigen. The indirect antibody capture IgM microplate ELISA revealed ≥90% concordance with the native antigen in detecting the CHIKV specific IgM antibodies whereas the recombinant antigen based MAC-ELISA showed 100% specificity. The recombinant antigens used in this study were effective and reliable targets for the diagnosis of CHIKV infection and also provide an alternative for native antigen use which is potentially biohazardous. © 2013 Wiley Periodicals, Inc.

  8. 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.

  9. Functional Assembly of Soluble and Membrane Recombinant Proteins of Mammalian NADPH Oxidase Complex.

    Science.gov (United States)

    Souabni, Hajer; Ezzine, Aymen; Bizouarn, Tania; Baciou, Laura

    2017-01-01

    Activation of phagocyte cells from an innate immune system is associated with a massive consumption of molecular oxygen to generate highly reactive oxygen species (ROS) as microbial weapons. This is achieved by a multiprotein complex, the so-called NADPH oxidase. The activity of phagocyte NADPH oxidase relies on an assembly of more than five proteins, among them the membrane heterodimer named flavocytochrome b 558 (Cytb 558 ), constituted by the tight association of the gp91 phox (also named Nox2) and p22 phox proteins. The Cytb 558 is the membrane catalytic core of the NADPH oxidase complex, through which the reducing equivalent provided by NADPH is transferred via the associated prosthetic groups (one flavin and two hemes) to reduce dioxygen into superoxide anion. The other major proteins (p47 phox , p67 phox , p40 phox , Rac) requisite for the complex activity are cytosolic proteins. Thus, the NADPH oxidase functioning relies on a synergic multi-partner assembly that in vivo can be hardly studied at the molecular level due to the cell complexity. Thus, a cell-free assay method has been developed to study the NADPH oxidase activity that allows measuring and eventually quantifying the ROS generation based on optical techniques following reduction of cytochrome c. This setup is a valuable tool for the identification of protein interactions, of crucial components and additives for a functional enzyme. Recently, this method was improved by the engineering and the production of a complete recombinant NADPH oxidase complex using the combination of purified proteins expressed in bacterial and yeast host cells. The reconstitution into artificial membrane leads to a fully controllable system that permits fine functional studies.

  10. 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.

  11. Recombinant human bone morphogenetic protein 2 in augmentation procedures: case reports.

    Science.gov (United States)

    Luiz, Jaques; Padovan, Luis Eduardo Marques; Claudino, Marcela

    2014-01-01

    To successfully rehabilitate edentulous patients using endosseous implants, there must be enough available bone. Several techniques have been proposed for augmentation of sites with insufficient bone volume. Although autogenous bone has long been considered the gold standard for such procedures, the limited availability of graft material and a high morbidity rate are potential disadvantages of this type of graft. An alternative is to use recombinant human bone morphogenetic protein 2 (rhBMP-2), which is able to support bone regeneration in the oral environment. These cases demonstrate the applicability of rhBMP-2 in maxillary sinus elevation and augmentation procedures in the maxilla to enable dental implant placement. The use of rhBMP-2 in alveolar augmentation procedures had several clinical benefits for these patients.

  12. Host Immunization with Recombinant Proteins to Screen Antigens for Tick Control.

    Science.gov (United States)

    Galay, Remil Linggatong; Miyata, Takeshi; Umemiya-Shirafuji, Rika; Mochizuki, Masami; Fujisaki, Kozo; Tanaka, Tetsuya

    2016-01-01

    Ticks (Parasitiformes: Ixodida) are known for their obligate blood feeding habit and their role in transmitting pathogens to various vertebrate hosts. Tick control using chemical acaricides is extensively used particularly in livestock management, but several disadvantages arise from resistance development of many tick species, and concerns on animal product and environmental contamination. Vaccination offers better protection and more cost-effective alternative to application of chemical acaricides, addressing their disadvantages. However, an ideal anti-tick vaccine targeting multiple tick species and all the tick stages is still wanting. Here, we describe the procedures involved in the evaluation of a vaccine candidate antigen against ticks at the laboratory level, from the preparation of recombinant proteins, administration to the rabbit host and monitoring of antibody titer, to tick infestation challenge and determination of the effects of immunization to ticks.

  13. 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.

  14. 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.

  15. Allergen extracts and recombinant proteins: comparison of efficiency of in vitro allergy diagnostics using multiplex assay on a biological microchip.

    Science.gov (United States)

    Smoldovskaya, Olga; Feyzkhanova, Guzel; Arefieva, Alla; Voloshin, Sergei; Ivashkina, Olga; Reznikov, Yuriy; Rubina, Alla

    2016-01-01

    Immunological test systems for diagnostics of type I hypersensitivity involve the following types of antigens: whole allergen extracts, individual highly purified proteins and their recombinant analogues. The goal of this study was to compare the results obtained with whole allergen extracts (birch pollen, cat dander, and timothy grass pollen) and their respective recombinant proteins in biochip-based immunoassay. Multiplex fluorescent immunoassay of 139 patients' blood serum samples was carried out using biological microchips (biochips). sIgE concentrations for the chosen allergens and their recombinant components were measured. ROC analysis was used for comparison of the results and determination of diagnostic accuracy. The results for the birch pollen extract and its recombinant allergens have shown that the diagnostic accuracy of the methods utilizing the whole allergen extract, its major component Bet v 1 and the combination of major and minor components (Bet v 1 and Bet v 2) was the same. Values for diagnostic accuracy for the cat dander extract and its major recombinant component Fel d 1 were equal. In contrast with birch pollen and cat dander allergens, using of recombinant components of timothy grass pollen (Phl p 1, Phl p 5, Phl p 7 and Phl p 12) did not allow reaching the diagnostic accuracy of using natural extract. Multiplex analysis of samples obtained from patients with allergy to birch pollen and cat dander using biological microchips has shown that comparable accuracy was observed for the assay with natural extracts and recombinant allergens. In the case of timothy grass allergen, using the recombinant components may be insufficient.

  16. Expression and Purification of Recombinant Proteins in Escherichia coli with a His6 or 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.

  17. Eimeria maxima microneme protein 2 delivered as DNA vaccine and recombinant protein induces immunity against experimental homogenous challenge.

    Science.gov (United States)

    Huang, Jingwei; Zhang, Zhenchao; Li, Menghui; Song, Xiaokai; Yan, Ruofeng; Xu, Lixin; Li, Xiangrui

    2015-10-01

    E. maxima is one of the seven species of Eimeria that infects chicken. Until now, only a few antigenic genes of E. maxima have been reported. In the present study, the immune protective effects against E. maxima challenge of recombinant protein and DNA vaccine encoding EmMIC2 were evaluated. Two-week-old chickens were randomly divided into five groups. The experimental group of chickens was immunized with 100 μg DNA vaccine pVAX1-MIC2 or 200 μg rEmMIC2 protein while the control group of chickens was injected with pVAX1 plasmid or sterile PBS. The results showed that the anti-EmMIC2 antibody titers of both rEmMIC2 protein and pVAX1-MIC2 groups were significantly higher as compared to PBS and pVAX1 control (Pmaxima challenge and it could be an effective antigen candidate for the development of new vaccines against E. maxima. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  18. 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.

  19. 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.

  20. Autoantibody detection in type 2 autoimmune hepatitis using a chimera recombinant protein.

    Science.gov (United States)

    Vitozzi, Susana; Lapierre, Pascal; Djilali-Saiah, Idriss; Alvarez, Fernando

    2002-04-01

    Autoantibodies against cytochrome P450 2D6 (CYP2D6), known as anti-liver/kidney microsome type 1 (LKM1) and/or anti-human formiminotransferase cyclodeaminase, formally known as anti-liver cytosol type 1 (LC1) define type 2 autoimmune hepatitis (AIH). The aims of this work are to develop a sensitive and specific test to detect anti-LKM1 and/or anti-LC1 autoantibodies and to establish the prevalence of anti-LC1. Sera from children with type 2 AIH (n=48) and those from a control group (n=100) were evaluated for anti-LKM1 and anti-LC1 by Enzyme-Linked Immunosorbent Assay (ELISA) and Western blotting. Each serum sample was assayed for reactivity against formiminotransferase cyclodeaminase and CYP2D6 alone or as part of a recombinant chimera protein. By ELISA with recombinant chimera protein, 50 serum samples were positive, 48 from patients with type 2 AIH and 2 from patients with chronic hepatitis C. Twenty-five of 48 (52%) patients studied were positive for both CYP2D6 and LC1 autoantibodies. Anti-LC1, either as the only marker or associated with anti-LKM1, was positive in 34/48 (71%). By Western blotting, anti-LC1 was found in 27/48 (56%) patients. This ELISA technique has proven to be antigen-specific and more sensitive than Western blot for the detection of anti-LC1 and anti-LKM1 autoantibodies. The prevalence of anti-LC1 (71%) confirms it as an important immunomarker in type 2 AIH.

  1. 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.

  2. A review of machine learning methods to predict the solubility of overexpressed recombinant proteins in Escherichia coli.

    Science.gov (United States)

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

    2014-05-08

    Over the last 20 years in biotechnology, the production of recombinant proteins has been a crucial bioprocess in both biopharmaceutical and research arena in terms of human health, scientific impact and economic volume. Although logical strategies of genetic engineering have been established, protein overexpression is still an art. In particular, heterologous expression is often hindered by low level of production and frequent fail due to opaque reasons. The problem is accentuated because there is no generic solution available to enhance heterologous overexpression. For a given protein, the extent of its solubility can indicate the quality of its function. Over 30% of synthesized proteins are not soluble. In certain experimental circumstances, including temperature, expression host, etc., protein solubility is a feature eventually defined by its sequence. Until now, numerous methods based on machine learning are proposed to predict the solubility of protein merely from its amino acid sequence. In spite of the 20 years of research on the matter, no comprehensive review is available on the published methods. This paper presents an extensive review of the existing models to predict protein solubility in Escherichia coli recombinant protein overexpression system. The models are investigated and compared regarding the datasets used, features, feature selection methods, machine learning techniques and accuracy of prediction. A discussion on the models is provided at the end. This study aims to investigate extensively the machine learning based methods to predict recombinant protein solubility, so as to offer a general as well as a detailed understanding for researches in the field. Some of the models present acceptable prediction performances and convenient user interfaces. These models can be considered as valuable tools to predict recombinant protein overexpression results before performing real laboratory experiments, thus saving labour, time and cost.

  3. Identification of a heterologous cellulase and its N-terminus that can guide recombinant proteins out of Escherichia coli.

    Science.gov (United States)

    Gao, Dongfang; Wang, Shengjun; Li, Haoran; Yu, Huili; Qi, Qingsheng

    2015-04-10

    The Gram-negative bacterium Escherichia coli has been widely used as a cell factory for the production of proteins and specialty chemicals because it is the best characterized host with many available expression and regulation systems. However, recombinant proteins produced in Escherichia coli are generally intracellular and often found in the form of inclusion bodies. Extracellular production of proteins is advantageous compared with intracellular production because extracellular proteins can be purified more easily and can avoid protease attack, which results in higher product quality. In this study, we found a catalytic domain of a cellulase (Cel-CD) and its N-terminus can be employed as carriers for extracellular production of recombinant proteins. In this report, we identified the catalytic domain of a cellulase (Cel-CD) from Bacillus sp. that can be secreted into the medium from recombinant E. coli BL21 (DE3) in large quantities without its native signal peptide. By subcellular location analysis, we proved that the secretion was a two-step process and the N-terminal sequence of the full length Cel-CD played a crucial function in secretion. Both the Cel-CD and its N-terminal sequence can serve as carriers for efficient extracellular production of select target proteins. Fusion of heterologous proteins with N20 from Cel-CD can carry the target proteins out of the cells with a concentration from 101 to 691 mg/L in flask cultivation. The extracellular recombinant proteins with a relative high purity. The results suggested that this system has a potential application in plant biomass conversion and industrial production of enzymes and therapeutic proteins.

  4. [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).

  5. Advances in Mammalian Cell Line Development Technologies for Recombinant Protein Production

    Directory of Open Access Journals (Sweden)

    Say Kong Ng

    2013-04-01

    Full Text Available From 2006 to 2011, an average of 15 novel recombinant protein therapeutics have been approved by US Food and Drug Administration (FDA annually. In addition, the expiration of blockbuster biologics has also spurred the emergence of biosimilars. The increasing numbers of innovator biologic products and biosimilars have thus fuelled the demand of production cell lines with high productivity. Currently, mammalian cell line development technologies used by most biopharmaceutical companies are based on either the methotrexate (MTX amplification technology or the glutamine synthetase (GS system. With both systems, the cell clones obtained are highly heterogeneous, as a result of random genome integration by the gene of interest and the gene amplification process. Consequently, large numbers of cell clones have to be screened to identify rare stable high producer cell clones. As such, the cell line development process typically requires 6 to 12 months and is a time, capital and labour intensive process. This article reviews established advances in protein expression and clone screening which are the core technologies in mammalian cell line development. Advancements in these component technologies are vital to improve the speed and efficiency of generating robust and highly productive cell line for large scale production of protein therapeutics.

  6. 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.

  7. Optimal protein library design using recombination or point mutations based on sequence-based scoring functions.

    Science.gov (United States)

    Pantazes, Robert J; Saraf, Manish C; Maranas, Costas D

    2007-08-01

    In this paper, we introduce and test two new sequence-based protein scoring systems (i.e. S1, S2) for assessing the likelihood that a given protein hybrid will be functional. By binning together amino acids with similar properties (i.e. volume, hydrophobicity and charge) the scoring systems S1 and S2 allow for the quantification of the severity of mismatched interactions in the hybrids. The S2 scoring system is found to be able to significantly functionally enrich a cytochrome P450 library over other scoring methods. Given this scoring base, we subsequently constructed two separate optimization formulations (i.e. OPTCOMB and OPTOLIGO) for optimally designing protein combinatorial libraries involving recombination or mutations, respectively. Notably, two separate versions of OPTCOMB are generated (i.e. model M1, M2) with the latter allowing for position-dependent parental fragment skipping. Computational benchmarking results demonstrate the efficacy of models OPTCOMB and OPTOLIGO to generate high scoring libraries of a prespecified size.

  8. Induction of Multifunctional Broadly Reactive T Cell Responses by a Plasmodium vivax Circumsporozoite Protein Recombinant Chimera.

    Science.gov (United States)

    Cabrera-Mora, Monica; Fonseca, Jairo Andres; Singh, Balwan; Oliveira-Ferreira, Joseli; Lima-Junior, Josué da Costa; Calvo-Calle, J Mauricio; Moreno, Alberto

    2015-09-01

    Plasmodium vivax is the most widespread species of Plasmodium, causing up to 50% of the malaria cases occurring outside sub-Saharan Africa. An effective vaccine is essential for successful control and potential eradication. A well-characterized vaccine candidate is the circumsporozoite protein (CSP). Preclinical and clinical trials have shown that both antibodies and cellular immune responses have been correlated with protection induced by immunization with CSP. On the basis of our reported approach of developing chimeric Plasmodium yoelii proteins to enhance protective efficacy, we designed PvRMC-CSP, a recombinant chimeric protein based on the P. vivax CSP (PvCSP). In this engineered protein, regions of the PvCSP predicted to contain human T cell epitopes were genetically fused to an immunodominant B cell epitope derived from the N-terminal region I and to repeat sequences representing the two types of PvCSP repeats. The chimeric protein was expressed in soluble form with high yield. As the immune response to PvCSP has been reported to be genetically restricted in the murine model, we tested the immunogenicity of PvRMC-CSP in groups of six inbred strains of mice. PvRMC-CSP was able to induce robust antibody responses in all the mouse strains tested. Synthetic peptides representing the allelic forms of the P. vivax CSP were also recognized to a similar extent regardless of the mouse strain. Furthermore, the immunization regimen induced high frequencies of multifunctional CD4(+) and CD8(+) PvRMC-CSP-specific T cells. The depth and breadth of the immune responses elicited suggest that immunization with PvRMC-CSP can circumvent the genetic restriction of the immune response to P. vivax CSP. Interestingly, PvRMC-CSP was also recognized by naturally acquired antibodies from individuals living in areas where malaria is endemic. These features make PvRMC-CSP a promising vaccine candidate for further development. Copyright © 2015, American Society for Microbiology. All

  9. 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.

  10. 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.

  11. Evaluation of Th1/Th2-Related Immune Response against Recombinant Proteins of Brucella abortus Infection in Mice.

    Science.gov (United States)

    Im, Young Bin; Park, Woo Bin; Jung, Myunghwan; Kim, Suk; Yoo, Han Sang

    2016-06-28

    Brucellosis is a zoonotic disease caused by Brucella, a genus of gram-negative bacteria. Cytokines have key roles in the activation of innate and acquired immunities. Despite several research attempts to reveal the immune responses, the mechanism of Brucella infection remains unclear. Therefore, immune responses were analyzed in mice immunized with nine recombinant proteins. Cytokine production profiles were analyzed in the RAW 264.7 cells and naive splenocytes after stimulation with three recombinant proteins, metal-dependent hydrolase (r0628), bacterioferritin (rBfr), and thiamine transporter substrate-binding protein (rTbpA). Immune responses were analyzed by ELISA and ELISpot assay after immunization with proteins in mice. The production levels of NO, TNF-α, and IL-6 were time-dependently increased after having been stimulated with proteins in the RAW 264.7 cells. In naive splenocytes, the production of IFN-γ and IL-2 was increased after stimulation with the proteins. It was concluded that two recombinant proteins, r0628 and rTbpA, showed strong immunogenicity that was induced with Th1-related cytokines IFN-γ, IL-2, and TNF-α more than Th2-related cytokines IL-6, IL-4, and IL-5 in vitro. Conversely, a humoral immune response was activated by increasing the number of antigen-secreting cells specifically. Furthermore, these could be candidate diagnosis antigens for better understanding of brucellosis.

  12. 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.

  13. 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

  14. 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 virus * recombinant protein * triple gene block * polyclonal antibodies Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.817, year: 2006

  15. Identification of immunogenic proteins and evaluation of four recombinant proteins as potential vaccine antigens from Vibrio anguillarum in flounder (Paralichthys olivaceus).

    Science.gov (United States)

    Xing, Jing; Xu, Hongsen; Wang, Yang; Tang, Xiaoqian; Sheng, Xiuzhen; Zhan, Wenbin

    2017-05-31

    Vibrio anguillarum is a severe bacterial pathogen that can infect a wide range of fish species. Identification of immunogenic proteins and development of vaccine are essential for disease prevention. In this study, immunogenic proteins were screened and identified from V. anguillarum, and then protective efficacy of the immunogenic proteins was evaluated. Immunogenic proteins in V. anguillarum whole cell were detected by Western blotting (WB) using immunized flounder (Paralichthys olivaceus) serum, and then identified by Mass spectrometry (MS). The recombinant proteins of four identified immunogenic proteins were produced and immunized to fish, and then percentages of surface membrane immunoglobulin-positive (sIg+) cells in peripheral blood lymphocytes (PBL), total antibodies, antibodies against V. anguillarum, antibodies against recombinant proteins and relative percent survival (RPS) were measured, respectively. The results showed that five immunogenic proteins, VAA, Groel, OmpU, PteF and SpK, were identified; their recombinant proteins, rOmpU, rGroel, rSpK and rVAA, could induce the proliferation of sIg+ cells in PBL and production of total antibodies, antibodies against V. anguillarum and antibodies against the recombinant proteins; their protection against V. anguillarum showed 64.86%, 72.97%, 21.62% and 78.38% RPS, respectively. The results revealed that the immunoproteomic technique using fish anti-V. anguillarum serum provided an efficient way to screen the immunogenic protein for vaccine antigen. Moreover, the rVAA, rGroel and rOmpU had potential to be vaccine candidates against V. anguillarum infection. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Vaccination with Clostridium perfringens recombinant proteins in combination with Montanide™ ISA 71 VG adjuvant increases protection against experimental necrotic enteritis in commercial broiler chickens

    Science.gov (United States)

    This study was performed to compare four Clostridium perfringens recombinant proteins as vaccine candidates using the Montanide™ ISA 71 VG adjuvant in an experimental model of necrotic enteritis. Broiler chickens were immunized with clostridial recombinant proteins with ISA 71 VG, and intestinal le...

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

    Science.gov (United States)

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

    1999-01-01

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

  18. The evaluation of lyophilized polymer matrices for administering recombinant human bone morphogenetic protein-2.

    Science.gov (United States)

    Duggirala, S S; Rodgers, J B; DeLuca, P P

    1996-07-01

    Novel unitary devices, prepared by lyophilization of viscous solutions of sodium carboxymethylcellulose (CMC) and methylcellulose (MC), were evaluated as sustained-release delivery systems for recombinant human bone morphogenetic protein-2 (rhBMP-2). In vitro characterization of the unitary devices, which contained rhBMP-2-loaded poly (d,l lactide-co-glycolide) (PLGA) bioerodible particles (BEPs), was conducted over a 2-month period. Determinations included buffer uptake, mass and molecular weight loss and rhBMP-2 release from the unitary devices. CMC devices imbibed approximately 16 times their weight of buffer, while with MC, equilibrium uptake was approximately 6 times the dry weight of the devices. Overall mass loss percentages were approximately 55 and 35%, respectively, for CMC and MC devices. rhBMP-2 release from the devices was essentially a triphasic process: an initial phase during which "free" protein (rhBMP-2 present on the surface and within the pores of the PLGA BEPs) was released, a lag period during which no release was discerned, and then release of "bound" rhBMP-2 (protein adsorbed to the BEPs). The release of bound protein correlated with the mass loss of the polymer which began after 3 weeks. Release from the unitary devices was lower than that from the BEPs alone, due to a retardation effect of the gelled CMC/MC polymers. In rabbits in which full-thickness cranial bone defects were created, the implants were well tolerated and induced significant new bone growth during an 8-week evaluation period. The CMC devices appear to have induced bone earlier (at 2 weeks), but this did not affect eventual 8-week results. CMC devices without rhBMP-2 appeared to provide some bone conduction, in contrast to the blank MC devices.

  19. 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

    Saccharomyces cerevisiae , expression cassettes carrying foreign genes integrate highly specifically at the targeted sites in the genome. In contrast, cassettes often integrate at random genomic positions in nonconventional yeasts, such as Pichia pastoris (syn. Komagataella phaffii ). Hence, cells from the same transformation event often behave differently, with significant clonal variation necessitating the screening of large numbers of strains. The importance of this study is that we systematically investigated the influence of integration events in more than 700 strains. Our findings provide novel insight into clonal variation in P. pastoris and, thus, how to avoid pitfalls and obtain reliable results. The underlying mechanisms may also play a role in other yeasts and hence could be generally relevant for recombinant yeast protein production strains. Copyright © 2018 American Society for Microbiology.

  20. Evaluation of novel inducible promoter/repressor systems for recombinant protein expression in Lactobacillus plantarum.

    Science.gov (United States)

    Heiss, Silvia; Hörmann, Angelika; Tauer, Christopher; Sonnleitner, Margot; Egger, Esther; Grabherr, Reingard; Heinl, Stefan

    2016-03-10

    Engineering lactic acid bacteria (LAB) is of growing importance for food and feed industry as well as for in vivo vaccination or the production of recombinant proteins in food grade organisms. Often, expression of a transgene is only desired at a certain time point or period, e.g. to minimize the metabolic burden for the host cell or to control the expression time span. For this purpose, inducible expression systems are preferred, though cost and availability of the inducing agent must be feasible. We selected the plasmid free strain Lactobacillus plantarum 3NSH for testing and characterization of novel inducible promoters/repressor systems. Their feasibility in recombinant protein production was evaluated. Expression of the reporter protein mCherry was monitored with the BioLector(®) micro-fermentation system. Reporter gene mCherry expression was compared under the control of different promoter/repressor systems: PlacA (an endogenous promoter/repressor system derived from L. plantarum 3NSH), PxylA (a promoter/repressor system derived from Bacillus megaterium DSMZ 319) and PlacSynth (synthetic promoter and codon-optimized repressor gene based on the Escherichia coli lac operon). We observed that PlacA was inducible solely by lactose, but not by non-metabolizable allolactose analoga. PxylA was inducible by xylose, yet showed basal expression under non-induced conditions. Growth on galactose (as compared to exponential growth phase on glucose) reduced basal mCherry expression at non-induced conditions. PlacSynth was inducible with TMG (methyl β-D-thiogalactopyranoside) and IPTG (isopropyl β-D-1-thiogalactopyranoside), but also showed basal expression without inducer. The promoter PlacSynth was used for establishment of a dual plasmid expression system, based on T7 RNA polymerase driven expression in L. plantarum. Comparative Western blot supported BioLector(®) micro-fermentation measurements. Conclusively, overall expression levels were moderate (compared to a

  1. An agmatine-inducible system for the expression of recombinant proteins in Enterococcus faecalis.

    Science.gov (United States)

    Linares, Daniel M; Perez, Marta; Ladero, Victor; Del Rio, Beatriz; Redruello, Begoña; Martin, M Cruz; Fernandez, María; Alvarez, Miguel A

    2014-12-04

    Scientific interest in Enterococcus faecalis has increased greatly over recent decades. Some strains are involved in food fermentation and offer health benefits, whereas others are vancomycin-resistant and cause infections that are difficult to treat. The limited availability of vectors able to express cloned genes efficiently in E. faecalis has hindered biotechnological studies on the bacterium's regulatory and pathogenicity-related genes. The agmatine deiminase (AGDI) pathway of E. faecalis, involved in the conversion of agmatine into putrescine, is driven by a response inducer gene aguR. This study describes that the exposure to the induction factor (agmatine) results in the transcription of genes under the control of the aguB promoter, including the aguBDAC operon. A novel E. faecalis expression vector, named pAGEnt, combining the aguR inducer gene and the aguB promoter followed by a cloning site and a stop codon was constructed. pAGEnt was designed for the overexpression and purification of a protein fused to a 10-amino-acid His-tag at the C-terminus. The use of GFP as a reporter of gene expression in E. faecalis revealed that under induction with 60 mM agmatine, fluorescence reached 40 arbitrary units compared to 0 in uninduced cells. pAGEnt vector can be used for the overexpression of recombinant proteins under the induction of agmatine in E. faecalis, with a close correlation between agmatine concentration and fluorescence when GFP was used as reporter.

  2. 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.

  3. 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.

  4. Phase 3 study of recombinant factor VIII Fc fusion protein in severe hemophilia A

    Science.gov (United States)

    Mahlangu, Johnny; Powell, Jerry S.; Ragni, Margaret V.; Chowdary, Pratima; Josephson, Neil C.; Pabinger, Ingrid; Hanabusa, Hideji; Gupta, Naresh; Kulkarni, Roshni; Fogarty, Patrick; Perry, David; Shapiro, Amy; Pasi, K. John; Apte, Shashikant; Nestorov, Ivan; Jiang, Haiyan; Li, Shuanglian; Neelakantan, Srividya; Cristiano, Lynda M.; Goyal, Jaya; Sommer, Jurg M.; Dumont, Jennifer A.; Dodd, Nigel; Nugent, Karen; Vigliani, Gloria; Luk, Alvin; Brennan, Aoife

    2014-01-01

    This phase 3 pivotal study evaluated the safety, efficacy, and pharmacokinetics of a recombinant FVIII Fc fusion protein (rFVIIIFc) for prophylaxis, treatment of acute bleeding, and perioperative hemostatic control in 165 previously treated males aged ≥12 years with severe hemophilia A. The study had 3 treatment arms: arm 1, individualized prophylaxis (25-65 IU/kg every 3-5 days, n = 118); arm 2, weekly prophylaxis (65 IU/kg, n = 24); and arm 3, episodic treatment (10-50 IU/kg, n = 23). A subgroup compared recombinant FVIII (rFVIII) and rFVIIIFc pharmacokinetics. End points included annualized bleeding rate (ABR), inhibitor development, and adverse events. The terminal half-life of rFVIIIFc (19.0 hours) was extended 1.5-fold vs rFVIII (12.4 hours; P < .001). Median ABRs observed in arms 1, 2, and 3 were 1.6, 3.6, and 33.6, respectively. In arm 1, the median weekly dose was 77.9 IU/kg; approximately 30% of subjects achieved a 5-day dosing interval (last 3 months on study). Across arms, 87.3% of bleeding episodes resolved with 1 injection. Adverse events were consistent with those expected in this population; no subjects developed inhibitors. rFVIIIFc was well-tolerated, had a prolonged half-life compared with rFVIII, and resulted in low ABRs when dosed prophylactically 1 to 2 times per week. This trial was registered at www.clinicaltrials.gov as #NCT01181128. PMID:24227821

  5. A new tagging system for production of recombinant proteins in Drosophila S2 cells using the third domain of the urokinase receptor

    DEFF Research Database (Denmark)

    Gårdsvoll, Henrik; Hansen, Line V; Jørgensen, Thomas J D

    2007-01-01

    The use of protein fusion tag technology greatly facilitates detection, expression and purification of recombinant proteins, and the demands for new and more effective systems are therefore expanding. We have used a soluble truncated form of the third domain of the urokinase receptor...... as a convenient C-terminal fusion partner for various recombinant extracellular human proteins used in basic cancer research. The stability of this cystein-rich domain, which structure adopts a three-finger fold, provides an important asset for its applicability as a fusion tag for expression of recombinant...... chromatography procedure using the immobilized anti-uPAR monoclonal antibody R2. An optional enterokinase cleavage site is included between the various recombinant proteins and the linker region of the tag, which enables generation of highly pure preparations of tag-free recombinant proteins. Using this system...

  6. Selectable high-yield recombinant protein production in human cells using a GFP/YFP nanobody affinity support.

    Science.gov (United States)

    Schellenberg, Matthew J; Petrovich, Robert M; Malone, Christine C; Williams, R Scott

    2018-03-25

    Recombinant protein expression systems that produce high yields of pure proteins and multi-protein complexes are essential to meet the needs of biologists, biochemists, and structural biologists using X-ray crystallography and cryo-electron microscopy. An ideal expression system for recombinant human proteins is cultured human cells where the correct translation and chaperone machinery are present. However, compared to bacterial expression systems, human cell cultures present several technical challenges to their use as an expression system. We developed a method that utilizes a YFP fusion-tag to generate recombinant proteins using suspension-cultured HEK293F cells. YFP is a dual-function tag that enables direct visualization and fluorescence-based selection of high expressing clones for and rapid purification using a high-stringency, high-affinity anti-GFP/YFP nanobody support. We demonstrate the utility of this system by expressing two large human proteins, TOP2α (340 KDa dimer) and a TOP2β catalytic core (260 KDa dimer). This robustly and reproducibly yields >10 mg/L liter of cell culture using transient expression or 2.5 mg/L using stable expression. Published 2018. This article is a US Government work and is in the public domain in the USA.

  7. Recombinant protein expression of Moringa oleifera lectin in methylotrophic yeast as active coagulant for sustainable high turbid water treatment.

    Science.gov (United States)

    Abd Wahid, Muhamad Azhar; Megat Mohd Noor, Megat Johari; Goto, Masafumi; Sugiura, Norio; Othman, Nor'azizi; Zakaria, Zuriati; Ahmad Mohammed, Thamer; Jusoh, Ahmad; Hara, Hirofumi

    2017-08-01

    The natural coagulant Moringa oleifera lectin (MoL) as cationic protein is a promising candidate in coagulation process of water treatment plant. Introducing the gene encoding MoL into a host, Pichia pastoris, to secrete soluble recombinant protein is assessed in this study. Initial screening using PCR confirmed the insertion of MoL gene, and SDS-PAGE analysis detected the MoL protein at 8 kDa. Cultured optimization showed the highest MoL protein at 520 mg/L was observed at 28 °C for 144 h of culturing by induction in 1% methanol. Approximately, 0.40 mg/mL of recombinant MoL protein showed 95 ± 2% turbidity removal of 1% kaolin suspension. In 0.1% kaolin suspension, the concentration of MoL at 10 μg/mL exhibits the highest turbidity reduction at 68 ± 1%. Thus, recombinant MoL protein from P. pastoris is an effective coagulant for water treatment.

  8. Overproduction of single-stranded-DNA-binding protein specifically inhibits recombination of UV-irradiated bacteriophage DNA in Escherichia coli

    International Nuclear Information System (INIS)

    Moreau, P.L.

    1988-01-01

    Overproduction of single-stranded DNA (ssDNA)-binding protein (SSB) in uvr Escherichia coli mutants results in a wide range of altered phenotypes. (i) Cell survival after UV irradiation is decreased; (ii) expression of the recA-lexA regulon is slightly reduced after UV irradiation, whereas it is increased without irradiation; and (iii) recombination of UV-damaged lambda DNA is inhibited, whereas recombination of nonirradiated DNA is unaffected. These results are consistent with the idea that in UV-damaged bacteria, SSB is first required to allow the formation of short complexes of RecA protein and ssDNA that mediate cleavage of the LexA protein. However, in a second stage, SSB should be displaced from ssDNA to permit the production of longer RecA-ssDNA nucleoprotein filaments that are required for strand pairing and, hence, recombinational repair. Since bacteria overproducing SSB appear identical in physiological respects to recF mutant bacteria, it is suggested that the RecF protein (alone or with other proteins of the RecF pathway) may help RecA protein to release SSB from ssDNA

  9. Purification of inclusion bodies using PEG precipitation under denaturing conditions to produce recombinant therapeutic proteins from Escherichia coli.

    Science.gov (United States)

    Chen, Huanhuan; Li, Ninghuan; Xie, Yueqing; Jiang, Hua; Yang, Xiaoyi; Cagliero, Cedric; Shi, Siwei; Zhu, Chencen; Luo, Han; Chen, Junsheng; Zhang, Lei; Zhao, Menglin; Feng, Lei; Lu, Huili; Zhu, Jianwei

    2017-07-01

    It has been documented that the purification of inclusion bodies from Escherichia coli by size exclusion chromatography (SEC) may benefit subsequent refolding and recovery of recombinant proteins. However, loading volume and the high cost of the column limits its application in large-scale manufacturing of biopharmaceutical proteins. We report a novel process using polyethylene glycol (PEG) precipitation under denaturing conditions to replace SEC for rapid purification of inclusion bodies containing recombinant therapeutic proteins. Using recombinant human interleukin 15 (rhIL-15) as an example, inclusion bodies of rhIL-15 were solubilized in 7 M guanidine hydrochloride, and rhIL-15 was precipitated by the addition of PEG 6000. A final concentration of 5% (w/v) PEG 6000 was found to be optimal to precipitate target proteins and enhance recovery and purity. Compared to the previously reported S-200 size exclusion purification method, PEG precipitation was easier to scale up and achieved the same protein yields and quality of the product. PEG precipitation also reduced manufacturing time by about 50 and 95% of material costs. After refolding and further purification, the rhIL-15 product was highly pure and demonstrated a comparable bioactivity with a rhIL-15 reference standard. Our studies demonstrated that PEG precipitation of inclusion bodies under denaturing conditions holds significant potential as a manufacturing process for biopharmaceuticals from E. coli protein expression systems.

  10. Engineering of a Potent Recombinant Lectin-Toxin Fusion Protein to Eliminate Human Pluripotent Stem Cells.

    Science.gov (United States)

    Tateno, Hiroaki; Saito, Sayoko

    2017-07-10

    The use of human pluripotent stem cells (hPSCs) such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) in regenerative medicine is hindered by their tumorigenic potential. Previously, we developed a recombinant lectin-toxin fusion protein of the hPSC-specific lectin rBC2LCN, which has a 23 kDa catalytic domain (domain III) of Pseudomonas aeruginosa exotoxin A (rBC2LCN-PE23). This fusion protein could selectively eliminate hPSCs following its addition to the cell culture medium. Here we conjugated rBC2LCN lectin with a 38 kDa domain of exotoxin A containing domains Ib and II in addition to domain III (PE38). The developed rBC2LCN-PE38 fusion protein could eliminate 50% of 201B7 hPSCs at a concentration of 0.003 μg/mL (24 h incubation), representing an approximately 556-fold higher activity than rBC2LCN-PE23. Little or no effect on human fibroblasts, human mesenchymal stem cells, and hiPSC-derived hepatocytes was observed at concentrations lower than 1 μg/mL. Finally, we demonstrate that rBC2LCN-PE38 selectively eliminates hiPSCs from a mixed culture of hiPSCs and hiPSC-derived hepatocytes. Since rBC2LCN-PE38 can be prepared from soluble fractions of E. coli culture at a yield of 9 mg/L, rBC2LCN-PE38 represents a practical reagent to remove human pluripotent stem cells residing in cultured cells destined for transplantation.

  11. Solitary BioY Proteins Mediate Biotin Transport into Recombinant Escherichia coli

    Science.gov (United States)

    Finkenwirth, Friedrich; Kirsch, Franziska

    2013-01-01

    Energy-coupling factor (ECF) transporters form a large group of vitamin uptake systems in prokaryotes. They are composed of highly diverse, substrate-specific, transmembrane proteins (S units), a ubiquitous transmembrane protein (T unit), and homo- or hetero-oligomeric ABC ATPases. Biotin transporters represent a special case of ECF-type systems. The majority of the biotin-specific S units (BioY) is known or predicted to interact with T units and ABC ATPases. About one-third of BioY proteins, however, are encoded in organisms lacking any recognizable T unit. This finding raises the question of whether these BioYs function as transporters in a solitary state, a feature ascribed to certain BioYs in the past. To address this question in living cells, an Escherichia coli K-12 derivative deficient in biotin synthesis and devoid of its endogenous high-affinity biotin transporter was constructed as a reference strain. This organism is particularly suited for this purpose because components of ECF transporters do not naturally occur in E. coli K-12. The double mutant was viable in media containing either high levels of biotin or a precursor of the downstream biosynthetic path. Importantly, it was nonviable on trace levels of biotin. Eight solitary bioY genes of proteobacterial origin were individually expressed in the reference strain. Each of the BioYs conferred biotin uptake activity on the recombinants, which was inferred from uptake assays with [3H]biotin and growth of the cells on trace levels of biotin. The results underscore that solitary BioY transports biotin across the cytoplasmic membrane. PMID:23836870

  12. A new impedance based approach to test the activity of recombinant protein--Semaphorins as a test case.

    Science.gov (United States)

    Birger, Anastasya; Besser, Elazar; Reubinoff, Benjamin; Behar, Oded

    2015-10-01

    The biological activity of a recombinant protein is routinely measured using a bioassay such as an enzyme assay. However, many proteins have no enzymatic activity and in many cases it is difficult to devise a simple and reliable approach to test their activity. Semaphorins, Ephrins, Slits, Netrins or amylin-assisted proteins have numerous activities affecting many systems and cell types in the human body. Most of them are also able to induce rapid cytoskeleton changes at least in some cell types. We assumed therefore, that such proteins might be tested based on their ability to modulate the cytoskeleton. Here we tested a number of semaphorins in an impedance based label-free platform that allows for dynamic monitoring of subtle morphological and adhesive changes. This system has proved to be a very fast, sensitive and effective way to monitor and determine the activity of such proteins. Furthermore we showed that it is possible to customize a cell-protein system by transfecting the cells with specific receptors and test the cell response following the addition of the recombinant ligand protein. Since other protein families such as Ephrins and Netrins can also influence the cytoskeleton of some cells, this approach may be applicable to a large number of proteins. Copyright © 2015 Elsevier GmbH. All rights reserved.

  13. Sequence Identification, Recombinant Production, and Analysis of the Self-Assembly of Egg Stalk Silk Proteins from Lacewing Chrysoperla carnea.

    Science.gov (United States)

    Neuenfeldt, Martin; Scheibel, Thomas

    2017-06-13

    Egg stalk silks of the common green lacewing Chrysoperla carnea likely comprise at least three different silk proteins. Based on the natural spinning process, it was hypothesized that these proteins self-assemble without shear stress, as adult lacewings do not use a spinneret. To examine this, the first sequence identification and determination of the gene expression profile of several silk proteins and various transcript variants thereof was conducted, and then the three major proteins were recombinantly produced in Escherichia coli encoded by their native complementary DNA (cDNA) sequences. Circular dichroism measurements indicated that the silk proteins in aqueous solutions had a mainly intrinsically disordered structure. The largest silk protein, which we named ChryC1, exhibited a lower critical solution temperature (LCST) behavior and self-assembled into fibers or film morphologies, depending on the conditions used. The second silk protein, ChryC2, self-assembled into nanofibrils and subsequently formed hydrogels. Circular dichroism and Fourier transform infrared spectroscopy confirmed conformational changes of both proteins into beta sheet rich structures upon assembly. ChryC3 did not self-assemble into any morphology under the tested conditions. Thereby, through this work, it could be shown that recombinant lacewing silk proteins can be produced and further used for studying the fiber formation of lacewing egg stalks.

  14. Recombinant proteins incorporating short non-native extensions may display increased aggregation propensity as detected by high resolution NMR spectroscopy

    International Nuclear Information System (INIS)

    Zanzoni, Serena; D’Onofrio, Mariapina; Molinari, Henriette; Assfalg, Michael

    2012-01-01

    Highlights: ► Bile acid binding proteins from different constructs retain structural integrity. ► NMR 15 N-T 1 relaxation data of BABPs show differences if LVPR extension is present. ► Deviations from a 15 N-T 1 /molecular-weight calibration curve indicate aggregation. -- Abstract: The use of a recombinant protein to investigate the function of the native molecule requires that the former be obtained with the same amino acid sequence as the template. However, in many cases few additional residues are artificially introduced for cloning or purification purposes, possibly resulting in altered physico-chemical properties that may escape routine characterization. For example, increased aggregation propensity without visible protein precipitation is hardly detected by most analytical techniques but its investigation may be of great importance for optimizing the yield of recombinant protein production in biotechnological and structural biology applications. In this work we show that bile acid binding proteins incorporating the common C-terminal LeuValProArg extension display different hydrodynamic properties from those of the corresponding molecules without such additional amino acids. The proteins were produced enriched in nitrogen-15 for analysis via heteronuclear NMR spectroscopy. Residue-specific spin relaxation rates were measured and related to rotational tumbling time and molecular size. While the native-like recombinant proteins show spin-relaxation rates in agreement with those expected for monomeric globular proteins of their mass, our data indicate the presence of larger adducts for samples of proteins with very short amino acid extensions. The used approach is proposed as a further screening method for the quality assessment of biotechnological protein products.

  15. Recombinant proteins incorporating short non-native extensions may display increased aggregation propensity as detected by high resolution NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Zanzoni, Serena; D' Onofrio, Mariapina; Molinari, Henriette [Department of Biotechnology, University of Verona, 37134 Verona (Italy); Assfalg, Michael, E-mail: michael.assfalg@univr.it [Department of Biotechnology, University of Verona, 37134 Verona (Italy)

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer Bile acid binding proteins from different constructs retain structural integrity. Black-Right-Pointing-Pointer NMR {sup 15}N-T{sub 1} relaxation data of BABPs show differences if LVPR extension is present. Black-Right-Pointing-Pointer Deviations from a {sup 15}N-T{sub 1}/molecular-weight calibration curve indicate aggregation. -- Abstract: The use of a recombinant protein to investigate the function of the native molecule requires that the former be obtained with the same amino acid sequence as the template. However, in many cases few additional residues are artificially introduced for cloning or purification purposes, possibly resulting in altered physico-chemical properties that may escape routine characterization. For example, increased aggregation propensity without visible protein precipitation is hardly detected by most analytical techniques but its investigation may be of great importance for optimizing the yield of recombinant protein production in biotechnological and structural biology applications. In this work we show that bile acid binding proteins incorporating the common C-terminal LeuValProArg extension display different hydrodynamic properties from those of the corresponding molecules without such additional amino acids. The proteins were produced enriched in nitrogen-15 for analysis via heteronuclear NMR spectroscopy. Residue-specific spin relaxation rates were measured and related to rotational tumbling time and molecular size. While the native-like recombinant proteins show spin-relaxation rates in agreement with those expected for monomeric globular proteins of their mass, our data indicate the presence of larger adducts for samples of proteins with very short amino acid extensions. The used approach is proposed as a further screening method for the quality assessment of biotechnological protein products.

  16. Polysome profiling of mAb producing CHO cell lines links translational control of cell proliferation and recombinant mRNA loading onto ribosomes with global and recombinant protein synthesis.

    Science.gov (United States)

    Godfrey, Charlotte L; Mead, Emma J; Daramola, Olalekan; Dunn, Sarah; Hatton, Diane; Field, Ray; Pettman, Gary; Smales, C Mark

    2017-08-01

    mRNA translation is a key process determining growth, proliferation and duration of a Chinese hamster ovary (CHO) cell culture and influences recombinant protein synthesis rate. During bioprocessing, CHO cells can experience stresses leading to reprogramming of translation and decreased global protein synthesis. Here we apply polysome profiling to determine reprogramming and translational capabilities in host and recombinant monoclonal antibody-producing (mAb) CHO cell lines during batch culture. Recombinant cell lines with the fastest cell specific growth rates were those with the highest global translational efficiency. However, total ribosomal capacity, determined from polysome profiles, did not relate to the fastest growing or highest producing mAb cell line, suggesting it is the ability to utilise available machinery that determines protein synthetic capacity. Cell lines with higher cell specific productivities tended to have elevated recombinant heavy chain transcript copy numbers, localised to the translationally active heavy polysomes. The highest titre cell line was that which sustained recombinant protein synthesis and maintained high recombinant transcript copy numbers in polysomes. Investigation of specific endogenous transcripts revealed a number that maintained or reprogrammed into heavy polysomes, identifying targets for potential cell engineering or those with 5' untranslated regions that might be utilised to enhance recombinant transcript translation. © 2017 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Identifying and engineering promoters for high level and sustainable therapeutic recombinant protein production in cultured mammalian cells.

    Science.gov (United States)

    Ho, Steven C L; Yang, Yuansheng

    2014-08-01

    Promoters are essential on plasmid vectors to initiate transcription of the transgenes when generating therapeutic recombinant proteins expressing mammalian cell lines. High and sustained levels of gene expression are desired during therapeutic protein production while gene expression is useful for cell engineering. As many finely controlled promoters exhibit cell and product specificity, new promoters need to be identified, optimized and carefully evaluated before use. Suitable promoters can be identified using techniques ranging from simple molecular biology methods to modern high-throughput omics screenings. Promoter engineering is often required after identification to either obtain high and sustained expression or to provide a wider range of gene expression. This review discusses some of the available methods to identify and engineer promoters for therapeutic recombinant protein expression in mammalian cells.

  18. Development of a reverse genetics system to generate a recombinant Ebola virus Makona expressing a green fluorescent protein

    Energy Technology Data Exchange (ETDEWEB)

    Albariño, César G., E-mail: calbarino@cdc.gov; Wiggleton Guerrero, Lisa; Lo, Michael K.; Nichol, Stuart T.; Towner, Jonathan S.

    2015-10-15

    Previous studies have demonstrated the potential application of reverse genetics technology in studying a broad range of aspects of viral biology, including gene regulation, protein function, cell entry, and pathogenesis. Here, we describe a highly efficient reverse genetics system used to generate recombinant Ebola virus (EBOV) based on a recent isolate from a human patient infected during the 2014–2015 outbreak in Western Africa. We also rescued a recombinant EBOV expressing a fluorescent reporter protein from a cleaved VP40 protein fusion. Using this virus and an inexpensive method to quantitate the expression of the foreign gene, we demonstrate its potential usefulness as a tool for screening antiviral compounds and measuring neutralizing antibodies. - Highlights: • Recombinant Ebola virus (EBOV) derived from Makona variant was rescued. • New protocol for viral rescue allows 100% efficiency. • Modified EBOV expresses a green fluorescent protein from a VP40-fused protein. • Modified EBOV was tested as tool to screen antiviral compounds and measure neutralizing antibodies.

  19. Development of a reverse genetics system to generate a recombinant Ebola virus Makona expressing a green fluorescent protein

    International Nuclear Information System (INIS)

    Albariño, César G.; Wiggleton Guerrero, Lisa; Lo, Michael K.; Nichol, Stuart T.; Towner, Jonathan S.

    2015-01-01

    Previous studies have demonstrated the potential application of reverse genetics technology in studying a broad range of aspects of viral biology, including gene regulation, protein function, cell entry, and pathogenesis. Here, we describe a highly efficient reverse genetics system used to generate recombinant Ebola virus (EBOV) based on a recent isolate from a human patient infected during the 2014–2015 outbreak in Western Africa. We also rescued a recombinant EBOV expressing a fluorescent reporter protein from a cleaved VP40 protein fusion. Using this virus and an inexpensive method to quantitate the expression of the foreign gene, we demonstrate its potential usefulness as a tool for screening antiviral compounds and measuring neutralizing antibodies. - Highlights: • Recombinant Ebola virus (EBOV) derived from Makona variant was rescued. • New protocol for viral rescue allows 100% efficiency. • Modified EBOV expresses a green fluorescent protein from a VP40-fused protein. • Modified EBOV was tested as tool to screen antiviral compounds and measure neutralizing antibodies

  20. Silk-Silk Interactions between Silkworm Fibroin and Recombinant Spider Silk Fusion Proteins Enable the Construction of Bioactive Materials.

    Science.gov (United States)

    Nilebäck, Linnea; Chouhan, Dimple; Jansson, Ronnie; Widhe, Mona; Mandal, Biman B; Hedhammar, My

    2017-09-20

    Natural silk is easily accessible from silkworms and can be processed into different formats suitable as biomaterials and cell culture matrixes. Recombinant DNA technology enables chemical-free functionalization of partial silk proteins through fusion with peptide motifs and protein domains, but this constitutes a less cost-effective production process. Herein, we show that natural silk fibroin (SF) can be used as a bulk material that can be top-coated with a thin layer of the recombinant spider silk protein 4RepCT in fusion with various bioactive motifs and domains. The coating process is based on a silk assembly to achieve stable interactions between the silk types under mild buffer conditions. The assembly process was studied in real time by quartz crystal microbalance with dissipation. Coatings, electrospun mats, and microporous scaffolds were constructed from Antheraea assama and Bombyx mori SFs. The morphology of the fibroin materials before and after coating with recombinant silk proteins was analyzed by scanning electron microscopy and atomic force microscopy. SF materials coated with various bioactive 4RepCT fusion proteins resulted in directed antibody capture, enzymatic activity, and improved cell attachment and spreading, respectively, compared to pristine SF materials. The herein-described procedure allows a fast and easy route for the construction of bioactive materials.

  1. Generation of Recombinant Oropouche Viruses Lacking the Nonstructural Protein NSm or NSs.

    Science.gov (United States)

    Tilston-Lunel, Natasha L; Acrani, Gustavo Olszanski; Randall, Richard E; Elliott, Richard M

    2015-12-23

    Oropouche virus (OROV) is a midge-borne human pathogen with a geographic distribution in South America. OROV was first isolated in 1955, and since then, it has been known to cause recurring outbreaks of a dengue-like illness in the Amazonian regions of Brazil. OROV, however, remains one of the most poorly understood emerging viral zoonoses. Here we describe the successful recovery of infectious OROV entirely from cDNA copies of its genome and generation of OROV mutant viruses lacking either the NSm or the NSs coding region. Characterization of the recombinant viruses carried out in vitro demonstrated that the NSs protein of OROV is an interferon (IFN) antagonist as in other NSs-encoding bunyaviruses. Additionally, we demonstrate the importance of the nine C-terminal amino acids of OROV NSs in IFN antagonistic activity. OROV was also found to be sensitive to IFN-α when cells were pretreated; however, the virus was still capable of replicating at doses as high as 10,000 U/ml of IFN-α, in contrast to the family prototype BUNV. We found that OROV lacking the NSm protein displayed characteristics similar to those of the wild-type virus, suggesting that the NSm protein is dispensable for virus replication in the mammalian and mosquito cell lines that were tested. Oropouche virus (OROV) is a public health threat in Central and South America, where it causes periodic outbreaks of dengue-like illness. In Brazil, OROV is the second most frequent cause of arboviral febrile illness after dengue virus, and with the current rates of urban expansion, more cases of this emerging viral zoonosis could occur. To better understand the molecular biology of OROV, we have successfully rescued the virus along with mutants. We have established that the C terminus of the NSs protein is important in interferon antagonism and that the NSm protein is dispensable for virus replication in cell culture. The tools described in this paper are important in terms of understanding this important yet

  2. Nanodiamond enhances immune responses in mice against recombinant HA/H7N9 protein.

    Science.gov (United States)

    Pham, Ngoc Bich; Ho, Thuong Thi; Nguyen, Giang Thu; Le, Thuy Thi; Le, Ngoc Thu; Chang, Huan-Cheng; Pham, Minh Dinh; Conrad, Udo; Chu, Ha Hoang

    2017-10-05

    The continuing spread of the newly emerged H7N9 virus among poultry in China, as well as the possibility of human-to-human transmission, has attracted numerous efforts to develop an effective vaccine against H7N9. The use of nanoparticles in vaccinology is inspired by the fact that most pathogens have a dimension within the nano-size range and therefore can be processed efficiently by the immune system, which leads to a potent immune response. Herein, we report a facile approach to increase antigen size to achieve not only fast but also effective responses against the recombinant HA/H7N9 protein via a simple conjugation of the protein onto the surface of nanodiamond particles. In this study, trimeric Haemagglutinin (H7) that is transiently expressed in N. benthamiana was purified using affinity chromatography, and its trimeric state was revealed successfully by the cross-linking reaction. The trimeric H7 solution was subsequently mixed with a nanodiamond suspension in different ratios. The successful conjugation of the trimeric H7 onto the surface of nanodiamond particles was demonstrated by the changes in size and Zeta-potential of the particles before and after protein coating, Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and Western-blot analysis. Next, biofunction of the protein-nanodiamond conjugates was screened using a haemagglutination assay. A mixture containing 5 µg of trimeric H7 and 60 µg of nanodiamond corresponds to a ratio of 1:12 (w/w) of agglutinated chicken red blood cells at HA titer of 1024, which is 512-fold higher than the HA titer of free trimeric H7. After the 2nd and 3rd immunization in mice, ELISA and Western blot analyses demonstrated that the physical mixture of trimeric H7 protein and nanodiamond (1:12, w/w) elicited statistically significant stronger H7-specific-IgG response demonstrated by higher amounts of H7N9-specific IgG (over 15.4-fold with P < 0.05 after the second immunization). These results

  3. Immobilized Metal Affinity Chromatography Co-Purifies TGF-β1 with Histidine-Tagged Recombinant Extracellular Proteins

    Science.gov (United States)

    Kaur, Jasvir; Reinhardt, Dieter P.

    2012-01-01

    Extracellular recombinant proteins are commonly produced using HEK293 cells as histidine-tagged proteins facilitating purification by immobilized metal affinity chromatography (IMAC). Based on gel analyses, this one-step purification typically produces proteins of high purity. Here, we analyzed the presence of TGF-β1 in such IMAC purifications using recombinant extracellular fibrillin-1 fragments as examples. Analysis of various purified recombinant fibrillin-1 fragments by ELISA consistently revealed the presence of picomolar concentrations of active and latent TGF-β1, but not of BMP-2. These quantities of TGF-β1 were not detectable by Western blotting and mass spectrometry. However, the amounts of TGF-β1 were sufficient to consistently trigger Smad2 phosphorylation in fibroblasts. The purification mechanism was analyzed to determine whether the presence of TGF-β1 in these protein preparations represents a specific or non-specific co-purification of TGF-β1 with fibrillin-1 fragments. Control purifications using conditioned medium from non-transfected 293 cells yielded similar amounts of TGF-β1 after IMAC. IMAC of purified TGF-β1 and the latency associated peptide showed that these proteins bound to the immobilized nickel ions. These data clearly demonstrate that TGF-β1 was co-purified by specific interactions with nickel, and not by specific interactions with fibrillin-1 fragments. Among various chromatographic methods tested for their ability to eliminate TGF-β1 from fibrillin-1 preparations, gel filtration under high salt conditions was highly effective. As various recombinant extracellular proteins purified in this fashion are frequently used for experiments that can be influenced by the presence of TGF-β1, these findings have far-reaching implications for the required chromatographic schemes and quality controls. PMID:23119075

  4. Generation of Nanobodies against SlyD and development of tools to eliminate this bacterial contaminant from recombinant proteins.

    Science.gov (United States)

    Hu, Yaozhong; Romão, Ema; Vertommen, Didier; Vincke, Cécile; Morales-Yánez, Francisco; Gutiérrez, Carlos; Liu, Changxiao; Muyldermans, Serge

    2017-09-01

    The gene for a protein domain, derived from a tumor marker, fused to His tag codons and under control of a T7 promotor was expressed in E. coli strain BL21 (DE3). The recombinant protein was purified from cell lysates through immobilized metal affinity chromatography and size-exclusion chromatography. A contaminating bacterial protein was consistently co-purified, even using stringent washing solutions containing 50 or 100 mM imidazole. Immunization of a dromedary with this contaminated protein preparation, and the subsequent generation and panning of the immune Nanobody library yielded several Nanobodies of which 2/3 were directed against the bacterial contaminant, reflecting the immunodominance of this protein to steer the dromedary immune response. Affinity adsorption of this contaminant using one of our specific Nanobodies followed by mass spectrometry identified the bacterial contaminant as FKBP-type peptidyl-prolyl cis-trans isomerase (SlyD) from E. coli. This SlyD protein contains in its C-terminal region 14 histidines in a stretch of 31 amino acids, which explains its co-purification on Ni-NTA resin. This protein is most likely present to varying extents in all recombinant protein preparations after immobilized metal affinity chromatography. Using our SlyD-specific Nb 5 we generated an immune-complex that could be removed either by immunocapturing or by size exclusion chromatography. Both methods allow us to prepare a recombinant protein sample where the SlyD contaminant was quantitatively eliminated. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Recombinant human parathyroid hormone related protein 1-34 and 1-84 and their roles in osteoporosis treatment.

    Directory of Open Access Journals (Sweden)

    Hua Wang

    Full Text Available Osteoporosis is a common disorder characterized by compromised bone strength that predisposes patients to increased fracture risk. Parathyroid hormone related protein (PTHrP is one of the candidates for clinical osteoporosis treatment. In this study, GST Gene Fusion System was used to express recombinant human PTHrP (hPTHrP 1-34 and 1-84. To determine whether the recombinant hPTHrP1-34 and 1-84 can enhance renal calcium reabsorption and promote bone formation, we examined effects of recombinant hPTHrP1-34 and 1-84 on osteogenic lineage commitment in a primary bone marrow cell culture system and on osteoporosis treatment. Results revealed that both of recombinant hPTHrP1-34 and 1-84 increased colony formation and osteogenic cell differentiation and mineralization in vitro; however, the effect of recombinant hPTHrP1-84 is a little stronger than that of hPTHrP1-34. Next, ovariectomy was used to construct osteoporosis animal model (OVX to test activities of these two recombinants in vivo. HPTHrP1-84 administration elevated serum calcium by up-regulating the expression of renal calcium transporters, which resulted in stimulation of osteoblastic bone formation. These factors contributed to augmented bone mass in hPTHrP1-84 treated OVX mice but did not affect bone resorption. There was no obvious bone mass alteration in hPTHrP1-34 treated OVX mice, which may be, at least partly, associated with shorter half-life of hPTHrP1-34 compared to hPTHrP1-84 in vivo. This study implies that recombinant hPTHrP1-84 is more effective than hPTHrP1-34 to enhance renal calcium reabsorption and to stimulate bone formation in vivo.

  6. Genetic interactions between the chromosome axis-associated protein Hop1 and homologous recombination determinants in Schizosaccharomyces pombe.

    Science.gov (United States)

    Brown, Simon David; Jarosinska, Olga Dorota; Lorenz, Alexander

    2018-03-17

    Hop1 is a component of the meiosis-specific chromosome axis and belongs to the evolutionarily conserved family of HORMA domain proteins. Hop1 and its orthologs in higher eukaryotes are a major factor in promoting double-strand DNA break formation and inter-homolog recombination. In budding yeast and mammals, they are also involved in a meiotic checkpoint kinase cascade monitoring the completion of double-strand DNA break repair. We used the fission yeast, Schizosaccharomyces pombe, which lacks a canonical synaptonemal complex to test whether Hop1 has a role beyond supporting the generation of double-strand DNA breaks and facilitating inter-homolog recombination events. We determined how mutants of homologous recombination factors genetically interact with hop1, studied the role(s) of the HORMA domain of Hop1, and characterized a bio-informatically predicted interactor of Hop1, Aho1 (SPAC688.03c). Our observations indicate that in fission yeast, Hop1 does require its HORMA domain to support wild-type levels of meiotic recombination and localization to meiotic chromatin. Furthermore, we show that hop1∆ only weakly interacts genetically with mutants of homologous recombination factors, and in fission yeast likely has no major role beyond break formation and promoting inter-homolog events. We speculate that after the evolutionary loss of the synaptonemal complex, Hop1 likely has become less important for modulating recombination outcome during meiosis in fission yeast, and that this led to a concurrent rewiring of genetic pathways controlling meiotic recombination.

  7. Maltose-binding protein enhances secretion of recombinant human granzyme B accompanied by in vivo processing of a precursor MBP fusion protein.

    Directory of Open Access Journals (Sweden)

    Benjamin Dälken

    Full Text Available BACKGROUND: The apoptosis-inducing serine protease granzyme B (GrB is an important factor contributing to lysis of target cells by cytotoxic lymphocytes. Expression of enzymatically active GrB in recombinant form is a prerequisite for functional analysis and application of GrB for therapeutic purposes. METHODS AND FINDINGS: We investigated the influence of bacterial maltose-binding protein (MBP fused to GrB via a synthetic furin recognition motif on the expression of the MBP fusion protein also containing an N-terminal α-factor signal peptide in the yeast Pichia pastoris. MBP markedly enhanced the amount of GrB secreted into culture supernatant, which was not the case when GrB was fused to GST. MBP-GrB fusion protein was cleaved during secretion by an endogenous furin-like proteolytic activity in vivo, liberating enzymatically active GrB without the need of subsequent in vitro processing. Similar results were obtained upon expression of a recombinant fragment of the ErbB2/HER2 receptor protein or GST as MBP fusions. CONCLUSIONS: Our results demonstrate that combination of MBP as a solubility enhancer with specific in vivo cleavage augments secretion of processed and functionally active proteins from yeast. This strategy may be generally applicable to improve folding and increase yields of recombinant proteins.

  8. Maltose-Binding Protein Enhances Secretion of Recombinant Human Granzyme B Accompanied by In Vivo Processing of a Precursor MBP Fusion Protein

    Science.gov (United States)

    Dälken, Benjamin; Jabulowsky, Robert A.; Oberoi, Pranav; Benhar, Itai; Wels, Winfried S.

    2010-01-01

    Background The apoptosis-inducing serine protease granzyme B (GrB) is an important factor contributing to lysis of target cells by cytotoxic lymphocytes. Expression of enzymatically active GrB in recombinant form is a prerequisite for functional analysis and application of GrB for therapeutic purposes. Methods and Findings We investigated the influence of bacterial maltose-binding protein (MBP) fused to GrB via a synthetic furin recognition motif on the expression of the MBP fusion protein also containing an N-terminal α-factor signal peptide in the yeast Pichia pastoris. MBP markedly enhanced the amount of GrB secreted into culture supernatant, which was not the case when GrB was fused to GST. MBP-GrB fusion protein was cleaved during secretion by an endogenous furin-like proteolytic activity in vivo, liberating enzymatically active GrB without the need of subsequent in vitro processing. Similar results were obtained upon expression of a recombinant fragment of the ErbB2/HER2 receptor protein or GST as MBP fusions. Conclusions Our results demonstrate that combination of MBP as a solubility enhancer with specific in vivo cleavage augments secretion of processed and functionally active proteins from yeast. This strategy may be generally applicable to improve folding and increase yields of recombinant proteins. PMID:21203542

  9. Immunoadjuvant activities of a recombinant chicken IL-12 in chickens vaccinated with Newcastle disease virus recombinant HN protein.

    Science.gov (United States)

    Su, Bor Sheu; Yin, Hsien Sheng; Chiu, Hua Hsien; Hung, Li Hsiang; Huang, Ji Ping; Shien, Jui Hung; Lee, Long Huw

    2011-08-05

    Recombinant fowlpox virus (rFPV/HN) expressing Newcastle disease virus (NDV) HN gene and rFPV/HN/chIL-12 co-expressing chicken IL-12 (chIL-12) and HN (rHN/chIL-12) genes have been characterized. rHN/chIL-12 or rchIL-12, expressed by our previous construct rFPV/chIL-12, co-administered with rHN was assessed for adjuvant activities of chIL-12. Chickens were vaccinated with various amounts of rHN/chIL-12 mixed with mineral oil (MO), intramuscularly. Levels of hemagglutination-inhibition (HI) antibody production depended on the concentration of the injected rHN or rHN/chIL-12. The lower HI antibody titers were obtained in chicken groups rHN/chIL-12/7-rHN/chIL-12/9, receiving 60ng rHN/8ng chIL-12 with MO, 30ng rHN/4ng chIL-12 with MO or 15ng rHN/2ng chIL-12 with MO, respectively, compared to those in chicken groups rHN/7-rHN/9, receiving rHN with MO alone. However, chickens in group rHN/chIL-12/7 or rHN/chIL-12/8 and rHN with MO alone showed the same effective protection. Chicken group rHN/chIL-12/9 was even more protective than that in group rHN/9. When rchIL-12 was co-injected with 15ng rHN plus MO, chickens produced low levels of HI antibody titers; while higher levels of IFN-γ production and an effective protection rate (83%) were obtained. On the other hand, low levels of IFN-γ production and low protection response (50%) were obtained in chickens injected with rHN with MO alone. Taken together, when the concentration of rHN decreased to certain levels, rchIL-12 reduced HI antibody production. The increase in the induction of IFN-γ production might suggest the enhancement of the cell-mediated immunity which conferred the protection from the NDV challenge. Copyright © 2011 Elsevier B.V. All rights reserved.

  10. Manufacturing process used to produce long-acting recombinant factor VIII Fc fusion protein.

    Science.gov (United States)

    McCue, Justin; Kshirsagar, Rashmi; Selvitelli, Keith; Lu, Qi; Zhang, Mingxuan; Mei, Baisong; Peters, Robert; Pierce, Glenn F; Dumont, Jennifer; Raso, Stephen; Reichert, Heidi

    2015-07-01

    Recombinant factor VIII Fc fusion protein (rFVIIIFc) is a long-acting coagulation factor approved for the treatment of hemophilia A. Here, the rFVIIIFc manufacturing process and results of studies evaluating product quality and the capacity of the process to remove potential impurities and viruses are described. This manufacturing process utilized readily transferable and scalable unit operations and employed multi-step purification and viral clearance processing, including a novel affinity chromatography adsorbent and a 15 nm pore size virus removal nanofilter. A cell line derived from human embryonic kidney (HEK) 293H cells was used to produce rFVIIIFc. Validation studies evaluated identity, purity, activity, and safety. Process-related impurity clearance and viral clearance spiking studies demonstrate robust and reproducible removal of impurities and viruses, with total viral clearance >8-15 log10 for four model viruses (xenotropic murine leukemia virus, mice minute virus, reovirus type 3, and suid herpes virus 1). Terminal galactose-α-1,3-galactose and N-glycolylneuraminic acid, two non-human glycans, were undetectable in rFVIIIFc. Biochemical and in vitro biological analyses confirmed the purity, activity, and consistency of rFVIIIFc. In conclusion, this manufacturing process produces a highly pure product free of viruses, impurities, and non-human glycan structures, with scale capabilities to ensure a consistent and adequate supply of rFVIIIFc. Copyright © 2015 Biogen. Published by Elsevier Ltd.. All rights reserved.

  11. Senataxin plays an essential role with DNA damage response proteins in meiotic recombination and gene silencing.

    Directory of Open Access Journals (Sweden)

    Olivier J Becherel

    2013-04-01

    Full Text Available Senataxin, mutated in the human genetic disorder ataxia with oculomotor apraxia type 2 (AOA2, plays an important role in maintaining genome integrity by coordination of transcription, DNA replication, and the DNA damage response. We demonstrate that senataxin is essential for spermatogenesis and that it functions at two stages in meiosis during crossing-over in homologous recombination and in meiotic sex chromosome inactivation (MSCI. Disruption of the Setx gene caused persistence of DNA double-strand breaks, a defect in disassembly of Rad51 filaments, accumulation of DNA:RNA hybrids (R-loops, and ultimately a failure of crossing-over. Senataxin localised to the XY body in a Brca1-dependent manner, and in its absence there was incomplete localisation of DNA damage response proteins to the XY chromosomes and ATR was retained on the axial elements of these chromosomes, failing to diffuse out into chromatin. Furthermore persistence of RNA polymerase II activity, altered ubH2A distribution, and abnormal XY-linked gene expression in Setx⁻/⁻ revealed an essential role for senataxin in MSCI. These data support key roles for senataxin in coordinating meiotic crossing-over with transcription and in gene silencing to protect the integrity of the genome.

  12. Recombinant protein production facility for fungal biomass-degrading enzymes using the yeast Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Mireille eHaon

    2015-09-01

    Full Text Available Filamentous fungi are the predominant source of lignocellulolytic enzymes used in industry for the transformation of plant biomass into high-value molecules and biofuels. The rapidity with which new fungal genomic and post-genomic data are being produced is vastly outpacing functional studies. This underscores the critical need for developing platforms dedicated to the recombinant expression of enzymes lacking confident functional annotation, a prerequisite to their functional and structural study. In the last decade, the yeast Pichia pastoris has become increasingly popular as a host for the production of fungal biomass-degrading enzymes, and particularly carbohydrate-active enzymes (CAZymes. This study aimed at setting-up a platform to easily and quickly screen the extracellular expression of biomass-degrading enzymes in Pichia pastoris. We first used three fungal glycoside hydrolases that we previously expressed using the protocol devised by Invitrogen to try different modifications of the original protocol. Considering the gain in time and convenience provided by the new protocol, we used it as basis to set-up the facility and produce a suite of fungal CAZymes (glycoside hydrolases, carbohydrate esterases and auxiliary activity enzyme families out of which more than 70% were successfully expressed. The platform tasks range from gene cloning to automated protein purifications and activity tests, and is open to the CAZyme users’ community.

  13. Development of oral cancer vaccine using recombinant Bifidobacterium displaying Wilms' tumor 1 protein.

    Science.gov (United States)

    Kitagawa, Koichi; Oda, Tsugumi; Saito, Hiroki; Araki, Ayame; Gonoi, Reina; Shigemura, Katsumi; Hashii, Yoshiko; Katayama, Takane; Fujisawa, Masato; Shirakawa, Toshiro

    2017-06-01

    Several types of vaccine-delivering tumor-associated antigens (TAAs) have been developed in basic and clinical research. Wilms' tumor 1 (WT1), identified as a gene responsible for pediatric renal neoplasm, is one of the most promising TAA for cancer immunotherapy. Peptide and dendritic cell-based WT1 cancer vaccines showed some therapeutic efficacy in clinical and pre-clinical studies but as yet no oral WT1 vaccine can be administrated in a simple and easy way. In the present study, we constructed a novel oral cancer vaccine using a recombinant Bifidobacterium longum displaying WT1 protein. B. longum 420 was orally administered into mice inoculated with WT1-expressing tumor cells for 4 weeks to examine anti-tumor effects. To analyze the WT1-specific cellular immune responses to oral B. longum 420, mice splenocytes were isolated and cytokine production and cytotoxic activities were determined. Oral administrations of B. longum 420 significantly inhibited WT1-expressing tumor growth and prolonged survival in mice. Immunohistochemical study and immunological assays revealed that B. longum 420 substantially induced tumor infiltration of CD4 + T and CD8 + T cells, systemic WT1-specific cytokine production, and cytotoxic activity mediated by WT1-epitope specific cytotoxic T lymphocytes, with no apparent adverse effects. Our novel oral cancer vaccine safely induced WT1-specific cellular immunity via activation of the gut mucosal immune system and achieved therapeutic efficacy with several practical advantages over existing non-oral vaccines.

  14. The business impact of an integrated continuous biomanufacturing platform for recombinant protein production.

    Science.gov (United States)

    Walther, Jason; Godawat, Rahul; Hwang, Chris; Abe, Yuki; Sinclair, Andrew; Konstantinov, Konstantin

    2015-11-10

    The biotechnology industry primarily uses batch technologies to manufacture recombinant proteins. The natural evolution of other industries has shown that transitioning from batch to continuous processing can yield significant benefits. A quantitative understanding of these benefits is critical to guide the implementation of continuous processing. In this manuscript, we use process economic modeling and Monte Carlo simulations to evaluate an integrated continuous biomanufacturing (ICB) platform and conduct risk-based valuation to generate a probabilistic range of net-present values (NPVs). For a specific ten-year product portfolio, the ICB platform reduces average cost by 55% compared to conventional batch processing, considering both capital and operating expenses. The model predicts that these savings can further increase by an additional 25% in situations with higher-than-expected product demand showing the upward potential of the ICB platform. The ICB platform achieves these savings and corresponding flexibility mainly due to process intensification in both upstream and downstream unit operations. This study demonstrates the promise of continuous bioprocessing while also establishing a novel framework to quantify financial benefits of other platform process technologies. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Evaluation of recombinant multi-epitope proteins for diagnosis of goat schistosomiasis by enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Lv, Chao; Hong, Yang; Fu, Zhiqiang; Lu, Ke; Cao, Xiaodan; Wang, Tao; Zhu, Chuangang; Li, Hao; Xu, Rui; Jia, Bingguang; Han, Qian; Dou, Xuefeng; Shen, Yuanxi; Zhang, Zuhang; Zai, Jinli; Feng, Jintao; Lin, Jiaojiao

    2016-03-09

    Schistosomiasis is a huge threat to human and animal health. Apart from bovines, goats play an important role in the transmission of schistosomiasis in some endemic areas of China. An accessible, quality-assured goat schistosomiasis diagnostic technique is needed. Recently, our laboratory identified two recombinant diagnostic antigens, SjPGM and SjRAD23 via an immuno-proteomic method. The application of these two recombinant antigens to develop a higher sensitivity and specificity technique for the sheep schistosomiasis diagnosis is urgently needed. Epitopes of SjPGM and SjRAD23 were predicted and three polypeptides, two from SjRAD23 and one from SjPGM, were selected. Recombinant plasmids containing two to three DNA sequences encoding predicted polypeptides or large hydrophilic region of Sj23 (LHD-Sj23) were constructed and expressed. Eight recombinant schistosome antigens including four multi-epitope proteins and four recombinant single-molecule antigens as well as SEA, were assessed by ELISA in 91 sera from schistosome-infected goats, 44 sera from non-infected goats, 37 sera from Orientobilharzia-infected goats, and 12 from Haemonchus contortus-infected goats. ELISA tests showed that three multi-epitope proteins had higher sensitivity than the four single-molecule antigens (rSjRAD23, rSjPGM, rBSjRAD23-1, rBSj23) and the multi-epitope protein rBSjPGM-BSjRAD23-1-BSj23 had the highest sensitivity (97.8 %, 89/91) and maintained good specificity (100 %, 44/44) as well as low cross-reactivity with haemonchosis (8.33 %, 3/12) and orientobilharziasis (13.51 %, 5/37) in the diagnosis of goat schistosomiasis. In contrast, when SEA was applied as a diagnosis antigen, it had 100 % (91/91) sensitivity, 75 % (33/44) specificity, 25 and 83.78 % cross-reactivity with haemonchosis (3/12) and orientobilharziasis (31/37), respectively. The application of recombinant multi-epitope proteins may increase the sensitivity of diagnosis technique and retain high specificity of single

  16. 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.

  17. Brucella melitensis VirB12 recombinant protein is a potential marker for serodiagnosis of human brucellosis.

    Science.gov (United States)

    Mirkalantari, Shiva; Zarnani, Amir-Hassan; Nazari, Mahboobeh; Irajian, Gholam Reza; Amirmozafari, Nour

    2017-03-03

    The numerous drawbacks of current serological tests for diagnosis of brucellosis which mainly results from cross reactivity with LPS from other gram-negative bacteria have generated an increasing interest to find more specific non-LPS antigens. Previous studies had indicated that Brucella VirB12 protein, a cell surface protein and component of type IV secretion system, induces antibody response during animal infection. However, this protein has not yet been tested as a serological diagnostic marker in human brucellosis. Recombinant VirB12 protein was prepared and evaluated the efficacy of it in an indirect enzyme-linked immunosorbent assay (ELISA) for brucellosis with sera collected from different region of Iran and the results were compared with a commercial ELISA kit. Sera from human brucellosis patients strongly reacted to the purified recombinant VirB12. The sensitivity, specificity, accuracy, negative predictive value and positive predictive value of recombinant VirB12-based ELISA related to the commercial-ELISA method were 87.8, 94, 90, 80 and 96.6% respectively. We concluded that antigenic VirB12 have a property value that can be considered as a candidate for using in serodiagnostic tests for human brucellosis.

  18. Toxoplasma gondii: biochemical and biophysical characterization of recombinant soluble dense granule proteins GRA2 and GRA6.

    Science.gov (United States)

    Bittame, Amina; Effantin, Grégory; Pètre, Graciane; Ruffiot, Pauline; Travier, Laetitia; Schoehn, Guy; Weissenhorn, Winfried; Cesbron-Delauw, Marie-France; Gagnon, Jean; Mercier, Corinne

    2015-03-27

    The most prominent structural feature of the parasitophorous vacuole (PV) in which the intracellular parasite Toxoplasma gondii proliferates is a membranous nanotubular network (MNN), which interconnects the parasites and the PV membrane. The MNN function remains unclear. The GRA2 and GRA6 proteins secreted from the parasite dense granules into the PV have been implicated in the MNN biogenesis. Amphipathic alpha-helices (AAHs) predicted in GRA2 and an alpha-helical hydrophobic domain predicted in GRA6 have been proposed to be responsible for their membrane association, thereby potentially molding the MMN in its structure. Here we report an analysis of the recombinant proteins (expressed in detergent-free conditions) by circular dichroism, which showed that full length GRA2 displays an alpha-helical secondary structure while recombinant GRA6 and GRA2 truncated of its AAHs are mainly random coiled. Dynamic light scattering and transmission electron microscopy showed that recombinant GRA6 and truncated GRA2 constitute a homogenous population of small particles (6-8 nm in diameter) while recombinant GRA2 corresponds to 2 populations of particles (∼8-15 nm and up to 40 nm in diameter, respectively). The unusual properties of GRA2 due to its AAHs are discussed. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Repression of the DCL2 and DCL4 genes in Nicotiana benthamiana plants for the transient expression of recombinant proteins.

    Science.gov (United States)

    Matsuo, Kouki; Matsumura, Takeshi

    2017-08-01

    The production of recombinant proteins in plants has many advantages, including safety and reduced costs. However, this technology still faces several issues, including low levels of production. The repression of RNA silencing seems to be particularly important for improving recombinant protein production because RNA silencing effectively degrades transgene-derived mRNAs in plant cells. Therefore, to overcome this, we used RNA interference technology to develop DCL2- and DCL4-repressed transgenic Nicotiana benthamiana plants (ΔD2, ΔD4, and ΔD2ΔD4 plants), which had much lower levels of NbDCL2 and/or NbDCL4 mRNAs than wild-type plants. A transient gene expression assay showed that the ΔD2ΔD4 plants accumulated larger amounts of green fluorescent protein (GFP) and human acidic fibroblast growth factor (aFGF) than ΔD2, ΔD4, and wild-type plants. Furthermore, the levels of GFP and aFGF mRNAs were also higher in ΔD2ΔD4 plants than in ΔD2, ΔD4, and wild-type plants. These findings demonstrate that ΔD2ΔD4 plants express larger amounts of recombinant proteins than wild-type plants, and so would be useful for recombinant protein production. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  20. Transfection of primary brain capillary endothelial cells for protein synthesis and secretion of recombinant erythropoietin: a strategy to enable protein delivery to the brain.

    Science.gov (United States)

    Burkhart, Annette; Andresen, Thomas Lars; Aigner, Achim; Thomsen, Louiza Bohn; Moos, Torben

    2017-07-01

    Treatment of chronic disorders affecting the central nervous system (CNS) is complicated by the inability of drugs to cross the blood-brain barrier (BBB). Non-viral gene therapy applied to brain capillary endothelial cells (BCECs) denotes a novel approach to overcome the restraints in this passage, 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 into the cell culture medium both luminally and abluminally, and despite lower levels of EPO reaching the abluminal chamber, the amount of recombinant EPO was sufficient to evolve a biological effect on astrocytes cultured at the abluminal side in terms of upregulated gene expression of brain-derived neurotropic factor (BDNF). In conclusion, non-viral gene therapy to RBECs leads to protein secretion and signifies a method for therapeutic proteins to target cells inside the CNS otherwise omitted due to the BBB.

  1. Recombinant protein expression for structural biology in HEK 293F suspension cells: a novel and accessible approach.

    Science.gov (United States)

    Portolano, Nicola; Watson, Peter J; Fairall, Louise; Millard, Christopher J; Milano, Charles P; Song, Yun; Cowley, Shaun M; Schwabe, John W R

    2014-10-16

    The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293 F cells.

  2. 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

  3. Fusion protein-based biofilm fabrication composed of recombinant azurin–myoglobin for dual-level biomemory application

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Taek [Research Institute for Basic Science, Sogang University, Heukseok-dong, Dongjak-gu, Seoul 156-756 (Korea, Republic of); Chung, Yong-Ho; Yoon, Jinho [Department of Chemical and Biomolecular Engineering, Sogang University, Heukseok-dong, Dongjak-gu, 35 Baekbeom-ro (Sinsu-dong), Mapo-gu, Seoul 121-742 (Korea, Republic of); Min, Junhong [School of Integrative Engineering, Chung-Ang University, Heukseok-dong, Dongjak-gu, Seoul 156-756 (Korea, Republic of); Choi, Jeong-Woo, E-mail: jwchoi@sogang.ac.kr [Department of Chemical and Biomolecular Engineering, Sogang University, Heukseok-dong, Dongjak-gu, 35 Baekbeom-ro (Sinsu-dong), Mapo-gu, Seoul 121-742 (Korea, Republic of)

    2014-11-30

    Graphical abstract: - Highlights: • We developed the fusion protein-based biofilm on the inorganic surface. • For making the fusion protein, the recombinant azurin and the myoglobin was conjugated by the native chemical ligation method. • The developed fusion protein shows unique electrochemical property. • The proposed fusion protein biofilm appears to be a good method for dual-level biomemory device. - Abstract: In the present study, a fusion protein-based biofilm composed of a recombinant azurin–myoglobin (Azu-Myo) has been developed and confirmed its original electrochemical property for dual-level biomemory device application. For this purpose, the azurin was modified with cysteine residues for direct immobilization and conjugation. Then, the recombinant azurin was conjugated with the myoglobin via a sulfo-SMCC bifunctional linker using the chemical ligation method (CLM). The SDS-PAGE and UV–vis spectroscopy were performed to examine the fusion protein conjugates. The prepared Azu-Myo fusion protein was self-assembled onto Au substrate for the biofilm fabrication. Then, the atomic force microscopy (AFM) was used to confirm the immobilization and the surface-enhanced Raman spectroscopy (SERS) was carried out to the surface analysis. Also, the cyclic voltammetry (CV) was carried out to observe an electrochemical property of fabricated biofilm. As a result, the two pair of redox potential values was obtained for dual-level biomemory device application. Then, the dual-level biomemory function was verified by the multi-potential chronoamperometry (MPCA). The results indicate a new fabrication method and material combination for advances in bioelectronic device development.

  4. Yeast expressed recombinant Hemagglutinin protein of Novel H1N1 elicits neutralising antibodies in rabbits and mice

    Directory of Open Access Journals (Sweden)

    Athmaram TN

    2011-11-01

    Full Text Available Abstract Currently available vaccines for the pandemic Influenza A (H1N1 2009 produced in chicken eggs have serious impediments viz limited availability, risk of allergic reactions and the possible selection of sub-populations differing from the naturally occurring virus, whereas the cell culture derived vaccines are time consuming and may not meet the demands of rapid global vaccination required to combat the present/future pandemic. Hemagglutinin (HA based subunit vaccine for H1N1 requires the HA protein in glycosylated form, which is impossible with the commonly used bacterial expression platform. Additionally, bacterial derived protein requires extensive purification and refolding steps for vaccine applications. For these reasons an alternative heterologous system for rapid, easy and economical production of Hemagglutinin protein in its glycosylated form is required. The HA gene of novel H1N1 A/California/04/2009 was engineered for expression in Pichia pastoris as a soluble secreted protein. The full length HA- synthetic gene having α-secretory tag was integrated into P. pastoris genome through homologous recombination. The resultant Pichia clones having multiple copy integrants of the transgene expressed full length HA protein in the culture supernatant. The Recombinant yeast derived H1N1 HA protein elicited neutralising antibodies both in mice and rabbits. The sera from immunised animals also exhibited Hemagglutination Inhibition (HI activity. Considering the safety, reliability and also economic potential of Pichia expression platform, our preliminary data indicates the feasibility of using this system as an alternative for large-scale production of recombinant influenza HA protein in the face of influenza pandemic threat.

  5. Fusion protein-based biofilm fabrication composed of recombinant azurin–myoglobin for dual-level biomemory application

    International Nuclear Information System (INIS)

    Lee, Taek; Chung, Yong-Ho; Yoon, Jinho; Min, Junhong; Choi, Jeong-Woo

    2014-01-01

    Graphical abstract: - Highlights: • We developed the fusion protein-based biofilm on the inorganic surface. • For making the fusion protein, the recombinant azurin and the myoglobin was conjugated by the native chemical ligation method. • The developed fusion protein shows unique electrochemical property. • The proposed fusion protein biofilm appears to be a good method for dual-level biomemory device. - Abstract: In the present study, a fusion protein-based biofilm composed of a recombinant azurin–myoglobin (Azu-Myo) has been developed and confirmed its original electrochemical property for dual-level biomemory device application. For this purpose, the azurin was modified with cysteine residues for direct immobilization and conjugation. Then, the recombinant azurin was conjugated with the myoglobin via a sulfo-SMCC bifunctional linker using the chemical ligation method (CLM). The SDS-PAGE and UV–vis spectroscopy were performed to examine the fusion protein conjugates. The prepared Azu-Myo fusion protein was self-assembled onto Au substrate for the biofilm fabrication. Then, the atomic force microscopy (AFM) was used to confirm the immobilization and the surface-enhanced Raman spectroscopy (SERS) was carried out to the surface analysis. Also, the cyclic voltammetry (CV) was carried out to observe an electrochemical property of fabricated biofilm. As a result, the two pair of redox potential values was obtained for dual-level biomemory device application. Then, the dual-level biomemory function was verified by the multi-potential chronoamperometry (MPCA). The results indicate a new fabrication method and material combination for advances in bioelectronic device development

  6. Vaccine potential of recombinant saposin-like protein 2 against Fasciolosis gigantica in mice.

    Science.gov (United States)

    Kueakhai, Pornanan; Changklungmoa, Narin; Riengrojpitak, Suda; Chaichanasak, Pannigan; Meemon, Krai; Chaithirayanon, Kulathida; Chantree, Pathanin; Sansri, Veerawat; Itagaki, Tadashi; Sobhon, Prasert

    2013-11-12

    Saposin-like protein 2 (SAP-2) is a protein that adult of Fasciola spp. use to lyse plasma membrane of red blood cells, so that their contents can be digested by proteases for the parasites' nutrients. Thus SAP-2 is a plausible target for vaccination against these parasites. Recombinant Fasciola gigantica saposin-like protein 2 (rFgSAP-2) was expressed in Escherichia coli BL21 (DE3). A vaccination was performed in ICR mice (n=10) by subcutaneous injection with 50μg of rFgSAP-2 combined with Freund's adjuvant. At 2 weeks after the second boost, mice were infected with 30 F. gigantica metacercariae by oral route. The percentages of protection of rFgSAP-2 vaccine against F. gigantica were estimated to be 76.4-78.5% when compared with non vaccinated-infected and adjuvant-infected controls, respectively. The antibodies in immune sera of vaccinated mice were shown by immuno-blotting to react with native FgSAP-2 in the extract of 2- and 4-week-old juvenile parasites. By determining the levels of IgG1 and IgG2a in the immune sera, which are indicative of Th2 and Th1 immune responses, it was found that both Th1 and Th2 humoral immune response were significantly increased in rFgSAP-2 immunized group compared with the control groups, with higher levels of Th2 (IgG1) than Th1 (IgG2a). The levels of serum aspartate aminotransferase (AST) and alanine transaminase (ALT) in rFgSAP-2-immunized group showed no significant difference from those of the non-immunized and infected group, indicating that early juvenile parasites induced liver parenchyma damage, even though the numbers of worm recoveries were significantly different. This study indicates that rFgSAP-2 has a high potential as a vaccine candidate against F. gigantica in mice, and this potential will be tested in larger economic animals. Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. Toxoplasma gondii: Biochemical and biophysical characterization of recombinant soluble dense granule proteins GRA2 and GRA6

    International Nuclear Information System (INIS)

    Bittame, Amina; Effantin, Grégory; Pètre, Graciane; Ruffiot, Pauline; Travier, Laetitia; Schoehn, Guy; Weissenhorn, Winfried; Cesbron-Delauw, Marie-France; Gagnon, Jean; Mercier, Corinne

    2015-01-01

    The most prominent structural feature of the parasitophorous vacuole (PV) in which the intracellular parasite Toxoplasma gondii proliferates is a membranous nanotubular network (MNN), which interconnects the parasites and the PV membrane. The MNN function remains unclear. The GRA2 and GRA6 proteins secreted from the parasite dense granules into the PV have been implicated in the MNN biogenesis. Amphipathic alpha-helices (AAHs) predicted in GRA2 and an alpha-helical hydrophobic domain predicted in GRA6 have been proposed to be responsible for their membrane association, thereby potentially molding the MMN in its structure. Here we report an analysis of the recombinant proteins (expressed in detergent-free conditions) by circular dichroism, which showed that full length GRA2 displays an alpha-helical secondary structure while recombinant GRA6 and GRA2 truncated of its AAHs are mainly random coiled. Dynamic light scattering and transmission electron microscopy showed that recombinant GRA6 and truncated GRA2 constitute a homogenous population of small particles (6–8 nm in diameter) while recombinant GRA2 corresponds to 2 populations of particles (∼8–15 nm and up to 40 nm in diameter, respectively). The unusual properties of GRA2 due to its AAHs are discussed. - Highlights: • Toxoplasma gondii: soluble GRA2 forms 2 populations of particles. • T. gondii: the dense granule protein GRA2 folds intrinsically as an alpha-helix. • T. gondii: monomeric soluble GRA6 forms particles of 6–8 nm in diameter. • T. gondii: monomeric soluble GRA6 is random coiled. • Unusual biophysical properties of the dense granule protein GRA2 from T. gondii

  8. Toxoplasma gondii: Biochemical and biophysical characterization of recombinant soluble dense granule proteins GRA2 and GRA6

    Energy Technology Data Exchange (ETDEWEB)

    Bittame, Amina [CNRS, UMR 5163, 38042 Grenoble (France); Université Grenoble Alpes, 38042 Grenoble (France); Effantin, Grégory [Université Grenoble Alpes, Institut de Biologie Structurale (IBS), 38044 Grenoble (France); CNRS, IBS, 38044 Grenoble (France); CEA, IBS, 38044 Grenoble (France); Unit for Virus Host-Cell Interactions (UVHCI), UMI 3265 (UJF-EMBL-CNRS), 38027 Grenoble (France); Pètre, Graciane; Ruffiot, Pauline; Travier, Laetitia [CNRS, UMR 5163, 38042 Grenoble (France); Université Grenoble Alpes, 38042 Grenoble (France); Schoehn, Guy; Weissenhorn, Winfried [Université Grenoble Alpes, Institut de Biologie Structurale (IBS), 38044 Grenoble (France); CNRS, IBS, 38044 Grenoble (France); CEA, IBS, 38044 Grenoble (France); Unit for Virus Host-Cell Interactions (UVHCI), UMI 3265 (UJF-EMBL-CNRS), 38027 Grenoble (France); Cesbron-Delauw, Marie-France; Gagnon, Jean [CNRS, UMR 5163, 38042 Grenoble (France); Université Grenoble Alpes, 38042 Grenoble (France); Mercier, Corinne, E-mail: corinne.mercier@ujf-grenoble.fr [CNRS, UMR 5163, 38042 Grenoble (France); Université Grenoble Alpes, 38042 Grenoble (France)

    2015-03-27

    The most prominent structural feature of the parasitophorous vacuole (PV) in which the intracellular parasite Toxoplasma gondii proliferates is a membranous nanotubular network (MNN), which interconnects the parasites and the PV membrane. The MNN function remains unclear. The GRA2 and GRA6 proteins secreted from the parasite dense granules into the PV have been implicated in the MNN biogenesis. Amphipathic alpha-helices (AAHs) predicted in GRA2 and an alpha-helical hydrophobic domain predicted in GRA6 have been proposed to be responsible for their membrane association, thereby potentially molding the MMN in its structure. Here we report an analysis of the recombinant proteins (expressed in detergent-free conditions) by circular dichroism, which showed that full length GRA2 displays an alpha-helical secondary structure while recombinant GRA6 and GRA2 truncated of its AAHs are mainly random coiled. Dynamic light scattering and transmission electron microscopy showed that recombinant GRA6 and truncated GRA2 constitute a homogenous population of small particles (6–8 nm in diameter) while recombinant GRA2 corresponds to 2 populations of particles (∼8–15 nm and up to 40 nm in diameter, respectively). The unusual properties of GRA2 due to its AAHs are discussed. - Highlights: • Toxoplasma gondii: soluble GRA2 forms 2 populations of particles. • T. gondii: the dense granule protein GRA2 folds intrinsically as an alpha-helix. • T. gondii: monomeric soluble GRA6 forms particles of 6–8 nm in diameter. • T. gondii: monomeric soluble GRA6 is random coiled. • Unusual biophysical properties of the dense granule protein GRA2 from T. gondii.

  9. The Effect of Recombinant Human MG53 Protein on Tourniquet-induced Ischemia Reperfusion Injury in Rat Muscle

    Science.gov (United States)

    2014-06-01

    blind to the treatment , and the prevalence of damaged fibers was quantitated from 10 10x images from each muscle . Approximately 800 fibers were counted...therapeutic cell membrane repair in treatment of muscular dystrophy . Sci Transl Med. 2012; 4(139):139ra185. 11. Weisleder N, Lin P, Zhao X, Orange M, Zhu H...The effect of recombinant human MG53 protein on tourniquet- induced ischemia reperfusion injury in rat muscle Benjamin T. Corona, Ph.D.1, Koyal Garg

  10. Cloning, expression, and purification of recombinant protein MPT-64 from a virulent strain of Mycobacterium bovis in a prokaryotic system.

    Science.gov (United States)

    Tashakkori, Maryam Mohammadi; Tebianian, Majid; Tabatabaei, Mohammad; Mosavari, Nader

    2016-12-01

    Tuberculosis (TB) is a zoonotic infectious disease common to humans and animals that is caused by the rod-shaped acid-fast bacterium Mycobacterium bovis. Rapid and sensitive detection of TB is promoted by specific antigens. Virulent strains of the TB complex from M. bovis contain 16 regions of difference (RD) in their genome that encode important proteins, including major protein of Mycobacterium Tuberculosis 64 (MBT-64, which is a primary immune-stimulating antigen encoded by RD-2. In this study, we cloned, expressed, and purified MPT-64 as a potent M. bovis antigen in a prokaryotic system for use in future diagnostic studies. The antigenic region of the Mpt64 gene was investigated by bioinformatics methods, cloned into the PQE-30 plasmid, and expressed in Escherichia coli M15 cells, followed by isopropyl β-d-1-thiogalactopyranoside induction. The expressed protein was analyzed sodium dodecyl sulfate polyacrylamide gel electrophoresis and purified using a nickel-affinity column. Biological activity was confirmed by western blot using specific antibodies. Our data verified the successful cloning of the Mpt64 gene (687-bp segment) via the expression vector and purification of recombinant MPT-64 as a 24-kDa protein. These results indicated successful expression and purification of recombinant MPT-64 protein in a prokaryotic system. This protein can be used for serological diagnosis, improved detection of pathogenicity and non-pathogenicity between infected cattle, and for verification of suspected cases of bovine TB. Copyright © 2016.

  11. Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers, Additives and Detergents Solutions for Recombinant Protein Production.

    Directory of Open Access Journals (Sweden)

    Raheem Ullah

    Full Text Available Proteases are widely used to remove affinity and solubility tags from recombinant proteins to avoid potential interference of these tags with the structure and function of the fusion partner. In recent years, great interest has been seen in use of the human rhinovirus 3C protease owing to its stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally employed for purification and stabilization of proteins, hence, necessitate their removal by tedious and time-consuming procedures before proteolysis can occur. To address this issue, we examined the effect of elution buffers used for common affinity based purifications, salt ions, stability/solubility and reducing agents, and detergents on the activity of the human rhinovirus 3C protease using three different fusion proteins at 4°C, a temperature of choice for purification of many proteins. The results show that the human rhinovirus 3C protease performs better at 4°C than the frequently used tobacco etch virus protease and its activity was insensitive to most of the experimental conditions tested. Though number of fusion proteins tested is limited, we expect that these finding will facilitate the use of the human rhinovirus 3C protease in recombinant protein production for pharmaceutical and biotechnological applications.

  12. Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers, Additives and Detergents Solutions for Recombinant Protein Production.

    Science.gov (United States)

    Ullah, Raheem; Shah, Majid Ali; Tufail, Soban; Ismat, Fouzia; Imran, Muhammad; Iqbal, Mazhar; Mirza, Osman; Rhaman, Moazur

    2016-01-01

    Proteases are widely used to remove affinity and solubility tags from recombinant proteins to avoid potential interference of these tags with the structure and function of the fusion partner. In recent years, great interest has been seen in use of the human rhinovirus 3C protease owing to its stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally employed for purification and stabilization of proteins, hence, necessitate their removal by tedious and time-consuming procedures before proteolysis can occur. To address this issue, we examined the effect of elution buffers used for common affinity based purifications, salt ions, stability/solubility and reducing agents, and detergents on the activity of the human rhinovirus 3C protease using three different fusion proteins at 4°C, a temperature of choice for purification of many proteins. The results show that the human rhinovirus 3C protease performs better at 4°C than the frequently used tobacco etch virus protease and its activity was insensitive to most of the experimental conditions tested. Though number of fusion proteins tested is limited, we expect that these finding will facilitate the use of the human rhinovirus 3C protease in recombinant protein production for pharmaceutical and biotechnological applications.

  13. A successful strategy for the recovering of active P21, an insoluble recombinant protein of Trypanosoma cruzi

    Science.gov (United States)

    Santos, Marlus Alves Dos; Teixeira, Francesco Brugnera; Moreira, Heline Hellen Teixeira; Rodrigues, Adele Aud; Machado, Fabrício Castro; Clemente, Tatiana Mordente; Brigido, Paula Cristina; Silva, Rebecca Tavares E.; Purcino, Cecílio; Gomes, Rafael Gonçalves Barbosa; Bahia, Diana; Mortara, Renato Arruda; Munte, Claudia Elisabeth; Horjales, Eduardo; da Silva, Claudio Vieira

    2014-03-01

    Structural studies of proteins normally require large quantities of pure material that can only be obtained through heterologous expression systems and recombinant technique. In these procedures, large amounts of expressed protein are often found in the insoluble fraction, making protein purification from the soluble fraction inefficient, laborious, and costly. Usually, protein refolding is avoided due to a lack of experimental assays that can validate correct folding and that can compare the conformational population to that of the soluble fraction. Herein, we propose a validation method using simple and rapid 1D 1H nuclear magnetic resonance (NMR) spectra that can efficiently compare protein samples, including individual information of the environment of each proton in the structure.

  14. Expression and Evaluation of Recombinant Plasmodium knowlesi Merozoite Surface Protein-3 (MSP-3 for Detection of Human Malaria.

    Directory of Open Access Journals (Sweden)

    Jeremy Ryan De Silva

    Full Text Available Malaria remains a major health threat in many parts of the globe and causes high mortality and morbidity with 214 million cases of malaria occurring globally in 2015. Recent studies have outlined potential diagnostic markers and vaccine candidates one of which is the merozoite surface protein (MSP-3. In this study, novel recombinant Plasmodium knowlesi MSP-3 was cloned, expressed and purified in an Escherichia coli system. Subsequently, the recombinant protein was evaluated for its sensitivity and specificity. The recombinant pkMSP-3 protein reacted with sera from patients with P. knowlesi infection in both Western blot (61% and ELISA (100%. Specificity-wise, pkMSP-3 did not react with healthy donor sera in either assay and only reacted with a few non-malarial parasitic patient sera in the ELISA assay (3 of 49. In conclusion, sensitivity and specificity of pkMSP-3 was found to be high in the ELISA and Western Blot assay and thus utilising both assays in tandem would provide the best sero-diagnostic result for P. knowlesi infection.

  15. Two potential recombinant rabies vaccines expressing canine parvovirus virion protein 2 induce immunogenicity to canine parvovirus and rabies virus.

    Science.gov (United States)

    Luo, Jun; Shi, Hehe; Tan, Yeping; Niu, Xuefeng; Long, Teng; Zhao, Jing; Tian, Qin; Wang, Yifei; Chen, Hao; Guo, Xiaofeng

    2016-08-17

    Both rabies virus (RABV) and canine parvovirus (CPV) cause lethal diseases in dogs. In this study, both high egg passage Flury (HEP-Flury) strains of RABV and recombinant RABV carrying double RABV glycoprotein (G) gene were used to express the CPV virion protein 2 (VP2) gene, and were designated rHEP-VP2 and, rHEP-dG-VP2 respectively. The two recombinant RABVs maintained optimal virus titration according to their viral growth kinetics assay compared with the parental strain HEP-Flury. Western blotting indicated that G protein and VP2 were expressed in vitro. The expression of VP2 in Crandell feline kidney cells post-infection by rHEP-VP2 and rHEP-dG-VP2 was confirmed by indirect immunofluorescence assay with antibody against VP2. Immunogenicity of recombinant rabies viruses was tested in Kunming mice. Both rHEP-VP2 and rHEP-dG-VP2 induced high levels of rabies antibody compared with HEP-Flury. Mice immunized with rHEP-VP2 and rHEP-dG-VP2 both had a high level of antibodies against VP2, which can protect against CPV infection. A challenge experiment indicated that more than 80% mice immunized with recombinant RABVs survived after infection of challenge virus standard 24 (CVS-24). Together, this study showed that recombinant RABVs expressing VP2 induced protective immune responses to RABV and CPV. Therefore, rHEP-VP2 and rHEP-dG-VP2 might be potential combined vaccines for RABV and CPV. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. [Construction and prokaryotic expression of recombinant gene EGFRvIII HBcAg and immunogenicity analysis of the fusion protein].

    Science.gov (United States)

    Duan, Xiao-yi; Wang, Jian-sheng; Guo, You-min; Han, Jun-li; Wang, Quan-ying; Yang, Guang-xiao

    2007-01-01

    To construct recombinant prokaryotic expression plasmid pET28a(+)/c-PEP-3-c and evaluate the immunogenicity of the fusion protein. cDNA fragment encoding PEP-3 was obtained from pGEM-T Easy/PEP-3 and inserted into recombinant plasmid pGEMEX/HBcAg. Then it was subcloned in prokaryotic expression vector and transformed into E.coli BL21(DE3). The fusion protein was expressed by inducing IPTG and purified by Ni(2+)-NTA affinity chromatography. BALB/c mice were immunized with fusion protein and the antibody titre was determined by indirect ELISA. The recombinant gene was confirmed to be correct by restriction enzyme digestion and DNA sequencing. After prokaryotic expression, fusion protein existed in sediment and accounted for 56% of all bacterial lysate. The purified product accounted for 92% of all protein and its concentration was 8 g/L. The antibody titre in blood serum reached 1:16 000 after the fourth immunization and reached 1:2.56x10(5) after the sixth immunization. The titre of anti-PEP-3 antibody reached 1:1.28x10(5) and the titre of anti-HBcAg antibody was less than 1:4x10(3). Fusion gene PEP-3-HBcAg is highly expressed in E.coli BL21. The expressed fusion protein can induce neutralizing antibody with high titer and specificity, which lays a foundation for the study of genetically engineering vaccine for malignant tumors with the high expression of EGFRvIII.

  17. Effects of Recombinant Human Bone Morphogenetic Protein-2 on Vertical Bone Augmentation in a Canine Model.

    Science.gov (United States)

    Hsu, Yung-Ting; Al-Hezaimi, Khalid; Galindo-Moreno, Pablo; O'Valle, Francisco; Al-Rasheed, Abdulaziz; Wang, Hom-Lay

    2017-09-01

    Vertical bone augmentation (VBA) remains unpredictable and challenging for most clinicians. This study aims to compare hard tissue outcomes of VBA, with and without recombinant human bone morphogenetic protein (rhBMP)-2, under space-making titanium mesh in a canine model. Eleven male beagle dogs were used in the study. Experimental ridge defects were created to form atrophic ridges. VBA was performed via guided bone regeneration using titanium mesh and allografts. In experimental hemimandibles, rhBMP-2/absorbable collagen sponge was well mixed with allografts prior to procedures, whereas a control buffer was applied within controls. Dogs were euthanized after a 4-month healing period. Clinical and radiographic examinations were performed to assess ridge dimensional changes. In addition, specimens were used for microcomputed tomography (micro-CT) assessment and histologic analysis. Membrane exposure was found on five of 11 (45.5%) rhBMP-2-treated sites, whereas it was found on nine of 11 (81.8%) non-rhBMP-2-treated sites. Within 4 months of healing, rhBMP-2-treated sites showed better radiographic bone density, greater defect fill, and significantly more bone gain in ridge height (P 0.05). Under light microscope, predominant lamellar patterns were found in the specimen obtained from rhBMP-2 sites. With inherent limitations of the canine model and the concern of such a demanding surgical technique, current findings suggest that the presence of rhBMP-2 in a composite graft allows an increase of vertical gain, with formation of ectopic bone over the titanium mesh in comparison with non-rhBMP-2 sites.

  18. Effect of rTMP-GH recombinant fusion protein on thrombocytopoiesis in irradiation injured mice

    International Nuclear Information System (INIS)

    Xu Yang; Wang Junping; Chen Fang; Shen Mingqiang; Chen Mo; Wang Song; Ran Xinze; Su Yongping; Kai Li

    2009-01-01

    Objective: To investigate the in vivo effects of rTMP-GH recombinant fusion protein on thrombocytopoiesis in mice with thrombopenia inflicted by irradiation. Methods: BALB/C mice weighting around 20 g were irradiated with 5 Gy of 60 Co γ-ray irradiation to generate thrombopenia. The irradiation injured mice were injected with rTMP-GH or rhGH subcutaneously at the dose of 200 (μg ·kg -1 · d -1 for 7 days. From the 6 th day, the platelets in blood samples from vena caudalis were counted routinely, and the pathological changes of bone marrow were determined by morphological observation. Results: From the 10 th day, the levels of blood platelet in rTMP-GH treated mice were much higher than those of rhGH treatment group and normal saline (NS) control group, especially at the nadir (P < 0.01). On the 22 nd day, the platelet count has recovered up to 80% of normal level in rTMP-GH treatment group, while it has just recovered up to 30% in NS control group. Morphological observation showed that there was obvious reconstruction of bone marrow in mice treated with rTMP-GH, compared with NS group.The number of megarkaryoblasts and megakaryocytes in bone marrow of rTMP-GH treated mice (3.07 ± 0.32) was much higher than those of rhGH treatment group (2.20 ± 0.22, P < 0.05) and NS control group (0.87 ± 0.19, P <0.01). Conclusions: rTMP-GH has potent effects on the recovery of blood platelet by promoting megarkaryocytopoiesis in irradiation injuried mice. (authors)

  19. Locally limited inhibition of bone resorption and orthodontic relapse by recombinant osteoprotegerin protein.

    Science.gov (United States)

    Schneider, D A; Smith, S M; Campbell, C; Hayami, T; Kapila, S; Hatch, N E

    2015-04-01

    To determine minimal dose levels required for local inhibition of orthodontic relapse by recombinant OPG protein (OPG-Fc), while also determining effects of injected OPG-Fc on alveolar bone and long bone. The Department of Orthodontics and Pediatric Dentistry at the University of Michigan. Eighteen male Sprague Dawley rats. Maxillary molars were moved with nickel-titanium springs and then allowed to relapse in Sprague Dawley rats. Upon appliance removal, animals were injected with a single dose of 1.0 mg/kg OPG-Fc, 0.1 mg/kg OPG-Fc, or phosphate-buffered saline (vehicle) just distal to the molar teeth. Tooth movement measurements were made from stone casts, which were scanned and digitally measured. Alveolar tissues were examined by histology. Micro-computed tomography was used to quantify changes in alveolar and femur bone. Local injection of OPG-Fc inhibited molar but not incisor relapse, when compared to vehicle-injected animals. No significant differences in alveolar or femur bone were seen between the three treatment groups after 24 days of relapse. Our results demonstrate that a single local injection of OPG-Fc effectively inhibits orthodontic relapse, with minimal systemic bone metabolic effects. Our results also show that a single injection of OPG-Fc will influence tooth movement only in teeth close to the injection site. These findings indicate that OPG-Fc has potential as a safe and effective pharmacological means to locally control osteoclasts, for uses such as maintaining anchorage during orthodontic tooth movement and preventing orthodontic relapse in humans. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  20. A dimer of the lymphoid protein RAG1 recognizes the recombination signal sequence and the complex stably incorporates the high mobility group protein HMG2.

    Science.gov (United States)

    Rodgers, K K; Villey, I J; Ptaszek, L; Corbett, E; Schatz, D G; Coleman, J E

    1999-07-15

    RAG1 and RAG2 are the two lymphoid-specific proteins required for the cleavage of DNA sequences known as the recombination signal sequences (RSSs) flanking V, D or J regions of the antigen-binding genes. Previous studies have shown that RAG1 alone is capable of binding to the RSS, whereas RAG2 only binds as a RAG1/RAG2 complex. We have expressed recombinant core RAG1 (amino acids 384-1008) in Escherichia coli and demonstrated catalytic activity when combined with RAG2. This protein was then used to determine its oligomeric forms and the dissociation constant of binding to the RSS. Electrophoretic mobility shift assays show that up to three oligomeric complexes of core RAG1 form with a single RSS. Core RAG1 was found to exist as a dimer both when free in solution and as the minimal species bound to the RSS. Competition assays show that RAG1 recognizes both the conserved nonamer and heptamer sequences of the RSS. Zinc analysis shows the core to contain two zinc ions. The purified RAG1 protein overexpressed in E.coli exhibited the expected cleavage activity when combined with RAG2 purified from transfected 293T cells. The high mobility group protein HMG2 is stably incorporated into the recombinant RAG1/RSS complex and can increase the affinity of RAG1 for the RSS in the absence of RAG2.

  1. Production of Recombinant Protein Pap31 and Its Application for the Diagnosis of Bartonella bacilliformis Infection

    National Research Council Canada - National Science Library

    Taye, A; Chen, H; Duncan, K; Zhang, Z; Hendrix, L; Gonzalez, J; Ching, W

    2005-01-01

    .... The objective of this research is to develop a rapid serologic diagnostic test using recombinant antigens to overcome the limitations of the current standard IFA technique for laboratory diagnosis...

  2. Recombinant Protein Truncation Strategy for Inducing Bactericidal Antibodies to the Macrophage Infectivity Potentiator Protein of Neisseria meningitidis and Circumventing Potential Cross-Reactivity with Human FK506-Binding Proteins

    OpenAIRE

    Bielecka, Magdalena K.; Devos, Nathalie; Gilbert, Mélanie; Hung, Miao-Chiu; Weynants, Vincent; Heckels, John E.; Christodoulides, Myron

    2014-01-01

    A recombinant macrophage infectivity potentiator (rMIP) protein of Neisseria meningitidis induces significant serum bactericidal antibody production in mice and is a candidate meningococcal vaccine antigen. However, bioinformatics analysis of MIP showed some amino acid sequence similarity to human FK506-binding proteins (FKBPs) in residues 166 to 252 located in the globular domain of the protein. To circumvent the potential concern over generating antibodies that could recognize human protein...

  3. An efficient tag derived from the common epitope of tospoviral NSs proteins for monitoring recombinant proteins expressed in both bacterial and plant systems.

    Science.gov (United States)

    Cheng, Hao-Wen; Chen, Kuan-Chun; Raja, Joseph A J; Li, Jian-Xian; Yeh, Shyi-Dong

    2013-04-15

    NSscon (23 aa), a common epitope in the gene silencing suppressor NSs proteins of the members of the Watermelon silver mottle virus (WSMoV) serogroup, was previously identified. In this investigation, we expressed different green fluorescent protein (GFP)-fused deletions of NSscon in bacteria and reacted with NSscon monoclonal antibody (MAb). Our results indicated that the core 9 amino acids, "(109)KFTMHNQIF(117)", denoted as "nss", retain the reactivity of NSscon. In bacterial pET system, four different recombinant proteins labeled with nss, either at N- or C-extremes, were readily detectable without position effects, with sensitivity superior to that for the polyhistidine-tag. When the nss-tagged Zucchini yellow mosaic virus (ZYMV) helper component-protease (HC-Pro) and WSMoV nucleocapsid protein were transiently expressed by agroinfiltration in tobacco, they were readily detectable and the tag's possible efficacy for gene silencing suppression was not noticed. Co-immunoprecipitation of nss-tagged and non-tagged proteins expressed from bacteria confirmed the interaction of potyviral HC-Pro and coat protein. Thus, we conclude that this novel nss sequence is highly valuable for tagging recombinant proteins in both bacterial and plant expression systems. Copyright © 2013 Elsevier B.V. All rights reserved.

  4. A G-protein-coupled chemokine receptor: A putative insertion site for a multi-pathogen recombinant capripoxvirus vaccine strategy.

    Science.gov (United States)

    Cêtre-Sossah, Catherine; Dickmu, Simon; Kwiatek, Olivier; Albina, Emmanuel

    2017-09-01

    Capripoxviruses (CaPVs) have been shown to be ideal viral vectors for the development of recombinant multivalent vaccines to enable delivery of immunogenic genes from ruminant pathogens. So far, the viral thymidine kinase (TK) gene is the only gene used to generate recombinants. A putative non-essential gene encoding a G-protein-coupled chemokine receptor subfamily homologue (GPCR) was targeted as an additional insertion site. Peste des petits ruminants (PPR) was chosen as a disease model. A new recombinant CaPV expressing the viral attachment hemagglutinin (H) of the PPR virus (PPRV) in the GPCR insertion site (rKS1-HPPR-GPCR) was generated in the backbone North African isolate KS1 strain of lumpy skin disease virus (LSDV). Comparison with the recombinant CaPV expressing the H of PPRV in the TK gene (rKS1-HPPR-TK) shown to induce protection against both PPR and LSD in both sheep and goats was assessed. The suitability of the GPCR gene to be a putative additional insertion site in the CaPV genome is evaluated and discussed. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Construction and characterisation of a recombinant fowlpox virus that expresses the human papilloma virus L1 protein

    Directory of Open Access Journals (Sweden)

    Zanotto Carlo

    2011-11-01

    Full Text Available Abstract Background Human papilloma virus (HPV-16 is the most prevalent high-risk mucosal genotype. Virus-like-particle (VLP-based immunogens developed recently have proven to be successful as prophylactic HPV vaccines, but are still too expensive for developing countries. Although vaccinia viruses expressing the HPV-16 L1 protein (HPV-L1 have been studied, fowlpox-based recombinants represent efficient and safer vectors for immunocompromised hosts due to their ability to elicit a complete immune response and their natural host-range restriction to avian species. Methods A new fowlpox virus recombinant encoding HPV-L1 (FPL1 was engineered and evaluated for the correct expression of HPV-L1 in vitro, using RT-PCR, immunoprecipitation, Western blotting, electron microscopy, immunofluorescence, and real-time PCR assays. Results The FPL1 recombinant correctly expresses HPV-L1 in mammalian cells, which are non-permissive for the replication of this vector. Conclusion This FPL1 recombinant represents an appropriate immunogen for expression of HPV-L1 in human cells. The final aim is to develop a safe, immunogenic, and less expensive prophylactic vaccine against HPV.

  6. Evaluation of recombinant activated protein C for severe sepsis at a tertiary academic medical center

    Directory of Open Access Journals (Sweden)

    Anger KE

    2013-06-01

    Full Text Available Kevin E Anger,1 Jeremy R DeGrado,1 Bonnie C Greenwood,1 Steven A Cohen,2 Paul M Szumita1 1Department of Pharmacy, Brigham and Women’s Hospital, Boston, MA, USA; 2Department of Family Medicine and Population Health, Division of Epidemiology, Virginia Commonwealth University, Richmond, VA, USA Purpose: Early clinical trials of recombinant human activated protein C (rhAPC for severe sepsis excluded patients at high risk of bleeding. Recent literature suggests bleeding rates are higher in clinical practice and may be associated with worsened outcomes. Our objective was to evaluate baseline demographics; incidence, and risk factors for major bleeding; and mortality of patients receiving rhAPC for severe sepsis at our institution. Methods: A retrospective study was performed for all patients receiving rhAPC for treatment of severe sepsis at a tertiary academic medical center from January 2002 to June 2009. Demographic information, clinical variables, intensive care unit, and hospital outcomes were recorded. Results: Of the 156 patients that received rhAPC, 54 (34.6% did not meet institutional criteria for safe use at baseline due to bleeding precaution or contraindication. Twenty-three (14.7% patients experienced a major bleeding event. Multivariate analysis demonstrated baseline International Normalized Ratio ≥2.5 (odds ratio [OR] 3.68, 95% confidence interval [CI]: 1.28–10.56; P = 0.03 and platelet count ≤100 × 103/mm3 (OR 2.86, 95% CI: 1.07–7.67; P = 0.01 as significant predictors of a major bleed. Overall hospital mortality was 57.7%. Multivariate analysis demonstrated the presence of ≥3 organ dysfunctions (OR 2.46, 95% CI: 1.19–5.09; P < 0.05 and medical intensive care unit admission (OR 1.99, 95% CI: 1.00–3.98; P = 0.05 were independent variables associated with hospital mortality. Conclusion: Patients receiving rhAPC at our institution had higher APACHE II scores, mortality, and major bleeding events than published

  7. Oral Administration of Recombinant Saccharomyces boulardii Expressing Ovalbumin-CPE Fusion Protein Induces Antibody Response in Mice.

    Science.gov (United States)

    Bagherpour, Ghasem; Ghasemi, Hosnie; Zand, Bahare; Zarei, Najmeh; Roohvand, Farzin; Ardakani, Esmat M; Azizi, Mohammad; Khalaj, Vahid

    2018-01-01

    Saccharomyces boulardii , a subspecies of Saccharomyces cerevisiae , is a well-known eukaryotic probiotic with many benefits for human health. In the present study, a recombinant strain of S. boulardii was prepared to use as a potential oral vaccine delivery vehicle. In this sense, a ura3 auxotroph strain of S. boulardii CNCM I-745 (known as S. cerevisiae HANSEN CBS 5926, Yomogi ® ) was generated using CRISPR/Cas9 methodology. Then a gene construct encoding a highly immunogenic protein, ovalbumin (OVA), was prepared and transformed into the ura3 - S. boulardii . To facilitate the transport of the recombinant immunogen across the intestinal barrier, a claudin-targeting sequence from Clostridium perfringens enterotoxin (CPE) was added to the C-terminus of the expression cassette. The recombinant S. boulardii strain expressing the OVA-CPE fusion protein was then administered orally to a group of mice, and serum IgG and fecal IgA levels were evaluated by ELISA. Our results demonstrated that anti-OVA IgG in serum significantly increased in test group ( P boulardii or PBS), and the fecal IgA titer was significantly higher in test group ( P boulardii strain expressing the similar construct lacking C-terminal CPE was also administered orally. The result showed an increased level of serum IgG in group receiving yeasts expressing the CPE negative construct compared to control groups; however, the fecal IgA levels did not increase significantly. In conclusion, our findings indicated that the yeast S. boulardii , as a delivery vehicle with possible immunomodulatory effects, and c-CPE, as a targeting tag, synergistically assist to stimulate systemic and local immunity. This proposed recombinant S. boulardii system might be useful in the expression of other antigenic peptides, making it as a promising tool for oral delivery of vaccines or therapeutic proteins.

  8. Identification, characterization, and synthesis of peptide epitopes and a recombinant six-epitope protein for Trichomonas vaginalis serodiagnosis

    Directory of Open Access Journals (Sweden)

    Alderete JF

    2013-08-01

    Full Text Available J F Alderete, Calvin J NeaceSchool of Molecular Biosciences, College of Veterinary Medicine, Washington State University, Pullman, WA, USAAbstract: There is a need for a rapid, accurate serodiagnostic test useful for both women and men infected by Trichomonas vaginalis, which causes the number one sexually transmitted infection (STI. Women and men exposed to T. vaginalis make serum antibody to fructose-1,6-bisphosphate aldolase (ALD, α-enolase (ENO, and glyceraldehyde-3-phosphate dehydrogenase (GAP. We identified, by epitope mapping, the common and distinct epitopes of each protein detected by the sera of women patients with trichomonosis and by the sera of men highly seropositive to the immunogenic protein α-actinin (positive control sera. We analyzed the amino acid sequences to determine the extent of identity of the epitopes of each protein with other proteins in the databanks. This approach identified epitopes unique to T. vaginalis, indicating these peptide-epitopes as possible targets for a serodiagnostic test. Individual or combinations of 15-mer peptide epitopes with low to no identity with other proteins were reactive with positive control sera from both women and men but were unreactive with negative control sera. These analyses permitted the synthesis of a recombinant His6 fusion protein of 111 amino acids with an Mr of ~13.4 kDa, which consisted of 15-mer peptides of two distinct epitopes each for ALD, ENO, and GAP. This recombinant protein was purified by affinity chromatography. This composite protein was detected by enzyme-linked immunosorbent assay (ELISA, dot blots, and immunoblots, using positive control sera from women and men. These data indicate that it is possible to identify epitopes and that either singly, in combination, or as a composite protein represent targets for a point-of-care serodiagnostic test for T. vaginalis.Keywords: diagnostics, point-of-care, targets, trichomonosis

  9. Cloning, Expression and Purification of the Recombinant HIV-1 Tat-Nef Fusion Protein in Prokaryotic Expression System

    Directory of Open Access Journals (Sweden)

    Somayeh Kadkhodayan

    2016-07-01

    Full Text Available Abstract Background: Nef is one of the HIV-1 critical proteins, because it is essential for viral replication and AIDS disease progression and induction of immune response against it can partially inhibit viral infection. Moreover, a domain of the HIV-1 Trans-Activator of Transcription (Tat, 48-60 aa could act as a cell penetrating peptide (CPP. In current study, cloning and expression of Tat-Nef fusion protein was performed in E. coli for the first time. The protein expression was confirmed by western blot analysis and was purified using reverse staining method. Materials and Methods: In this experimental study, primarily, cloning of Tat-Nef fusion gene was done in pGEX6p2 expression vector. Then, the expression of Tat-Nef recombinat protein in E.coli BL21 (DE3 strain was performed by using IPTG inducer. The protein expression was confirmed by SDS-PAGE and western blotting using anti-Nef monoclonal antibody. Then, the recombinant fusion protein was purified from gel using reverse staining method. Results: The results of PCR analysis and enzyme digestion showed a clear band of ~ 726 bp in agarose gel indicating the correct Tat-Nef fusion cloning in pGEX6p2 prokaryotic expression vector. In addition, a 54 kDa band of Tat-Nef on SDS-PAGE revealed Tat-Nef protein expression that western blot analysis using anti-Nef monoclonal antibody confirmed it. Conclusion: The purified Tat-Nef recombinant fusion protein will be used as an antigen for protein vaccine design against HIV infection.

  10. Production of a Recombinant Dengue Virus 2 NS5 Protein and Potential Use as a Vaccine Antigen.

    Science.gov (United States)

    Alves, Rúbens Prince Dos Santos; Pereira, Lennon Ramos; Fabris, Denicar Lina Nascimento; Salvador, Felipe Scassi; Santos, Robert Andreata; Zanotto, Paolo Marinho de Andrade; Romano, Camila Malta; Amorim, Jaime Henrique; Ferreira, Luís Carlos de Souza

    2016-06-01

    Dengue fever is caused by any of the four known dengue virus serotypes (DENV1 to DENV4) that affect millions of people worldwide, causing a significant number of deaths. There are vaccines based on chimeric viruses, but they still are not in clinical use. Anti-DENV vaccine strategies based on nonstructural proteins are promising alternatives to those based on whole virus or structural proteins. The DENV nonstructural protein 5 (NS5) is the main target of anti-DENV T cell-based immune responses in humans. In this study, we purified a soluble recombinant form of DENV2 NS5 expressed in Escherichia coli at large amounts and high purity after optimization of expression conditions and purification steps. The purified DENV2 NS5 was recognized by serum from DENV1-, DENV2-, DENV3-, or DENV4-infected patients in an epitope-conformation-dependent manner. In addition, immunization of BALB/c mice with NS5 induced high levels of NS5-specific antibodies and expansion of gamma interferon- and tumor necrosis factor alpha-producing T cells. Moreover, mice immunized with purified NS5 were partially protected from lethal challenges with the DENV2 NGC strain and with a clinical isolate (JHA1). These results indicate that the recombinant NS5 protein preserves immunological determinants of the native protein and is a promising vaccine antigen capable of inducing protective immune responses. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  11. Purification of recombinant budgerigar fledgling disease virus VP1 capsid protein and its ability for in vitro capsid assembly

    Science.gov (United States)

    Rodgers, R. E.; Chang, D.; Cai, X.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    A recombinant system for the major capsid VP1 protein of budgerigar fledgling disease virus has been established. The VP1 gene was inserted into a truncated form of the pFlag-1 vector and expressed in Escherichia coli. The budgerigar fledgling disease virus VP1 protein was purified to near homogeneity by immunoaffinity chromatography. Fractions containing highly purified VP1 were pooled and found to constitute 3.3% of the original E. coli-expressed VP1 protein. Electron microscopy revealed that the VP1 protein was isolated as pentameric capsomeres. Electron microscopy also revealed that capsid-like particles were formed in vitro from purified VP1 capsomeres with the addition of Ca2+ ions and the removal of chelating and reducing agents.

  12. Recombinant rabies virus expressing the H protein of canine distemper virus protects dogs from the lethal distemper challenge.

    Science.gov (United States)

    Wang, Feng-Xue; Zhang, Shu-Qin; Zhu, Hong-Wei; Yang, Yong; Sun, Na; Tan, Bin; Li, Zhen-Guang; Cheng, Shi-Peng; Fu, Zhen F; Wen, Yong-Jun

    2014-12-05

    The rabies virus (RV) vector LBNSE expressing foreign antigens have shown considerable promise as vaccines against viral and bacteria diseases, which is effective and safe. We produced a new RV-based vaccine vehicle expressing 1.824 kb hemagglutinin (H) gene of the canine distemper virus (CDV) by reverse genetics technology. The recombinant virus LBNSE-CDV-H retained growth properties similar to those of vector LBNSE both in BSR and mNA cell culture. The H gene of CDV was expressed and detected by immunostaining. To compare the immunogenicity of LBNSE-CDV-H, dogs were immunized with each of these recombinant viruses by intramuscular (i.m.). The dogs were bled at third weeks after the immunization for the measurement of virus neutralizing antibody (VNA) and then challenged with virulent virus (ZJ 7) at fourth weeks. The parent virus (LBNSE) without expression of any foreign molecules was included for comparison. Dogs inoculated with LBNSE-CDV-H showed no any signs of disease and exhibited seroconversion against both RV and CDV H protein. The LBNSE-CDV-H did not cause disease in dogs and conferred protection from challenge with a lethal wild type CDV strain, demonstrating its potential value for wildlife conservation efforts. Together, these studies suggest that recombinant RV expressing H protein from CDV stimulated high levels of adaptive immune responses (VNA), and protected all dogs challenge infection. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. EFFICIENCY OF RECOMBINANT TNF-BINDING PROTEIN FROM VARIOLA VIRUS IN A MODEL OF COLLAGEN-INDUCED ARTHRITIS

    Directory of Open Access Journals (Sweden)

    D. D. Tsyrendorzhiev

    2013-01-01

    Full Text Available Abstract. This paper presents the results of the research on the effectiveness of recombinant TNF-binding protein of variola virus (VARV-CrmB in a model of collagen-induced arthritis (CIA in mice (CBAxC57Bl6 F1. The introduction of VARV-CrmB and polyclonal antibody to recombinant mouse TNF (poly-AbMuTNF led to an improvement of clinical manifestations of CIA by reducing the swelling and increasing the mobility of mice limbs. The introduction of VARV-CrmB and poly-AbMuTNF reduced the number of neutrophilic granulocytes and granulocytic precursors. The introduction of VARV-CrmB and poly-AbMuTNF into mice decreased collagenolysis in the blood serum and the content of glycosaminoglycans at the early stages of experimentation. Treatment with VARV-CrmB and poly-AbMuTNF of mice with CIA significantly decreased the chemiluminescence response of blood leukocytes. VARV-CrmB exerted more pronounced inhibitory effect on the production of reactive oxygen metabolites by blood leukocytes of mice with CIA than poly-AbMuTNF. Improvement of clinical condition of the mice with CIA has a more prolonged effect following introduction of the VARV-CrmB than after injection of poly-AbMuTNF. The results suggest the recombinant viral protein VARVCrmB to be a new potential TNF-antagonist.

  14. Recombinant proteins of Zaire ebolavirus induce potent humoral and cellular immune responses and protect against live virus infection in mice.

    Science.gov (United States)

    Lehrer, Axel T; Wong, Teri-Ann S; Lieberman, Michael M; Humphreys, Tom; Clements, David E; Bakken, Russell R; Hart, Mary Kate; Pratt, William D; Dye, John M

    2018-05-24

    Infections with filoviruses in humans are highly virulent, causing hemorrhagic fevers which result in up to 90% mortality. In addition to natural infections, the ability to use these viruses as bioterrorist weapons is of significant concern. Currently, there are no licensed vaccines or therapeutics available to combat these infections. The pathogenesis of disease involves the dysregulation of the host's immune system, which results in impairment of the innate and adaptive immune responses, with subsequent development of lymphopenia, thrombocytopenia, hemorrhage, and death. Questions remain with regard to the few survivors of infection, who manage to mount an effective adaptive immune response. These questions concern the humoral and cellular components of this response, and whether such a response can be elicited by an appropriate prophylactic vaccine. The data reported herein describe the production and evaluation of a recombinant subunit Ebola virus vaccine candidate consisting of insect cell expressed Zaire ebolavirus (EBOV) surface glycoprotein (GP) and the matrix proteins VP24 and VP40. The recombinant subunit proteins are shown to be highly immunogenic in mice, yielding both humoral and cellular responses, as well as highly efficacious, providing up to 100% protection against a lethal challenge with live virus. These results demonstrate proof of concept for such a recombinant non-replicating vaccine candidate in the mouse model of EBOV which helps to elucidate immune correlates of protection and warrants further development. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. 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.

  16. Immune responses induced by recombinant Bacillus subtilis expressing the spike protein of transmissible gastroenteritis virus in pigs.

    Science.gov (United States)

    Mou, Chunxiao; Zhu, Liqi; Xing, Xianping; Lin, Jian; Yang, Qian

    2016-07-01

    Transmissible gastroenteritis (TGE) causes severe diarrhea in suckling piglets, results in enormous economic loss in swine-producing areas of the world. To develop an effective, safe, and convenient vaccine for the prevention of TGE, we have constructed a recombinant Bacillus subtilis strain (B. subtilis CotGSG) displaying the transmissible gastroenteritis virus (TGEV) spike (S) protein and discussed its immune function to intestinal submucosal dendritic cells (DCs). Our results showed that the recombinant B. subtilis had the ability to recruit more DCs to sample B. subtilis CotGSG, migrate to MLNs, and induce immune responses. Immunized piglets with B. subtilis CotGSG could significantly elevate the specific SIgA titers in feces, IgG titers and neutralizing antibodies in serum. Collectively, our results suggested that recombinant B. subtilis CotGSG expressing the TGEV S protein could effectively induce immune responses via DCs, and provided a perspective on potential novel strategy and approach that may be applicable to the development of the next generation of TGEV vaccines. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Single step purification of recombinant proteins using the metal ion-inducible autocleavage (MIIA) domain as linker for tag removal.

    Science.gov (United States)

    Ibe, Susan; Schirrmeister, Jana; Zehner, Susanne

    2015-08-20

    For fast and easy purification, proteins are typically fused with an affinity tag, which often needs to be removed after purification. Here, we present a method for the removal of the affinity tag from the target protein in a single step protocol. The protein VIC_001052 of the coral pathogen Vibrio coralliilyticus ATCC BAA-450 contains a metal ion-inducible autocatalytic cleavage (MIIA) domain. Its coding sequence was inserted into an expression vector for the production of recombinant fusion proteins. Following, the target proteins MalE and mCherry were produced as MIIA-Strep fusion proteins in Escherichia coli. The target proteins could be separated from the MIIA-Strep part simply by the addition of calcium or manganese(II) ions within minutes. The cleavage is not affected in the pH range from 5.0 to 9.0 or at low temperatures (6°C). Autocleavage was also observed with immobilized protein on an affinity column. The protein yield was similar to that achieved with a conventional purification protocol. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Expression of Hepatitis C Virus Core and E2 antigenic recombinant proteins and their use for development of diagnostic assays.

    Science.gov (United States)

    Ali, Amjad; Nisar, Muhammad; Idrees, Muhammad; Rafique, Shazia; Iqbal, Muhammad

    2015-05-01

    Early diagnosis of HCV infection is based on detection of antibodies against HCV proteins using recombinant viral antigens. The present study was designed to select, clone and express the antigenic regions of Core and E2 genes from local HCV-3a genotype and to utilize the antigenic recombinant proteins (Core & E2) to develop highly sensitive, specific and economical diagnostic assays for detection of HCV infection. The antigenic sites were determined within Core and E2 genes and were then cloned in pET-28a expression vector. The right orientation of the desired inserted fragments of Core and E2 were confirmed via sequencing prior to expression and were then transformed in BL21 (DE3) pLysS strains of E. coli and induced with 0.5mM Isopropyl-b-D-thiogalactopyranoside (IPTG) for the production of antigenic recombinant proteins. The produced truncated antigens were then purified by Nickel affinity chromatography and were confirmed by western blotting, immunoblotting and enzyme-linked immunosorbent assay (ELISA). The expressed Core and E2 recombinant antigens were used to develop immunoblotting assay for the detection of anti-HCV antibodies in sera. With immunoblotting, a total of 93-HCV infected sera and 35-HCV negative individuals were tested for the presence of anti-HCV antibodies to the Core and E2 antigens. Recombinant antigen showed 100% reactivity against HCV infected sera, with no cross reactivity against HCV-negative sera. The immunoblot assay mixture of recombinant antigens (Core+E2) showed a strong reaction intensity in the test area (TA) as compared to the individual truncated Core and E2 recombinant antigens. In the in-house ELISA assay, mixed Core and E2 recombinant antigens showed 100% reactivity against a standardized panel of 150-HCV-positive sera and non reactivity against a standardized panel of 150 HCV-negative sera while also being non reactive to sera positive for other viral infections. The antigenic recombinant antigens also were tested for the

  19. Evaluation of recombinant porin (rOmp2a) protein as a potential antigen candidate for serodiagnosis of Human Brucellosis.

    Science.gov (United States)

    Pathak, Prachi; Kumar, Ashu; Thavaselvam, Duraipandian

    2017-07-11

    Brucellosis is an important zoonotic disease caused by different Brucella species and human brucellosis is commonly prevalent in different states of India. Among various Brucella species, B. melitensis is most pathogenic to human and included as category B biothreat which can cause infection through aerosol, cut, wounds in skin and contact with infected animals. The diagnosis of human brucellosis is very important for proper treatment and management of disease as there is no vaccine available for human use. The present study was designed to clone, express and purify immunodominant recombinant omp2a (rOmp2a) porin protein of B. melitensis and to evaluate this new antigen candidate for specific serodiagnosis of human brucellosis by highly sensitive iELISA (indirect enzyme linked immunosorbent assay). Omp2a gene of B. melitensis 16 M strain was cloned and expressed in pET-SUMO expression system. The recombinant protein was purified under denaturing conditions using 8 M urea. The purified recombinant protein was confirmed by western blotting by reacting with anti-HIS antibody. The sero-reactivity of the recombinant protein was also checked by reacting with antisera of experimentally infected mice with B. melitensis 16 M at different time points. Serodiagnostic potential of recombinant porin antigen was tested against 185 clinical serum samples collected from regions endemic to brucellosis in southern part of India by iELISA. The samples were grouped into five groups. Group 1 contained cultured confirmed positive serum samples of brucellosis (n = 15), group 2 contained sera samples from positive cases of brucellosis previously tested by conventional methods of RBPT (n = 28) and STAT (n = 26), group 3 contained sera samples negative by RBPT(n = 36) and STAT (n = 32), group 4 contained sera samples of other febrile illness and PUO case (n = 35) and group 5 contained confirmed negative sera samples from healthy donors (n = 23). The rOmp2a was found to be

  20. Vaccination with Recombinant Baculovirus Expressing Ranavirus Major Capsid Protein Induces Protective Immunity in Chinese Giant Salamander, Andrias davidianus

    Directory of Open Access Journals (Sweden)

    Xiaoyuan Zhou

    2017-07-01

    Full Text Available The Chinese giant salamander iridovirus (CGSIV, belonging to the genus Ranavirus in the family Iridoviridae, is the causative agent of an emerging infectious disease causing high mortality of more than 90% and economic losses in Chinese giant salamanders in China. In this study, a recombinant baculovirus-based vaccine expressing the CGSIV major capsid protein (MCP was developed and its protective immunity in Chinese giant salamanders was evaluated. The recombinant Autographa californica nucleopolyhedrosis virus (AcNPV, expressing CGSIV MCP, designated as AcNPV-MCP, was generated with the highest titers of 1 × 108 plaque forming units/mL (PFU/mL and confirmed by Western blot and indirect immunofluorescence (IIF assays. Western blot analysis revealed that the expressed MCP reacted with mouse anti-MCP monoclonal antibodies at the band of about 53 kDa. The results of IIF indicated that the MCP was expressed in the infected Spodoptera frugiperda 9 (Sf9 cells with the recombinant baculovirus, and the Chinese giant salamander muscle cells also transduced with the AcNPV-MCP. Immunization with the recombinant baculovirus of AcNPV-MCP elicited robust specific humoral immune responses detected by ELISA and neutralization assays and potent cellular immune responses in Chinese giant salamanders. Importantly, the effective immunization conferred highly protective immunity for Chinese giant salamanders against CGSIV challenge and produced a relative percent of survival rate of 84%. Thus, the recombinant baculovirus expressing CGSIV MCP can induce significant immune responses involving both humoral and cell-mediated immunity in Chinese giant salamanders and might represent a potential baculovirus based vaccine candidate for Chinese giant salamanders against CGSIV.

  1. Expression and characterization of recombinant leptospiral outer membrane protein LipL32 from Leptospira interrogans serovar autumnalis.

    Science.gov (United States)

    Boonsathorn, Naphatsawan; Konghom, Ganokrot; Mongkolsiri, Kaveewan; Jirapongwattana, Chanin; Balachandra, Kruavon; Naigowit, Pimjai; Sawanpanyalert, Pathom

    2009-01-01

    Leptospira interrogans serovar autumnalis, a causative agent of leptospirosis in Thailand, was isolated from a patient for DNA extraction and amplification of LipL32 gene by polymerase chain reaction (PCR). The 782 bp PCR product was obtained, which was inserted into pAE plasmid with polyhistidine (His6 tag) to construct pAE-LipL32. This recombinant plasmid was transfected into E. coli BL21 (DE3). His6-LipL32 was purified by Ni-NTA affinity chromatography. The recombinant protein was used as antigen for testing with sera from leptospirosis and syphilis patients by dot-ELISA technique. It reacted positively with leptospirosis patient sera and negatively with syphilis and healthy sera.

  2. Construction of a recombinant viral vector containing part of the nucleocapsid protein gene of newcastle disease virus

    Energy Technology Data Exchange (ETDEWEB)

    Bader, D.E.

    1995-09-01

    This report describes the procedures used to clone a 673 base pair gene fragment of the major nucleocapsid protein gene of Newcastle disease virus into a viral vector molecule for the purpose of maintaining a stable, long-term, renewable source of this target sequence for gene probe studies. The gene fragment was prepared by reverse-transcription polymerase chain reaction of Newcastle disease virus RNA and was cloned into the viral DNA vector Ml3mp18 RF to produce a recombinant DNA molecule. The cloned fragment was shown to be present in the recombinant clones based on (i) clonal selection on indicator plates; (ii) restriction enzyme analysis; (iii) gene probe analysis and (iv) nested PCR amplification.

  3. Poly(norepinephrine)-coated open tubular column for the separation of proteins and recombination human erythropoietin by capillary electrochromatography.

    Science.gov (United States)

    Xiao, Xue; Zhang, Yamin; Wu, Jia; Jia, Li

    2017-12-01

    Recombinant human erythropoietin is an important therapeutic protein with high economic interest due to the benefits provided by its clinical use for the treatment of anemias associated with chronic renal failure and chemotherapy. In this work, a poly(norepinephrine)-coated open tubular column was successfully prepared based on the self-polymerization of norepinephrine under mild alkaline condition, the favorable film forming and easy adhesive properties of poly(norepinephrine). The poly(norepinephrine) coating was characterized by scanning electron microscopy and measurement of the electro-osmotic flow. The thickness of the coating was about 431 nm. The electrochromatographic performance of the poly(norepinephrine)-coated open tubular column was evaluated by separation of proteins. Some basic and acidic proteins including two variants of bovine serum albumin and two variants of β-lactoglobulin achieved separation in the poly(norepinephrine)-coated open tubular column. More importantly, the column demonstrated separation ability for the glycoforms of recombinant human erythropoietin. In addition, the column demonstrated good repeatability with the run-to-run, day-to-day, and column-to-column relative standard deviations of migration times of proteins less than 3.40%. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Recombinant Protein Containing B-Cell Epitopes of Different Loxosceles Spider Toxins Generates Neutralizing Antibodies in Immunized Rabbits.

    Science.gov (United States)

    Lima, Sabrina de Almeida; Guerra-Duarte, Clara; Costal-Oliveira, Fernanda; Mendes, Thais Melo; Figueiredo, Luís F M; Oliveira, Daysiane; Machado de Avila, Ricardo A; Ferrer, Valéria Pereira; Trevisan-Silva, Dilza; Veiga, Silvio S; Minozzo, João C; Kalapothakis, Evanguedes; Chávez-Olórtegui, Carlos

    2018-01-01

    Loxoscelism is the most important form of araneism in South America. The treatment of these accidents uses heterologous antivenoms obtained from immunization of production animals with crude loxoscelic venom. Due to the scarcity of this immunogen, new alternatives for its substitution in antivenom production are of medical interest. In the present work, three linear epitopes for Loxosceles astacin-like protease 1 (LALP-1) (SLGRGCTDFGTILHE, ENNTRTIGPFDYDSIMLYGAY, and KLYKCPPVNPYPGGIRPYVNV) and two for hyaluronidase (LiHYAL) (NGGIPQLGDLKAHLEKSAVDI and ILDKSATGLRIIDWEAWR) from Loxosceles intermedia spider venom were identified by SPOT-synthesis technique. One formerly characterized linear epitope (DFSGPYLPSLPTLDA) of sphingomyelinase D (SMase D) SMase-I from Loxosceles laeta was also chosen to constitute a new recombinant multiepitopic protein. These epitopes were combined with a previously produced chimeric multiepitopic protein (rCpLi) composed by linear and conformational B-cell epitopes from SMase D from L. intermedia venom, generating a new recombinant multiepitopic protein derived from loxoscelic toxins (rMEPLox). We demonstrated that rMEPLox is non-toxic and antibodies elicited in rabbits against this antigen present reactivity in ELISA and immunoblot assays with Brazilian L. intermedia, L. laeta, L. gaucho , and L. similis spider venoms. In vivo and in vitro neutralization assays showed that anti-rMEPLox antibodies can efficiently neutralize the sphingomyelinase, hyaluronidase, and metalloproteinase activity of L. intermedia venom. This study suggests that this multiepitopic protein can be a suitable candidate for experimental vaccination approaches or for antivenom production against Loxosceles spp. venoms.

  5. Immunization of Mice with Recombinant Brucella abortus Organic Hydroperoxide Resistance (Ohr) Protein Protects Against a Virulent Brucella abortus 544 Infection.

    Science.gov (United States)

    Hop, Huynh Tan; Reyes, Alisha Wehdnesday Bernardo; Simborio, Hannah Leah Tadeja; Arayan, Lauren Togonon; Min, Won Gi; Lee, Hu Jang; Lee, Jin Ju; Chang, Hong Hee; Kim, Suk

    2016-01-01

    In this study, the Brucella abortus ohr gene coding for an organic hydroperoxide resistance protein (Ohr) was cloned into a maltose fusion protein expression system (pMAL), inserted into Escherichia coli, and purified, and its immunogenicity was evaluated by western blot analysis using Brucella-positive mouse sera. The purified recombinant Ohr (rOhr) was treated with adjuvant and injected intraperitoneally into BALB/c mice. A protective immune response analysis revealed that rOhr induced a significant increase in both the IgG1 and IgG2a titers, and IgG2a reached a higher level than IgG1 after the second and third immunizations. Additionally, immunization with rOhr induced high production of IFN-γ as well as proinflammatory cytokines such as TNF, MCP-1, IL-12p70, and IL-6, but a lesser amount of IL-10, suggesting that rOhr predominantly elicited a cell-mediated immune response. In addition, immunization with rOhr caused a significantly higher degree of protection against a virulent B. abortus infection compared with a positive control group consisting of mice immunized with maltose-binding protein. These findings showed that B. abortus rOhr was able to induce both humoral and cell-mediated immunity in mice, which suggested that this recombinant protein could be a potential vaccine candidate for animal brucellosis.

  6. Evaluation of immunogenicity and protective efficacy of recombinant outer membrane proteins of Haemophilus parasuis serovar 5 in a murine model.

    Science.gov (United States)

    Li, Miao; Cai, Ru-Jian; Song, Shuai; Jiang, Zhi-Yong; Li, Yan; Gou, Hong-Chao; Chu, Pin-Pin; Li, Chun-Ling; Qiu, Hua-Ji

    2017-01-01

    Glässer's disease is an economically important infectious disease of pigs caused by Haemophilus parasuis. Few vaccines are currently available that could provide effective cross-protection against various serovars of H. parasuis. In this study, five OMPs (OppA, TolC, HxuC, LppC, and HAPS_0926) identified by bioinformatic approaches, were cloned and expressed as recombinant proteins. Antigenicity of the purified proteins was verified through Western blotting, and primary screening for protective potential was evaluated in vivo. Recombinant TolC (rTolC), rLppC, and rHAPS_0926 proteins showing marked protection of mice against H. parasuis infection, and were further evaluated individually or in combination. Mice treated with these three OMPs produced humoral and host cell-mediated responses, with a significant rise in antigen-specific IgG titer and lymphoproliferative response in contrast with the mock-immunized group. Significant increases were noted in CD4+, CD8+ T cells, and three cytokines (IL-2, IL-4, and IFN-γ) in vaccinated animals. The antisera against candidate antigens could efficiently impede bacterial survival in whole blood bactericidal assay against H. parasuis infection. The multi-protein vaccine induced more pronounced immune responses and offered better protection than individual vaccines. Our findings indicate that these three OMPs are promising antigens for the development of multi-component subunit vaccines against Glässer's disease.

  7. Recombinant Protein Containing B-Cell Epitopes of Different Loxosceles Spider Toxins Generates Neutralizing Antibodies in Immunized Rabbits

    Science.gov (United States)

    Lima, Sabrina de Almeida; Guerra-Duarte, Clara; Costal-Oliveira, Fernanda; Mendes, Thais Melo; Figueiredo, Luís F. M.; Oliveira, Daysiane; Machado de Avila, Ricardo A.; Ferrer, Valéria Pereira; Trevisan-Silva, Dilza; Veiga, Silvio S.; Minozzo, João C.; Kalapothakis, Evanguedes; Chávez-Olórtegui, Carlos

    2018-01-01

    Loxoscelism is the most important form of araneism in South America. The treatment of these accidents uses heterologous antivenoms obtained from immunization of production animals with crude loxoscelic venom. Due to the scarcity of this immunogen, new alternatives for its substitution in antivenom production are of medical interest. In the present work, three linear epitopes for Loxosceles astacin-like protease 1 (LALP-1) (SLGRGCTDFGTILHE, ENNTRTIGPFDYDSIMLYGAY, and KLYKCPPVNPYPGGIRPYVNV) and two for hyaluronidase (LiHYAL) (NGGIPQLGDLKAHLEKSAVDI and ILDKSATGLRIIDWEAWR) from Loxosceles intermedia spider venom were identified by SPOT-synthesis technique. One formerly characterized linear epitope (DFSGPYLPSLPTLDA) of sphingomyelinase D (SMase D) SMase-I from Loxosceles laeta was also chosen to constitute a new recombinant multiepitopic protein. These epitopes were combined with a previously produced chimeric multiepitopic protein (rCpLi) composed by linear and conformational B-cell epitopes from SMase D from L. intermedia venom, generating a new recombinant multiepitopic protein derived from loxoscelic toxins (rMEPLox). We demonstrated that rMEPLox is non-toxic and antibodies elicited in rabbits against this antigen present reactivity in ELISA and immunoblot assays with Brazilian L. intermedia, L. laeta, L. gaucho, and L. similis spider venoms. In vivo and in vitro neutralization assays showed that anti-rMEPLox antibodies can efficiently neutralize the sphingomyelinase, hyaluronidase, and metalloproteinase activity of L. intermedia venom. This study suggests that this multiepitopic protein can be a suitable candidate for experimental vaccination approaches or for antivenom production against Loxosceles spp. venoms. PMID:29666624

  8. Analysis of antigenic cross-reactivity between subgroup C avian pneumovirus and human metapneumovirus by using recombinant fusion proteins.

    Science.gov (United States)

    Luo, L; Sabara, M I; Li, Y

    2009-10-01

    Avian pneumovirus subgroup C (APV/C) has recently been reported to be more closely related to human metapneumovirus (hMPV) as determined by sequence analysis. To examine the antigenic relationship between APV/C and hMPV, the APV/C fusion (F) gene was cloned and expressed as an uncleaved glycoprotein in a baculovirus system. The reactivity of the APV/C F protein with antibodies against APV subgroups A, B, C, and hMPV was examined by Western blot analysis. The results showed that the expressed APV/C F protein was not only recognized by APV/C-specific antibodies but also by antibodies raised against hMPV. Previously expressed recombinant hMPV F protein also reacted with APV/C-specific antibodies, suggesting that there was significant antigenic cross-reactivity and a potential evolutionary relationship between hMPV and APV/C. Interestingly, the recombinant F proteins from APV/C and hMPV were not recognized by polyclonal antibodies specific to APV subgroups A and B.

  9. 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.

  10. INTERACTION BETWEEN DIFFERENT MOLECULAR FORMS OF IMMUNOGLOBULIN A AND RECOMBINANT DERIVATIVES POLYPEPTIDES OF BAC RECEPTOR PROTEINS FROM GROUP B STREPTOCOCCI

    Directory of Open Access Journals (Sweden)

    A. S. Korzhueva

    2008-01-01

    Full Text Available Abstract. The article concerns interactions between immunoglobulin A and recombinant P6, P7, P8 polypeptides, designed on the basis of externally localized Bac protein of the Group B streptococci, possessing IgA-binding activity.There is a current demand for immunochemical reagents that are strictly specific for IgA, in order to develop antigenic standards for detection of IgA levels in biological fluids, as well as for affinity purification of IgA and its fragments.To analyze an opportunity of the abovementioned application ways for these proteins, a special study was performed to assay an interaction capability of recombinant P6, P7, P8 polypeptides binding to Fc regions of different IgA forms (serum IgA, secretory IgA, subclasses of serum IgA – IgA1, IgA2. Selectivity of ligand binding was specially confirmed.It was found out that, among three presented polypeptides, the structure of recombinant P6 derivative proved to be optimal for IgA-binding ability of Bac protein.Structural features of IgA-binding fragments of Bac protein, i.e., binding site position on the IgA molecule (proximity to epitopes for three monoclonal antibodies, variability of the site structure, as well as resistance of binding site for P6, P7, P8 in IgA molecule against partial disulfide bonds reduction. (Med. Immunol., vol. 10, N 4-5, pp 327-336.

  11. A Neospora caninum vaccine using recombinant proteins fails to prevent foetal infection in pregnant cattle after experimental intravenous challenge.

    Science.gov (United States)

    Hecker, Yanina P; Cóceres, Verónica; Wilkowsky, Silvina E; Jaramillo Ortiz, José M; Morrell, Eleonora L; Verna, Andrea E; Ganuza, Agustina; Cano, Dora B; Lischinsky, Lilian; Angel, Sergio O; Zamorano, Patricia; Odeón, Anselmo C; Leunda, María R; Campero, Carlos M; Morein, Bror; Moore, Dadín P

    2014-12-15

    The aim of the present study was to evaluate the immunogenicity and protective efficacy of rNcSAG1, rNcHSP20 and rNcGRA7 recombinant proteins formulated with immune stimulating complexes (ISCOMs) in pregnant heifers against vertical transmission of Neospora caninum. Twelve pregnant heifers were divided into 3 groups of 4 heifers each, receiving different formulations before mating. Immunogens were administered twice subcutaneously: group A animals were inoculated with three recombinant proteins (rNcSAG1, rNcHSP20, rNcGRA7) formulated with ISCOMs; group B animals received ISCOM-MATRIX (without antigen) and group C received sterile phosphate-buffered saline (PBS) only. The recombinant proteins were expressed in Escherichia coli and purified nickel resin. All groups were intravenously challenged with the NC-1 strain of N. caninum at Day 70 of gestation and dams slaughtered at week 17 of the experiment. Heifers from group A developed specific antibodies against rNcSAG1, rNcHSP20 and rNcGRA7 prior to the challenge. Following immunization, an statistically significant increase of antibodies against rNcSAG1 and rNcHSP20 in all animals of group A was detected compared to animals in groups B and C at weeks 5, 13 and 16 (P0.001). There were no differences in IFN-γ production among the experimental groups at any time point (P>0.05). Transplacental transmission was determined in all foetuses of groups A, B and C by Western blot, immunohistochemistry and nested PCR. This work showed that rNcSAG1, rNcHSP20 and rNcGRA7 proteins while immunogenic in cattle failed to prevent the foetal infection in pregnant cattle challenged at Day 70 of gestation. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Multiple protein biomarker assessment for recombinant bovine somatotropin (rbST abuse in cattle.

    Directory of Open Access Journals (Sweden)

    Susann K J Ludwig

    Full Text Available Biomarker profiling, as a rapid screening approach for detection of hormone abuse, requires well selected candidate biomarkers and a thorough in vivo biomarker evaluation as previously done for detection of growth hormone doping in athletes. The bovine equivalent of growth hormone, called recombinant bovine somatotropin (rbST is (illegally administered to enhance milk production in dairy cows. In this study, first a generic sample pre-treatment and 4-plex flow cytometric immunoassay (FCIA were developed for simultaneous measurement of four candidate biomarkers selected from literature: insulin-like growth factor 1 (IGF-1, its binding protein 2 (IGFBP2, osteocalcin and endogenously produced antibodies against rbST. Next, bovine serum samples from two extensive controlled rbST animal treatment studies were used for in vivo validation and biomarker evaluation. Finally, advanced statistic tools were tested for the assessment of biomarker combination quality aiming to correctly identify rbST-treated animals. The statistical prediction tool k-nearest neighbours using a combination of the biomarkers osteocalcin and endogenously produced antibodies against rbST proved to be very reliable and correctly predicted 95% of the treated samples starting from the second rbST injection until the end of the treatment period and even thereafter. With the same biomarker combination, only 12% of untreated animals appeared false-positive. This reliability meets the requirements of Commission Decision 2002/657/EC for screening methods in veterinary control. From the results of this multidisciplinary study, it is concluded that the osteocalcin - anti-rbST-antibodies combination represent fit-for-purpose biomarkers for screening of rbST abuse in dairy cattle and can be reliably measured in both the developed 4-plex FCIA as well as in a cost-effective 2-plex microsphere-based binding assay. This screening method can be incorporated in routine veterinary monitoring

  13. 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

  14. Increased understanding of the biochemistry and biosynthesis of MUC2 and other gel-forming mucins through the recombinant expression of their protein domains.

    Science.gov (United States)

    Bäckström, Malin; Ambort, Daniel; Thomsson, Elisabeth; Johansson, Malin E V; Hansson, Gunnar C

    2013-06-01

    The gel-forming mucins are large and heavily O-glycosylated proteins which build up mucus gels. The recombinant production of full-length gel-forming mucins has not been possible to date. In order to study mucin biosynthesis and biochemistry, we and others have taken the alternative approach of constructing different recombinant proteins consisting of one or several domains of these large proteins and expressing them separately in different cell lines. Using this approach, we have determined that MUC2, the intestinal gel-forming mucin, dimerizes via its C-terminal cysteine-knot domain and also trimerizes via one of the N-terminal von Willebrand D domains. Both of these interactions are disulfide bond mediated. Via this assembly, a molecular network is built by which the mucus gel is formed. Here we discuss not only the functional understanding obtained from studies of the recombinant proteins, but also highlight the difficulties encountered when these proteins were produced recombinantly. We often found an accumulation of the proteins in the ER and consequently no secretion. This was especially apparent when the cysteine-rich domains of the N- and C-terminal parts of the mucins were expressed. Other proteins that we constructed were either not secreted or not expressed at all. Despite these problems, the knowledge of mucin biosynthesis and assembly has advanced considerably through the studies of these recombinant proteins.

  15. Fcγ1 fragment of IgG1 as a powerful affinity tag in recombinant Fc-fusion proteins: immunological, biochemical and therapeutic properties.

    Science.gov (United States)

    Soleimanpour, Saman; Hassannia, Tahereh; Motiee, Mahdieh; Amini, Abbas Ali; Rezaee, S A R

    2017-05-01

    Affinity tags are vital tools for the production of high-throughput recombinant proteins. Several affinity tags, such as the hexahistidine tag, maltose-binding protein, streptavidin-binding peptide tag, calmodulin-binding peptide, c-Myc tag, glutathione S-transferase and FLAG tag, have been introduced for recombinant protein production. The fragment crystallizable (Fc) domain of the IgG1 antibody is one of the useful affinity tags that can facilitate detection, purification and localization of proteins and can improve the immunogenicity, modulatory effects, physicochemical and pharmaceutical properties of proteins. Fcγ recombinant forms a group of recombinant proteins called Fc-fusion proteins (FFPs). FFPs are widely used in drug discovery, drug delivery, vaccine design and experimental research on receptor-ligand interactions. These fusion proteins have become successful alternatives to monoclonal antibodies for drug developments. In this review, the physicochemical, biochemical, immunological, pharmaceutical and therapeutic properties of recombinant FFPs were discussed as a new generation of bioengineering strategies.

  16. Tryptophan tags and de novo designed complementary affinity ligands for the expression and purification of recombinant proteins.

    Science.gov (United States)

    Pina, Ana Sofia; Carvalho, Sara; Dias, Ana Margarida G C; Guilherme, Márcia; Pereira, Alice S; Caraça, Luciana T; Coroadinha, Ana Sofia; Lowe, Christopher R; Roque, A Cecília A

    2016-11-11

    A common strategy for the production and purification of recombinant proteins is to fuse a tag to the protein terminal residues and employ a "tag-specific" ligand for fusion protein capture and purification. In this work, we explored the effect of two tryptophan-based tags, NWNWNW and WFWFWF, on the expression and purification of Green Fluorescence Protein (GFP) used as a model fusion protein. The titers obtained with the expression of these fusion proteins in soluble form were 0.11mgml -1 and 0.48mgml -1 for WFWFWF and NWNWNW, respectively. A combinatorial library comprising 64 ligands based on the Ugi reaction was prepared and screened for binding GFP-tagged and non-tagged proteins. Complementary ligands A2C2 and A3C1 were selected for the effective capture of NWNWNW and WFWFWF tagged proteins, respectively, in soluble forms. These affinity pairs displayed 10 6 M -1 affinity constants and Qmax values of 19.11±2.60ugg -1 and 79.39ugg -1 for the systems WFWFWF AND NWNWNW, respectively. GFP fused to the WFWFWF affinity tag was also produced as inclusion bodies, and a refolding-on column strategy was explored using the ligand A4C8, selected from the combinatorial library of ligands but in presence of denaturant agents. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. High-yield secretion of recombinant proteins expressed in tobacco cell culture with a designer glycopeptide tag: Process development.

    Science.gov (United States)

    Zhang, Ningning; Gonzalez, Maria; Savary, Brett; Xu, Jianfeng

    2016-03-01

    Low-yield protein production remains the most significant economic hurdle with plant cell culture technology. Fusions of recombinant proteins with hydroxyproline-O-glycosylated designer glycopeptide tags have consistently boosted secreted protein yields. This prompted us to study the process development of this technology aiming to achieve productivity levels necessary for commercial viability. We used a tobacco BY-2 cell culture expressing EGFP as fusion with a glycopeptide tag comprised of 32 repeat of "Ser-Pro" dipeptide, or (SP)32 , to study cell growth and protein secretion, culture scale-up, and establishment of perfusion cultures for continuous production. The BY-2 cells accumulated low levels of cell biomass (~7.5 g DW/L) in Schenk & Hildebrandt medium, but secreted high yields of (SP)32 -tagged EGFP (125 mg/L). Protein productivity of the cell culture has been stable for 6.0 years. The BY-2 cells cultured in a 5-L bioreactor similarly produced high secreted protein yield at 131 mg/L. Successful operation of a cell perfusion culture for 30 days was achieved under the perfusion rate of 0.25 and 0.5 day(-1) , generating a protein volumetric productivity of 17.6 and 28.9 mg/day/L, respectively. This research demonstrates the great potential of the designer glycopeptide technology for use in commercial production of valuable proteins with plant cell cultures. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Detection of Antibodies to U.S. Isolates of Avian Pneumovirus by a Recombinant Nucleocapsid Protein-Based Sandwich Enzyme-Linked Immunosorbent Assay

    OpenAIRE

    Gulati, Baldev R.; Munir, Shirin; Patnayak, Devi P.; Goyal, Sagar M.; Kapur, Vivek

    2001-01-01

    The nucleocapsid (N) protein of subgroup C (United States-specific) avian pneumovirus (APV/US) was expressed in Escherichia coli, and antibodies to the recombinant N protein were shown to specifically recognize the ≈47-kDa N protein of APV/US by Western immunoblot analysis. The recombinant APV/US N protein was used in a sandwich-capture enzyme-linked immunosorbent assay (ELISA), and the resulting assay was found to be more sensitive and specific than the routine indirect ELISA for the detecti...

  19. Properties of a mutant recA-encoded protein reveal a possible role for Escherichia coli recF-encoded protein in genetic recombination