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Sample records for recombinant proteins produced

  1. Cultivating Insect Cells To Produce Recombinant Proteins

    NASA Technical Reports Server (NTRS)

    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).

  2. Recombinant protein vaccines produced in insect cells.

    PubMed

    Cox, Manon M J

    2012-02-27

    The baculovirus-insect cell expression system is a well known tool for the production of complex proteins. The technology is also used for commercial manufacture of various veterinary and human vaccines. This review paper provides an overview of how this technology can be applied to produce a multitude of vaccine candidates. The key advantage of this recombinant protein manufacturing platform is that a universal "plug and play" process may be used for producing a broad range of protein-based prophylactic and therapeutic vaccines for both human and veterinary use while offering the potential for low manufacturing costs. Large scale mammalian cell culture facilities previously established for the manufacturing of monoclonal antibodies that have now become obsolete due to yield improvement could be deployed for the manufacturing of these vaccines. Alternatively, manufacturing capacity could be established in geographic regions that do not have any vaccine production capability. Dependent on health care priorities, different vaccines could be manufactured while maintaining the ability to rapidly convert to producing pandemic influenza vaccine when the need arises. PMID:22265860

  3. Producing Recombinant mTEX101; a Murine Testis Specific Protein

    PubMed Central

    Barzegar Yarmohammadi, Leila; Modarresi, Mohammad Hossein; Talebi, Saeed; Hadavi, Reza; Ostad Karampour, Mahyar; Mahmoudi, Ahmad Reza; Akhondi, Mohammad Mehdi; Rabbani, Hodjattallah; Jeddi-Tehrani, Mahmood

    2009-01-01

    Introduction Production of antibodies against specific proteins of testis germ cells is of great significance for the investigation of processes involved in spermatogenesis, study of infertility problems and determination of the probable role of these proteins as cancer-testis antigens. Murine Testis Specific Recombinant Protein 101 (mTEX101) is a 38kDa, GPI-anchored protein which is expressed in testis germ cells of adult mice but it seems to be absent in other tissues. The structure and function of mTEX101 is not completely understood yet, but it is speculated that it may transduce biochemical signals into the cytoplasm since mTEX101 does not have an intracellular domain but the precise mechanisms are still ambiguous. Materials and Methods RNA was extracted from three adult mice testis. The RNA was used in RT-PCR, employing a pair of specific primers for mTEX101 ORF region. TA-cloning technique was performed by the insertion of mTEX101 into a pGEM-T Easy Vector, followed by its subcloning into a His-tagged expression vector, pET-28a (+). The recombinant mTEX101 was then produced by transfection of the expression vector into BL 21 (DE3) E. coli strain. Results A recombinant protein, weighing 27kDa, was produced upon IPTG-induction of the bacterial host. The presence of mTEX101 protein was detected through Western blot analysis by anti-mTEX101 peptide antibodies. Conclusion We produced mTEX101 recombinant protein that could be used for the production of mono and polyclonal antibodies. PMID:23926468

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

    PubMed

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

    2014-11-01

    Microalgae have been used in food, cosmetic, and biofuel industries as a natural source of lipids, vitamins, pigments and antioxidants for a long time. Green microalgae, as potent photobioreactors, can be considered as an economical expression system to produce recombinant therapeutical proteins at large-scale due to low cost of production and scaling-up capitalization owning to the inexpensive medium requirement, fast growth rate, and the ease of manipulation. These microalgae possess all benefit eukaryotic expression systems including the ability of post-translational modifications required for proper folding and stability of active proteins. Among the many items regarded as recombinant protein production, this review compares the different expression systems with green microalgae like Dunaliella by viewing the nuclear/chloroplast transformation challenges/benefits, related selection markers/reporter genes, and crucial factors/strategies affecting the increase of foreign protein expression in microalgae transformants. Some important factors were discussed regarding the increase of protein yielding in microalgae transformants including: transformation-associated genotypic modifications, endogenous regulatory factors, promoters, codon optimization, enhancer elements, and milking of recombinant protein. PMID:25115849

  5. Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overview

    PubMed Central

    Gasser, Brigitte; Saloheimo, Markku; Rinas, Ursula; Dragosits, Martin; Rodríguez-Carmona, Escarlata; Baumann, Kristin; Giuliani, Maria; Parrilli, Ermenegilda; Branduardi, Paola; Lang, Christine; Porro, Danilo; Ferrer, Pau; Tutino, Maria Luisa; Mattanovich, Diethard; Villaverde, Antonio

    2008-01-01

    Different species of microorganisms including yeasts, filamentous fungi and bacteria have been used in the past 25 years for the controlled production of foreign proteins of scientific, pharmacological or industrial interest. A major obstacle for protein production processes and a limit to overall success has been the abundance of misfolded polypeptides, which fail to reach their native conformation. The presence of misfolded or folding-reluctant protein species causes considerable stress in host cells. The characterization of such adverse conditions and the elicited cell responses have permitted to better understand the physiology and molecular biology of conformational stress. Therefore, microbial cell factories for recombinant protein production are depicted here as a source of knowledge that has considerably helped to picture the extremely rich landscape of in vivo protein folding, and the main cellular players of this complex process are described for the most important cell factories used for biotechnological purposes. PMID:18394160

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

    SciTech Connect

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

    2009-07-31

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

  7. Recombinant protein production technology

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  8. A fast and simple method to eliminate Cpn60 from functional recombinant proteins produced by E. coli Arctic Express.

    PubMed

    Belval, Lorène; Marquette, Arnaud; Mestre, Pere; Piron, Marie-Christine; Demangeat, Gérard; Merdinoglu, Didier; Chich, Jean-François

    2015-05-01

    A frequent problem of recombinant protein production is their insolubility. To address this issue, engineered Escherichiacoli strains like Arctic Express that produce an exogenous chaperone facilitating protein folding, have been designed. A drawback is the frequent contamination of the protein by chaperones. A simple method, using urea at a sub-denaturing concentration, allows unbinding of Cpn60 from expressed protein. This method was successfully used to purify 2 proteins, an enzyme and a viral protein. The enzyme was fully active. The nature of interaction forces between enzyme and Cpn60 was investigated. The method is likely applicable to purify other proteins. PMID:25655203

  9. Native-sized recombinant spider silk protein produced in metabolically engineered Escherichia coli results in a strong fiber.

    PubMed

    Xia, Xiao-Xia; Qian, Zhi-Gang; Ki, Chang Seok; Park, Young Hwan; Kaplan, David L; Lee, Sang Yup

    2010-08-10

    Spider dragline silk is a remarkably strong fiber that makes it attractive for numerous applications. Much has thus been done to make similar fibers by biomimic spinning of recombinant dragline silk proteins. However, success is limited in part due to the inability to successfully express native-sized recombinant silk proteins (250-320 kDa). Here we show that a 284.9 kDa recombinant protein of the spider Nephila clavipes is produced and spun into a fiber displaying mechanical properties comparable to those of the native silk. The native-sized protein, predominantly rich in glycine (44.9%), was favorably expressed in metabolically engineered Escherichia coli within which the glycyl-tRNA pool was elevated. We also found that the recombinant proteins of lower molecular weight versions yielded inferior fiber properties. The results provide insight into evolution of silk protein size related to mechanical performance, and also clarify why spinning lower molecular weight proteins does not recapitulate the properties of native fibers. Furthermore, the silk expression, purification, and spinning platform established here should be useful for sustainable production of natural quality dragline silk, potentially enabling broader applications. PMID:20660779

  10. Co-expression of ferrochelatase allows for complete heme incorporation into recombinant proteins produced in E. coli

    PubMed Central

    Sudhamsu, Jawahar; Kabir, Mariam; Airola, Michael V.; Patel, Bhumit A.; Yeh, Syun-Ru; Rousseau, Dennis L.; Crane, Brian R.

    2010-01-01

    Over-expression of heme binding proteins in E. coli often results in sub-optimal heme incorporation and the amount of heme-bound protein produced usually varies with the protein of interest. Complete heme incorporation is important for biochemical characterization, spectroscopy, structural studies, and for the production of homogeneous commercial proteins with high activity. We have determined that recombinant proteins expressed in E. coli often contain less than a full complement of heme because they rather are partially incorporated with free-base porphyrin. Porphyrin-incorporated proteins have similar spectral characteristics as the desired heme-loaded targets, and thus are difficult to detect, even in purified samples. We present a straightforward and inexpensive solution to this problem that involves the co-expression of native ferrochelatase with the protein of interest. The method is shown to be effective for proteins that contain either Cys- or His- ligated hemes. PMID:20303407

  11. Site-specific chemical modification of recombinant proteins produced in mammalian cells by using the genetically encoded aldehyde tag.

    PubMed

    Wu, Peng; Shui, Wenqing; Carlson, Brian L; Hu, Nancy; Rabuka, David; Lee, Julia; Bertozzi, Carolyn R

    2009-03-01

    The properties of therapeutic proteins can be enhanced by chemical modification. Methods for site-specific protein conjugation are critical to such efforts. Here, we demonstrate that recombinant proteins expressed in mammalian cells can be site-specifically modified by using a genetically encoded aldehyde tag. We introduced the peptide sequence recognized by the endoplasmic reticulum (ER)-resident formylglycine generating enzyme (FGE), which can be as short as 6 residues, into heterologous proteins expressed in mammalian cells. Cotranslational modification of the proteins by FGE produced products bearing a unique aldehyde group. Proteins bearing this "aldehyde tag" were chemically modified by selective reaction with hydrazide- or aminooxy-functionalized reagents. We applied the technique to site-specific modification of monoclonal antibodies, the fastest growing class of biopharmaceuticals, as well as membrane-associated and cytosolic proteins expressed in mammalian cells. PMID:19202059

  12. High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

    PubMed Central

    Saez, Natalie J.; Nozach, Hervé; Blemont, Marilyne; Vincentelli, Renaud

    2014-01-01

    Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4 ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment. Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (www.venomics.eu), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive

  13. Recombinant Collagenlike Proteins

    NASA Technical Reports Server (NTRS)

    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.

  14. Human recombinant lysosomal enzymes produced in microorganisms.

    PubMed

    Espejo-Mojica, Ángela J; Alméciga-Díaz, Carlos J; Rodríguez, Alexander; Mosquera, Ángela; Díaz, Dennis; Beltrán, Laura; Díaz, Sergio; Pimentel, Natalia; Moreno, Jefferson; Sánchez, Jhonnathan; Sánchez, Oscar F; Córdoba, Henry; Poutou-Piñales, Raúl A; Barrera, Luis A

    2015-01-01

    Lysosomal storage diseases (LSDs) are caused by accumulation of partially degraded substrates within the lysosome, as a result of a function loss of a lysosomal protein. Recombinant lysosomal proteins are usually produced in mammalian cells, based on their capacity to carry out post-translational modifications similar to those observed in human native proteins. However, during the last years, a growing number of studies have shown the possibility to produce active forms of lysosomal proteins in other expression systems, such as plants and microorganisms. In this paper, we review the production and characterization of human lysosomal proteins, deficient in several LSDs, which have been produced in microorganisms. For this purpose, Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, and Ogataea minuta have been used as expression systems. The recombinant lysosomal proteins expressed in these hosts have shown similar substrate specificities, and temperature and pH stability profiles to those produced in mammalian cells. In addition, pre-clinical results have shown that recombinant lysosomal enzymes produced in microorganisms can be taken-up by cells and reduce the substrate accumulated within the lysosome. Recently, metabolic engineering in yeasts has allowed the production of lysosomal enzymes with tailored N-glycosylations, while progresses in E. coli N-glycosylations offer a potential platform to improve the production of these recombinant lysosomal enzymes. In summary, microorganisms represent convenient platform for the production of recombinant lysosomal proteins for biochemical and physicochemical characterization, as well as for the development of ERT for LSD. PMID:26071627

  15. Sequence analysis of the gene for a novel superantigen produced by Yersinia pseudotuberculosis and expression of the recombinant protein

    SciTech Connect

    Yasuhiko, Ito; Abe, Jun; Kohsaka, Takao

    1995-06-01

    We previously reported that the Gram-negative bacterium Yersinia pseudotuberculosis produces a superantigen (YPM, Y. pseudotuberculosis-derived mitogen) that expands T cells bearing V{beta}s 3, 9, 13.1, and 13.2 in an MHC class II-dependent manner. Based on the previously determined N-terminal 23 amino acids of YPM (T-D-Y-D-N-T-L-N-S-I-P-S-L-R-I-P-N-I-A-T-Y-T-G- (one-letter code)), we cloned the ypm gene and analyzed the nucleotide sequence. The gene encodes a 151-amino acid protein with a 20-amino acid signal peptide at its N terminus. The recombinant YPM expressed by the cloned gene exerted a mitogenic activity on human PBMC at a concentration of approximately 1 pg/ml. T cells bearing V{beta} 13.3 were preferentially expanded as well as T cells bearing the same V{beta} repertoires stimulated by native YPM. T cells were stimulated by the recombinant YPM in the presence of either fixed or unfixed HLA class II-transfected mouse fibroblasts. Furthermore, sequence diversity in the junctional region of the TCR {beta}-chain containing the V{beta}3 element could be observed after stimulation by the recombinant YPM. These results indicate that YPM belongs to the category of superantigens and should be included as a novel member. The amino acid sequence of the mature protein showed no significant homology to other superantigens derived from Gram-positive bacteria such as Staphylococcus aureus and Streptococcus pyogenes. This observation, together with the substantially smaller m.w. suggest that ypm must have evolved from a different ancestral gene. 67 refs., 7 figs., 5 tabs.

  16. Preparation of Specific Polyclonal Antibody Against the Recombinant Mutacin Produced by sfGFP Fusion Protein Technology

    PubMed Central

    Al-Homsi, Lamis; Al-Okla, Souad; Abbady, Abdul Q.

    2015-01-01

    Mutacin I, a bacteriocin produced by streptococcus mutans, displays an antimicrobial activity against many gram positive and some gram negative bacteria. Because of its medical importance, production of this short peptide in large scale for future applications is a significant challenge. This work described the improvement of a novel system to produce the recombinant mutacin using fusion protein technology. The short peptide was expressed directly as a fusion protein with a superfolder form of the green florescent protein (sfGFP), resulting in a high yield expression of soluble sfGFP-mutacin fusion protein (30 kDa) in the cytoplasm of E. coli. Mutacin was released from the fusion by enzymatic cleavage at the tobacco etch virus (TEV) protease recognition site and separated from the carrier sfGFP by nickel affinity and gel filtration chromatography. An additional advantage of this fusion system was tested in the generation of mutacin-specific polyclonal antibodies. Specific anti-mutacin IgGs were affinity purified, and were able to recognize the mutacin-sfGFP fusion protein or the cleaved forms of mutacin. Even though it was efficiently produced (25 mg/L) by this method, pure mutacin was devoid of antibiotic activity. Fourier transform infrared spectroscopy (FTIR) analysis revealed the absence of thioether bonds in the purified mutacin, which are critical for final structure and function of this antibiotic. Determining whether the activity of pure mutacin could be recovered by the reformation of such structures by chemical reaction needs more investigations. The development of this system will provide large quantities of mutacin for future studies and applications as broad spectrum antibacterial peptide. PMID:26668664

  17. Utilization of site-specific recombination for generating therapeutic protein producing cell lines.

    PubMed

    Campbell, Margie; Corisdeo, Susanne; McGee, Clair; Kraichely, Denny

    2010-07-01

    The AttSite Recombinase Technology from Intrexon, Blacksburg, VA, utilizes specific DNA sequences and proprietary recombinase enzymes to catalyze the insertion of a gene of interest at a specific location in the host cell genome. Using this technology, we have developed Chinese Hamster Ovary (CHO) cell lines that have incorporated attB recombination sites at highly transcriptionally active loci or 'hot spots' within the cell genome. Subsequently, these attB site containing host cell lines could then be used for the expression of future Centocor products. Candidate production cell lines would be generated by a simple recombination event. Since the therapeutic gene of interest would preferentially integrate into the pre-selected high-expressing attB site, candidate cell lines would consistently express high levels of the gene of interest. We have been able to demonstrate that the AttSite Recombinase Technology could be a valid approach for the development of high-expressing production cell lines. PMID:20300883

  18. A novel expression vector, designated as pHisJM, for producing recombinant His-fusion proteins.

    PubMed

    Masuda, Junko; Takayama, Eiji; Satoh, Ayano; Kojima-Aikawa, Kyoko; Suzuki, Kimihiro; Matsumoto, Isamu

    2004-10-01

    Compared to glutathione S -transferase (GST), tagging with hexahistidine residues (His) has several merits: low levels of toxicity and immunogenicity, a smaller size and no electric charge. We have constructed a novel expression vector, designated as pHisJM (EMBL/GenBank/DDJB accession no. AB116367), for producing recombinant His-fusion proteins. This vector was constructed by replacing GST and multiple cloning site (MCS) cassettes in pGEX-5X-3 with those of hexahistidine and MCS derived from pRSET C vector. Human annexin IV (Anx IV) was used as target protein. His-Anx IV fusion protein was expressed using pHisJM and gave a 40 kDa band when immuno-stained with anti-His mAb or anti-Anx IV mAb as predicted. To compare expression efficiency, a Anx IV cDNA inserted-pHisJM or pGEX-5X-3 was transformed into Escherichia coli DH5alpha, JM109, BL21 and BL21(DE3). Using pHisJM, Anx IV protein was highly expressed in all cell strains. In addition to the merits of using His-tag, pHisJM has several advantages: 1) it has high expression efficiency; 2) it can be used in any Escherichia coli strain; and 3) it can be used in a single strain of Escherichia coli in all steps from plasmid construction to the expression of the target gene. PMID:15604794

  19. Recombinant DNA production of spider silk proteins

    PubMed Central

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

    2013-01-01

    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. PMID:24119078

  20. Molecular farming of human cytokines and blood products from plants: challenges in biosynthesis and detection of plant-produced recombinant proteins.

    PubMed

    da Cunha, Nicolau B; Vianna, Giovanni R; da Almeida Lima, Thaina; Rech, Elíbio

    2014-01-01

    Plants have emerged as an attractive alternative to the traditional mammalian cell cultures or microbial cell-based systems system for the production of valuable recombinant proteins. Through recombinant DNA technology, plants can be engineered to produce large quantities of pharmaceuticals and industrial proteins of high quality at low costs. The recombinant production, by transgenic plants, of therapeutic proteins normally present in human plasma, such as cytokines, coagulation factors, anticoagulants, and immunoglobulins, represents a response to the ongoing challenges in meeting the demand for therapeutic proteins to treat serious inherited or acquired bleeding and immunological diseases. As the clinical utilization of fractionated plasma molecules is limited by high production costs, using recombinant biopharmaceuticals derived from plants represents a feasible alternative to provide efficient treatment. Plant-derived pharmaceuticals also reduce the potential risks to patients of infection with pathogens or unwanted immune responses due to immunogenic antigens. In this review, we summarize the recent advances in molecular farming of cytokines. We also examine the technological basis, upcoming challenges, and perspectives for the biosynthesis and detection of these molecules in different plant production platforms. PMID:24376137

  1. Fusion of the Dhfr/Mtx and IR/MAR Gene Amplification Methods Produces a Rapid and Efficient Method for Stable Recombinant Protein Production

    PubMed Central

    Miki, Daisuke; Shimizu, Noriaki

    2012-01-01

    Amplification of the dihydrofolate reductase gene (Dhfr) by methotrexate (Mtx) exposure is commonly used for recombinant protein expression in Chinese hamster ovary (CHO) cells. However, this method is both time- and labor-intensive, and the high-producing cells that are generated are frequently unstable in culture. Another gene amplification method is based on using a plasmid bearing a mammalian replication initiation region (IR) and a matrix attachment region (MAR), which result in the spontaneous initiation of gene amplification in transfected cells. The IR/MAR and Dhfr/Mtx methods of gene amplification are based on entirely different principles. In this study, we combine these two methods to yield a novel method, termed the IR/MAR-Dhfr fusion method, which was used to express three proteins, the Fc receptor, GFP, and recombinant antibody. The fusion method resulted in a dramatic increase in expression of all three proteins in two CHO sub-lines, DXB-11, and DG44. The IR/MAR-Dhfr fusion amplified the genes rapidly and efficiently, and produced larger amounts of antibody than the Dhfr/Mtx or IR/MAR methods alone. While the amplified structure produced by the Dhfr/Mtx method was highly unstable, and the antibody production rate rapidly decreased with the culture time of the cells, the IR/MAR-Dhfr fusion method resulted in stable amplification and generated clonal cells that produced large amounts of antibody protein over a long period of time. In summary, the novel IR/MAR-Dhfr fusion method enables isolation of stable cells that produce larger amounts of a target recombinant protein more rapidly and easily than either the Dhfr/Mtx or IR/MAR methods alone. PMID:23300841

  2. The pURI family of expression vectors: a versatile set of ligation independent cloning plasmids for producing recombinant His-fusion proteins.

    PubMed

    Curiel, José Antonio; de Las Rivas, Blanca; Mancheño, José Miguel; Muñoz, Rosario

    2011-03-01

    A family of restriction enzyme- and ligation-independent cloning vectors has been developed for producing recombinant His-tagged fusion proteins in Escherichia coli. These are based on pURI2 and pURI3 expression vectors which have been previously used for the successful production of recombinant proteins at the milligram scale. The newly designed vectors combines two different promoters (lpp(p)-5 and T7 RNA polymerase Ø10), two different endoprotease recognition sites for the His₆-tag removal (enterokinase and tobacco etch virus), different antibiotic selectable markers (ampicillin and erythromycin resistance), and different placements of the His₆-tag (N- and C-terminus). A single gene can be cloned and further expressed in the eight pURI vectors by using six nucleotide primers, avoiding the restriction enzyme and ligation steps. A unique NotI site was introduced to facilitate the selection of the recombinant plasmid. As a case study, the new vectors have been used to clone the gene coding for the phenolic acid decarboxylase from Lactobacillus plantarum. Interestingly, the obtained results revealed markedly different production levels of the target protein, emphasizing the relevance of the cloning strategy on soluble protein production yield. Efficient purification and tag removal steps showed that the affinity tag and the protease cleavage sites functioned properly. The novel family of pURI vectors designed for parallel cloning is a useful and versatile tool for the production and purification of a protein of interest. PMID:21055470

  3. Methanococcus vannielii selenium-binding protein (SeBP): Chemical reactivity of recombinant SeBP produced in Escherichia coli

    PubMed Central

    Patteson, Kemberly G.; Trivedi, Neel; Stadtman, Thressa C.

    2005-01-01

    A selenium-binding protein (SeBP) from Methanococcus vannielii was recently identified, and its gene was isolated and overexpressed in Escherichia coli [Self, W. T., Pierce, R. & Stadtman, T. C. (2004) IUBMB Life 56, 501–507]. SeBP and recombinant SeBP (rSeBP) migrated as ≈42-kDa species on native gels and as ≈33-kDa species on SDS gels. rSeBP consists of identical 8.8-kDa subunits, each containing a single cysteine residue. rSeBP isolated in the absence of reducing agents contained oxidized cysteine (89%) and very little bound selenium (0.05 eq or less per subunit). Complete reduction of the oxidized cysteine residues in rSeBP with Tris(2-carboxyethyl)phosphine required addition of a denaturant, such as 1 M guanidine-hydrochloride. With selenite as the selenium source and the isolated reduced protein as sole reductant, binding of one selenium per tetramer under anaerobic conditions required four cysteine thiol groups, one on each subunit. In the corresponding reaction, with reduced glutathione (GSH), equimolar amounts of selenodiglutathione (GSSeSG) and glutathione disulfide are formed from selenite and 4 GSH. At GSH-to-selenite ratios >4:1, conversion of GSSeSG to a perselenide derivative, GSSe–, occurs. However, with the reduced rSeBP as sole electron donor in the reaction with selenite, further conversion of the R-SSeS-R product apparently did not occur. Prior alkylation of the cysteine thiol groups in reduced rSeBP prevented selenite reduction and selenium binding under comparable conditions. PMID:16103372

  4. Use of a Chimeric Hsp70 to Enhance the Quality of Recombinant Plasmodium falciparum S-Adenosylmethionine Decarboxylase Protein Produced in Escherichia coli.

    PubMed

    Makhoba, Xolani Henry; Burger, Adélle; Coertzen, Dina; Zininga, Tawanda; Birkholtz, Lyn-Marie; Shonhai, Addmore

    2016-01-01

    S-adenosylmethionine decarboxylase (PfAdoMetDC) from Plasmodium falciparum is a prospective antimalarial drug target. The production of recombinant PfAdoMetDC for biochemical validation as a drug target is important. The production of PfAdoMetDC in Escherichia coli has been reported to result in unsatisfactory yields and poor quality product. The co-expression of recombinant proteins with molecular chaperones has been proposed as one way to improve the production of the former in E. coli. E. coli heat shock proteins DnaK, GroEL-GroES and DnaJ have previously been used to enhance production of some recombinant proteins. However, the outcomes were inconsistent. An Hsp70 chimeric protein, KPf, which is made up of the ATPase domain of E. coli DnaK and the substrate binding domain of P. falciparum Hsp70 (PfHsp70) has been previously shown to exhibit chaperone function when it was expressed in E. coli cells whose resident Hsp70 (DnaK) function was impaired. We proposed that because of its domain constitution, KPf would most likely be recognised by E. coli Hsp70 co-chaperones. Furthermore, because it possesses a substrate binding domain of plasmodial origin, KPf would be primed to recognise recombinant PfAdoMetDC expressed in E. coli. First, using site-directed mutagenesis, followed by complementation assays, we established that KPf with a mutation in the hydrophobic residue located in its substrate binding cavity was functionally compromised. We further co-expressed PfAdoMetDC with KPf, PfHsp70 and DnaK in E. coli cells either in the absence or presence of over-expressed GroEL-GroES chaperonin. The folded and functional status of the produced PfAdoMetDC was assessed using limited proteolysis and enzyme assays. PfAdoMetDC co-expressed with KPf and PfHsp70 exhibited improved activity compared to protein co-expressed with over-expressed DnaK. Our findings suggest that chimeric KPf may be an ideal Hsp70 co-expression partner for the production of recombinant plasmodial

  5. Use of a Chimeric Hsp70 to Enhance the Quality of Recombinant Plasmodium falciparum S-Adenosylmethionine Decarboxylase Protein Produced in Escherichia coli

    PubMed Central

    Makhoba, Xolani Henry; Burger, Adélle; Coertzen, Dina; Zininga, Tawanda; Birkholtz, Lyn-Marie; Shonhai, Addmore

    2016-01-01

    S-adenosylmethionine decarboxylase (PfAdoMetDC) from Plasmodium falciparum is a prospective antimalarial drug target. The production of recombinant PfAdoMetDC for biochemical validation as a drug target is important. The production of PfAdoMetDC in Escherichia coli has been reported to result in unsatisfactory yields and poor quality product. The co-expression of recombinant proteins with molecular chaperones has been proposed as one way to improve the production of the former in E. coli. E. coli heat shock proteins DnaK, GroEL-GroES and DnaJ have previously been used to enhance production of some recombinant proteins. However, the outcomes were inconsistent. An Hsp70 chimeric protein, KPf, which is made up of the ATPase domain of E. coli DnaK and the substrate binding domain of P. falciparum Hsp70 (PfHsp70) has been previously shown to exhibit chaperone function when it was expressed in E. coli cells whose resident Hsp70 (DnaK) function was impaired. We proposed that because of its domain constitution, KPf would most likely be recognised by E. coli Hsp70 co-chaperones. Furthermore, because it possesses a substrate binding domain of plasmodial origin, KPf would be primed to recognise recombinant PfAdoMetDC expressed in E. coli. First, using site-directed mutagenesis, followed by complementation assays, we established that KPf with a mutation in the hydrophobic residue located in its substrate binding cavity was functionally compromised. We further co-expressed PfAdoMetDC with KPf, PfHsp70 and DnaK in E. coli cells either in the absence or presence of over-expressed GroEL-GroES chaperonin. The folded and functional status of the produced PfAdoMetDC was assessed using limited proteolysis and enzyme assays. PfAdoMetDC co-expressed with KPf and PfHsp70 exhibited improved activity compared to protein co-expressed with over-expressed DnaK. Our findings suggest that chimeric KPf may be an ideal Hsp70 co-expression partner for the production of recombinant plasmodial

  6. Correction: BTI-Tnao38, a new cell line derived from Trichoplusia ni, is permissive for AcMNPV infection and produces high levels of recombinant proteins.

    PubMed

    Hashimoto, Yoshi; Zhang, Sheng; Zhang, Shiying; Chen, Yun-Ru; Blissard, Gary W

    2012-01-01

    After publication we discovered an error in the identification of the origin of the cell line reported in our article in BMC Biotechnology (2010, 10:50), entitled "Ao38, a new cell line from eggs of the black witch moth, Ascalapha odorata (Lepidoptera: Noctuidae), is permissive for AcMNPV infection and produces high levels of recombinant proteins". Upon analysis of primary A. odorata cultures, we found that they were contaminated with cells of Trichoplusia ni origin. The origin of the Ao38 cell line was determined as T. ni using three marker genes and the Ao38 cell line was renamed BTI-Tnao38. References to the origin of the cell line as Ascalapha odorata should be replaced with "a cell line of Trichoplusia ni origin". The absence of TNCL virus detection in the BTI-Tnao38 (Ao38) cell line was confirmed using a highly sensitive RT-PCR protocol capable of detecting TNCL virus RNA at approximately 0.018 copies/cell. Because of these observations, we have revised the title of the original article to "Correction: BTI-Tnao38, a new cell line derived from Trichoplusia ni, is permissive for AcMNPV infection and produces high levels of recombinant proteins" and two additional authors were added to reflect their contributions to the analysis of this cell line. PMID:22531032

  7. Recombinant protein production and streptomycetes.

    PubMed

    Anné, Jozef; Maldonado, Bárbara; Van Impe, Jan; Van Mellaert, Lieve; Bernaerts, Kristel

    2012-04-30

    The biopharmaceutical market has come a long way since 1982, when the first biopharmaceutical product, recombinant human insulin, was launched. Just over 200 biopharma products have already gained approval. The global market for biopharmaceuticals which is currently valued at over US$99 billion has been growing at an impressive compound annual growth rate over the previous years. To produce these biopharmaceuticals and other industrially important heterologous proteins, different prokaryotic and eukaryotic expression systems are used. All expression systems have some advantages as well as some disadvantages that should be considered in selecting which one to use. Choosing the best one requires evaluating the options--from yield to glycosylation, to proper folding, to economics of scale-up. No host cell from which all the proteins can be universally expressed in large quantities has been found so far. Therefore, it is important to provide a variety of host-vector expression systems in order to increase the opportunities to screen for the most suitable expression conditions or host cell. In this overview, we focus on Streptomyces lividans, a Gram-positive bacterium with a proven excellence in secretion capacity, as host for heterologous protein production. We will discuss its advantages and disadvantages, and how with systems biology approaches strains can be developed to better producing cell factories. PMID:21777629

  8. Improving recombinant protein purification yield

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  9. Functionalized silk assembled from a recombinant spider silk fusion protein (Z-4RepCT) produced in the methylotrophic yeast Pichia pastoris.

    PubMed

    Jansson, Ronnie; Lau, Cheuk H; Ishida, Takuya; Ramström, Margareta; Sandgren, Mats; Hedhammar, My

    2016-05-01

    Functional biological materials are a growing research area with potential applicability in medicine and biotechnology. Using genetic engineering, the possibility to introduce additional functions into spider silk-based materials has been realized. Recently, a recombinant spider silk fusion protein, Z-4RepCT, was produced intracellularly in Escherichia coli and could after purification self-assemble into silk-like fibers with ability to bind antibodies via the IgG-binding Z domain. In this study, the use of the methylotrophic yeast Pichia pastoris for production of Z-4RepCT has been investigated. Temperature, pH and production time were influencing the amount of soluble Z-4RepCT retrieved from the extracellular fraction. Purification of secreted Z-4RepCT resulted in a mixture of full-length and degraded silk proteins that failed to self-assemble into fibers. A position in the C-terminal domain of 4RepCT was identified as being subjected to proteolytic cleavage by proteases in the Pichia culture supernatant. Moreover, the C-terminal domain was subjected to glycosylation during production in P. pastoris. These observed alterations of the CT domain are suggested to contribute to the failure in fiber assembly. As alternative approach, Z-4RepCT retrieved from the intracellular fraction, which was less degraded, was used and shown to retain ability to assemble into silk-like fibers after enzymatic deglycosylation. PMID:26814048

  10. IL-10-IFN-γ Double Producers CD4+ T Cells Are Induced by Immunization with an Amastigote Stage Specific Derived Recombinant Protein of Trypanosoma Cruzi

    PubMed Central

    Flores-García, Yevel; Rosales-Encina, José Luis; Satoskar, Abhay R.; Talamás-Rohana, Patricia

    2011-01-01

    During the acute phase of infection, T. cruzi replicates extensively and releases immunomodulatory molecules that delay parasite-specific responses mediated by effector T cells. This mechanism of evasion allows the parasite to spread in the host. Parasite molecules that regulate the host immune response during Chagas'disease have not been fully identified. GPI-anchored mucins, glycoinositolphospholipids, and glycoproteins comprise some of the most abundant T. cruzi surface molecules. IL-10 IFN-γ-secreting CD4+ T cells are activated during chronic infections and are responsible for prolonged persistence of parasite and for host protection against severe inflammatory responses. In this work we evaluated the role of rMBP::SSP4 protein of T. cruzi, a recombinant protein derived from a GPI anchored antigen, SSP4, as an immunomodulator molecule, finding that it was able to induce high concentrations of IL-10 and IFN-γ both in vivo and in vitro; during this last condition, both cytokines were produced by IL-10-IFN-γ-secreting CD4+ T cells. PMID:21927578

  11. Flocculation increases the efficacy of depth filtration during the downstream processing of recombinant pharmaceutical proteins produced in tobacco.

    PubMed

    Buyel, Johannes F; Fischer, Rainer

    2014-02-01

    Flocculation is a cost-effective method that is used to improve the efficiency of clarification by causing dispersed particles to clump together, allowing their removal by sedimentation, centrifugation or filtration. The efficacy of flocculation for any given process depends on the nature and concentration of the particulates in the feed stream, the concentration, charge density and length of the flocculant polymer, the shear rate, the properties of the feed stream (e.g. pH and ionic strength) and the properties of the target products. We tested a range of flocculants and process conditions using a design of experiments approach to identify the most suitable polymers for the clarification step during the production of a HIV-neutralizing monoclonal antibody (2G12) and a fluorescent marker protein (DsRed) expressed in transgenic tobacco leaves. Among the 23 different flocculants we tested, the greatest reduction in turbidity was achieved with Polymin P, a branched, cationic polyethylenimine with a charge density of 13.0 meq/g. This flocculant reduced turbidity by more than 90% under a wide range of process conditions. We developed a model that predicted its performance under different process conditions, and this enabled us to increase the depth filter capacity three-sevenfold depending on the process scale, depth filter type and plant species. The costs of filter consumables were reduced by more than 50% compared with a process without flocculant, and there was no loss of recovery for either 2G12 or DsRed. PMID:24165151

  12. Immunosorbent Assay Based on Recombinant Hemagglutinin Protein Produced in a High-Efficiency Mammalian Expression System for Surveillance of Measles Immunity

    PubMed Central

    Bouche, Fabienne; Ammerlaan, Wim; Berthet, Francoise; Houard, Sophie; Schneider, Francois; Muller, Claude P.

    1998-01-01

    Recombinant hemagglutinin (H) protein of the measles virus (MV) was produced in mammalian cells with a high-yield expression system based on the Semliki Forest virus replicon. Crude membrane preparations of H protein-transfected BHK-21 cells were used to coat microtiter plates to measure specific immunoglobulin G antibodies in 228 serologically defined serum samples mainly from measles late-convalescent adults. The titers by the enzyme-linked immunosorbent assay for the H protein (H-ELISA) closely correlated with neutralization test (NT) titers (R2 = 0.66), hemagglutination inhibition test (HI) titers (R2 = 0.64), with the titers from a certified commercial ELISA based on whole MV-infected cells (MV-ELISA; R2 = 0.45). The correlations described above were better than those of the commercial MV-ELISA titers with the NT (R2 = 0.52) or HI (R2 = 0.48) titers. By using the 2nd International Standard for anti-measles serum, the detection level of the assay corresponds to 215 mIU/ml for undiluted serum, which corresponds to the estimated threshold for protective immunity. The specificity, accuracy, and positive predictive value were, in general, better for the H-ELISA than for a commercial MV-ELISA, independent of whether HI, NT, or HI and NT were used as “gold standards.” In contrast, the H-ELISA proved to be slightly less sensitive than the MV-ELISA (sensitivities, 98.6 versus 99.5%, respectively; P was not significant). The assays did not differ significantly in the number of serum samples with positive HI and NT results (n = 212) which measured false negative (H-ELISA, 2 of 212 [0.94%]; MV-ELISA, 1 of 212 [0.47%]), but the H-ELISA detected significantly more measles-susceptible individuals than the MV-ELISA (10 of 11 versus 3 of 11, respectively; P < 0.05) among the individuals whose sera had negative HI and NT results. Our data demonstrate that the H-protein preparation that we describe could be a cost-effective alternative to current whole-virus-based ELISAs for

  13. Utilizing Protein-lean Co-products from Corn Containing Recombinant Pharmaceutical Proteins for Ethanol Production

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein-lean fractions of corn (maize) containing recombinant (r) pharmaceutical proteins were used to produce fuel ethanol and residual r-proteins in the co-product, distillers dry grains with solubles (DDGS), were determined. Transgenic corn lines containing recombinant green fluorescence protein ...

  14. Polyclonal activation of naïve T cells by urease deficient-recombinant BCG that produced protein complex composed of heat shock protein 70, CysO and major membrane protein-II

    PubMed Central

    2014-01-01

    Background Mycobacterium bovis bacillus Calmette-Guérin (BCG) is known to be only partially effective in inhibiting M. tuberculosis (MTB) multiplication in human. A new recombinant (r) urease-deficient BCG (BCG-dHCM) that secretes protein composed of heat shock protein (HSP)70, MTB-derived CysO and major membrane protein (MMP)-II was produced for the efficient production of interferon gamma (IFN-γ) which is an essential element for mycobacteriocidal action and inhibition of neutrophil accumulation in lungs. Methods Human monocyte-derived dendritic cells (DC) and macrophages were differentiated from human monocytes, infected with BCG and autologous T cells-stimulating activity of different constructs of BCG was assessed. C57BL/6 mice were used to test the effectiveness of BCG for the production of T cells responsive to MTB-derived antigens (Ags). Results BCG-dHCM intracellularly secreted HSP70-CysO-MMP-II fusion protein, and activated DC by up-regulating Major Histcompatibility Complex (MHC), CD86 and CD83 molecules and enhanced various cytokines production from DC and macrophages. BCG-dHCM activated naïve T cells of both CD4 and CD8 subsets through DC, and memory type CD4+ T cells through macrophages in a manner dependent on MHC and CD86 molecules. These T cell activations were inhibited by the pre-treatment of Ag-presenting cells (APCs) with chloroquine. The single and primary BCG-dHCM-inoculation produced long lasting T cells responsive to in vitro secondarily stimulation with HSP70, CysO, MMP-II and H37Rv-derived cytosolic protein, and partially inhibited the replication of aerosol-challenged MTB. Conclusions The results indicate that introduction of different type of immunogenic molecules into a urease-deficient rBCG is useful for providing polyclonal T cell activating ability to BCG and for production of T cells responsive to secondary stimulation. PMID:24690183

  15. Extracellular secretion of recombinant proteins

    SciTech Connect

    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.

  16. Overview of the Purification of Recombinant Proteins

    PubMed Central

    Wingfield, Paul T.

    2015-01-01

    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 in this unit and elsewhere in the book. In the interim there has been a shift towards 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 from the other commonly used expression systems are mentioned and apart from cell breakage methods, the protein purification methods and strategies are essentially the same. PMID:25829302

  17. Transgenic silkworms produce recombinant human type III procollagen in cocoons.

    PubMed

    Tomita, Masahiro; Munetsuna, Hiroto; Sato, Tsutomu; Adachi, Takahiro; Hino, Rika; Hayashi, Masahiro; Shimizu, Katsuhiko; Nakamura, Namiko; Tamura, Toshiki; Yoshizato, Katsutoshi

    2003-01-01

    We describe the generation of transgenic silkworms that produce cocoons containing recombinant human collagen. A fusion cDNA was constructed encoding a protein that incorporated a human type III procollagen mini-chain with C-propeptide deleted, a fibroin light chain (L-chain), and an enhanced green fluorescent protein (EGFP). This cDNA was ligated downstream of the fibroin L-chain promoter and inserted into a piggyBac vector. Silkworm eggs were injected with the vectors, producing worms displaying EGFP fluorescence in their silk glands. The cocoons emitted EGFP fluorescence, indicating that the promoter and fibroin L-chain cDNAs directed the synthesized products to be secreted into cocoons. The presence of fusion proteins in cocoons was demonstrated by immunoblotting, collagenase-sensitivity tests, and amino acid sequencing. The fusion proteins from cocoons were purified to a single electrophoretic band. This study demonstrates the viability of transgenic silkworms as a tool for producing useful proteins in bulk. PMID:12483223

  18. Heterologous expression of newly identified galectin-8 from sea urchin embryos produces recombinant protein with lactose binding specificity and anti-adhesive activity

    PubMed Central

    Karakostis, Kostantinos; Costa, Caterina; Zito, Francesca; Matranga, Valeria

    2015-01-01

    Galectin family members specifically bind beta-galactoside derivatives and are involved in different cellular events, including cell communication, signalling, apoptosis, and immune responses. Here, we report a tandem-repeat type galectin from the Paracentrotus lividus sea urchin embryo, referred to as Pl-GAL-8. The 933nt sequence encodes a protein of 34.73 kDa, containing the conserved HFNPRF and WGxExR motifs in the two highly similar carbohydrate-recognition domains (CRD). The three-dimensional protein structure model of the N-CRD confirms the high evolutionary conservation of carbohydrate binding sites. The temporal gene expression is regulated during development and transcripts localize at the tip of the archenteron at gastrula stage, in a subset of the secondary mesenchyme cells that differentiate into blastocoelar (immune) cells. Functional studies using a recombinant Pl-GAL-8 expressed in bacteria demonstrate its hemo-agglutinating activity on human red blood cells through the binding to lactose, as well as its ability in inhibiting the adhesion of human Hep-G2 cells to the substrate. The recent implications in autoimmune diseases and inflammatory disorders make Gal-8 an attractive candidate for therapeutic purposes. Our results offer a solid basis for addressing the use of the new Pl-GAL-8 in functional and applicative studies, respectively in the developmental and biomedical fields. PMID:26640155

  19. Recombinant Human Factor IX Produced from Transgenic Porcine Milk

    PubMed Central

    Lee, Meng-Hwan; Lin, Yin-Shen; Tu, Ching-Fu; Yen, Chon-Ho

    2014-01-01

    Production of biopharmaceuticals from transgenic animal milk is a cost-effective method for highly complex proteins that cannot be efficiently produced using conventional systems such as microorganisms or animal cells. Yields of recombinant human factor IX (rhFIX) produced from transgenic porcine milk under the control of the bovine α-lactalbumin promoter reached 0.25 mg/mL. The rhFIX protein was purified from transgenic porcine milk using a three-column purification scheme after a precipitation step to remove casein. The purified protein had high specific activity and a low ratio of the active form (FIXa). The purified rhFIX had 11.9 γ-carboxyglutamic acid (Gla) residues/mol protein, which approached full occupancy of the 12 potential sites in the Gla domain. The rhFIX was shown to have a higher isoelectric point and lower sialic acid content than plasma-derived FIX (pdFIX). The rhFIX had the same N-glycosylation sites and phosphorylation sites as pdFIX, but had a higher specific activity. These results suggest that rhFIX produced from porcine milk is physiologically active and they support the use of transgenic animals as bioreactors for industrial scale production in milk. PMID:24955355

  20. Engineering Chinese hamster ovary (CHO) cells for producing recombinant proteins with simple glycoforms by zinc-finger nuclease (ZFN)-mediated gene knockout of mannosyl (alpha-1,3-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase (Mgat1).

    PubMed

    Sealover, Natalie R; Davis, Angela M; Brooks, Jeanne K; George, Henry J; Kayser, Kevin J; Lin, Nan

    2013-08-10

    While complex N-linked glycoforms are often desired in biotherapeutic protein production, proteins with simple, homogeneous glycan structure have implications for X-ray crystallography and for recombinant therapeutics targeted to the mannose receptor of antigen presenting cells. Mannosyl (alpha-1,3-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase (Mgat1, also called GnTI) adds N-acetylglucosamine to the Man5GlcNAc2 (Man5) N-glycan structure as part of complex N-glycan synthesis. Here, we report the use of zinc-finger nuclease (ZFN) genome editing technology to create Mgat1 disrupted Chinese hamster ovary (CHO) cell lines. These cell lines allow for the production of recombinant proteins with Man5 as the predominant N-linked glycosylation species. This method provides advantages over previously reported methods to create Mgat1-deficient cell lines. The use of ZFN-based genome editing eliminates potential regulatory concerns associated with random chemical mutagenesis, while retaining the robust growth and productivity characteristics of the parental cell lines. These Mgat1 disrupted cell lines may be used to produce mannose receptor-targeted therapeutic proteins. Cell line generation work can be performed in both Mgat1 disrupted and wild-type host cell lines to conduct X-ray crystallography studies of protein therapeutics in the same cell line used for production. PMID:23777858

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

    PubMed

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

    2012-01-01

    Molecular farming, long considered a promising strategy to produce valuable recombinant proteins not only for human and veterinary medicine, but also for agriculture and industry, now has some commercially available products. Various plant-based production platforms including whole-plants, aquatic plants, plant cell suspensions, and plant tissues (hairy roots) have been compared in terms of their advantages and limits. Effective recombinant strategies are summarized along with descriptions of scalable culture systems and examples of commercial progress and success. PMID:21924345

  2. Plant cell cultures for the production of recombinant proteins.

    PubMed

    Hellwig, Stephan; Drossard, Jürgen; Twyman, Richard M; Fischer, Rainer

    2004-11-01

    The use of whole plants for the synthesis of recombinant proteins has received a great deal of attention recently because of advantages in economy, scalability and safety compared with traditional microbial and mammalian production systems. However, production systems that use whole plants lack several of the intrinsic benefits of cultured cells, including the precise control over growth conditions, batch-to-batch product consistency, a high level of containment and the ability to produce recombinant proteins in compliance with good manufacturing practice. Plant cell cultures combine the merits of whole-plant systems with those of microbial and animal cell cultures, and already have an established track record for the production of valuable therapeutic secondary metabolites. Although no recombinant proteins have yet been produced commercially using plant cell cultures, there have been many proof-of-principle studies and several companies are investigating the commercial feasibility of such production systems. PMID:15529167

  3. Metabolic Adaptation in Transplastomic Plants Massively Accumulating Recombinant Proteins

    PubMed Central

    Bally, Julia; Job, Claudette; Belghazi, Maya; Job, Dominique

    2011-01-01

    Background Recombinant chloroplasts are endowed with an astonishing capacity to accumulate foreign proteins. However, knowledge about the impact on resident proteins of such high levels of recombinant protein accumulation is lacking. Methodology/Principal Findings Here we used proteomics to characterize tobacco (Nicotiana tabacum) plastid transformants massively accumulating a p-hydroxyphenyl pyruvate dioxygenase (HPPD) or a green fluorescent protein (GFP). While under the conditions used no obvious modifications in plant phenotype could be observed, these proteins accumulated to even higher levels than ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), the most abundant protein on the planet. This accumulation occurred at the expense of a limited number of leaf proteins including Rubisco. In particular, enzymes involved in CO2 metabolism such as nuclear-encoded plastidial Calvin cycle enzymes and mitochondrial glycine decarboxylase were found to adjust their accumulation level to these novel physiological conditions. Conclusions/Significance The results document how protein synthetic capacity is limited in plant cells. They may provide new avenues to evaluate possible bottlenecks in recombinant protein technology and to maintain plant fitness in future studies aiming at producing recombinant proteins of interest through chloroplast transformation. PMID:21966485

  4. Recombineering BAC transgenes for protein tagging.

    PubMed

    Ciotta, Giovanni; Hofemeister, Helmut; Maresca, Marcello; Fu, Jun; Sarov, Mihail; Anastassiadis, Konstantinos; Stewart, A Francis

    2011-02-01

    Protein tagging offers many advantages for proteomic and regulomic research, particularly due to the use of generic and highly sensitive methods that can be applied with reasonable throughput. Ideally, protein tagging is equivalent to having a high affinity antibody for every chosen protein. However, these advantages are compromised if the tagged protein is overexpressed, which is usually the case from cDNA expression vectors. BAC (bacterial artificial chromosome) transgenes present a way to express a chosen protein at physiological levels with all regulatory elements in their native configurations, including cell cycle, alternative splicing and microRNA regulation. Recombineering has become the method of choice for modifying large constructs like BACs. Here, we present a method for protein tagging by recombineering BACs, transfecting cells and evaluating tagged protein expression. PMID:20868752

  5. A Highly Efficient and Simple Construction Strategy for Producing Recombinant Baculovirus Bombyx mori Nucleopolyhedrovirus

    PubMed Central

    Liu, Xingjian; Wei, Yonglong; Li, Yinü; Li, Haoyang; Yang, Xin; Yi, Yongzhu; Zhang, Zhifang

    2016-01-01

    The silkworm baculovirus expression system is widely used to produce recombinant proteins. Several strategies for constructing recombinant viruses that contain foreign genes have been reported. Here, we developed a novel defective-rescue BmNPV Bacmid (reBmBac) expression system. A CopyControl origin of replication was introduced into the viral genome to facilitate its genetic manipulation in Escherichia coli and to ensure the preparation of large amounts of high quality reBmBac DNA as well as high quality recombinant baculoviruses. The ORF1629, cathepsin and chitinase genes were partially deleted or rendered defective to improve the efficiency of recombinant baculovirus generation and the expression of foreign genes. The system was validated by the successful expression of luciferase reporter gene and porcine interferon γ. This system can be used to produce batches of recombinant baculoviruses and target proteins rapidly and efficiently in silkworms. PMID:27008267

  6. A Highly Efficient and Simple Construction Strategy for Producing Recombinant Baculovirus Bombyx mori Nucleopolyhedrovirus.

    PubMed

    Liu, Xingjian; Wei, Yonglong; Li, Yinü; Li, Haoyang; Yang, Xin; Yi, Yongzhu; Zhang, Zhifang

    2016-01-01

    The silkworm baculovirus expression system is widely used to produce recombinant proteins. Several strategies for constructing recombinant viruses that contain foreign genes have been reported. Here, we developed a novel defective-rescue BmNPV Bacmid (reBmBac) expression system. A CopyControl origin of replication was introduced into the viral genome to facilitate its genetic manipulation in Escherichia coli and to ensure the preparation of large amounts of high quality reBmBac DNA as well as high quality recombinant baculoviruses. The ORF1629, cathepsin and chitinase genes were partially deleted or rendered defective to improve the efficiency of recombinant baculovirus generation and the expression of foreign genes. The system was validated by the successful expression of luciferase reporter gene and porcine interferon γ. This system can be used to produce batches of recombinant baculoviruses and target proteins rapidly and efficiently in silkworms. PMID:27008267

  7. Self-assembly of tunable protein suprastructures from recombinant oleosin

    PubMed Central

    Vargo, Kevin B.; Parthasarathy, Ranganath; Hammer, Daniel A.

    2012-01-01

    Using recombinant amphiphilic proteins to self-assemble suprastructures would allow precise control over surfactant chemistry and the facile incorporation of biological functionality. We used cryo-TEM to confirm self-assembled structures from recombinantly produced mutants of the naturally occurring sunflower protein, oleosin. We studied the phase behavior of protein self-assembly as a function of solution ionic strength and protein hydrophilic fraction, observing nanometric fibers, sheets, and vesicles. Vesicle membrane thickness correlated with increasing hydrophilic fraction for a fixed hydrophobic domain length. The existence of a bilayer membrane was corroborated in giant vesicles through the localized encapsulation of hydrophobic Nile red and hydrophilic calcein. Circular dichroism revealed that changes in nanostructural morphology in this family of mutants was unrelated to changes in secondary structure. Ultimately, we envision the use of recombinant techniques to introduce novel functionality into these materials for biological applications. PMID:22753512

  8. Recombinant protein blends: silk beyond natural design.

    PubMed

    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. PMID:26686863

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

    NASA Technical Reports Server (NTRS)

    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.

  10. Gene delivery into plant cells for recombinant protein production.

    PubMed

    Chen, Qiang; Lai, Huafang

    2015-01-01

    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. PMID:26075275

  11. Gene Delivery into Plant Cells for Recombinant Protein Production

    PubMed Central

    Chen, Qiang

    2015-01-01

    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. PMID:26075275

  12. Recombinant baculovirus vectors expressing glutathione-S-transferase fusion proteins.

    PubMed

    Davies, A H; Jowett, J B; Jones, I M

    1993-08-01

    Recombinant baculoviruses are a popular means of producing heterologous protein in eukaryotic cells. Purification of recombinant proteins away from the insect cell background can, however, remain an obstacle for many developments. Recently, prokaryotic fusion protein expression systems have been developed allowing single-step purification of the heterologous protein and specific proteolytic cleavage of the affinity tag moiety from the desired antigen. Here we report the introduction of these attributes to the baculovirus system. "Baculo-GEX" vectors enable baculovirus production of fusion proteins with the above advantages, but in a eukaryotic post-translational processing environment. Glutathione-S-transferase (GST) fusions are stable cytoplasmic proteins in insect cells and may therefore be released by sonication alone, avoiding the solubility problems and detergent requirements of bacterial systems. Thus large amounts of authentic antigen may be purified in a single, non-denaturing step. PMID:7763917

  13. Systemic delivery of recombinant proteins by genetically modified myoblasts

    SciTech Connect

    Barr, E.; Leiden, J.M. )

    1991-12-06

    The ability to stably deliver recombinant proteins to the systemic circulation would facilitate the treatment of a variety of acquired and inherited diseases. To explore the feasibility of the use of genetically engineered myoblasts as a recombinant protein delivery system, stable transfectants of the murine C2C12 myoblast cell line were produced that synthesize and secrete high levels of human growth hormone (hGH) in vitro. Mice injected with hGH-transfected myoblasts had significant levels of hGH in both muscle and serum that were stable for at least 3 weeks after injection. Histological examination of muscles injected with {beta}-galactosidase-expressing C2C12 myoblasts demonstrated that many of the injected cells had fused to form multinucleated myotubes. Thus, genetically engineered myoblasts can be used for the stable delivery of recombinant proteins into the circulation.

  14. Production of recombinant proteins in microalgae at pilot greenhouse scale.

    PubMed

    Gimpel, Javier A; Hyun, James S; Schoepp, Nathan G; Mayfield, Stephen P

    2015-02-01

    Recombinant protein production in microalgae chloroplasts can provide correctly folded proteins in significant quantities and potentially inexpensive costs compared to other heterologous protein production platforms. The best results have been achieved by using the psbA promoter and 5' untranslated region (UTR) to drive the expression of heterologous genes in a psbA-deficient, non-photosynthetic, algal host. Unfortunately, using such a strategy makes the system unviable for large scale cultivation using natural sunlight for photosynthetic growth. In this study we characterized eight different combinations of 5' regulatory regions and psbA coding sequences for their ability to restore photosynthesis in a psbA-deficient Chlamydomonas reinhardtii, while maintaining robust accumulation of a commercially viable recombinant protein driven by the psbA promoter/5'UTR. The recombinant protein corresponded to bovine Milk Amyloid A (MAA), which is present in milk colostrum and could be used to prevent infectious diarrhea in mammals. This approach allowed us to identify photosynthetic strains that achieved constitutive production of MAA when grown photosynthetically in 100 L bags in a greenhouse. Under these conditions, the maximum MAA expression achieved was 1.86% of total protein, which corresponded to 3.28 mg/L of culture medium. Within our knowledge, this is the first report of a recombinant protein being produced this way in microalgae. PMID:25116083

  15. Recombinant therapeutic proteins: production platforms and challenges.

    PubMed

    Dingermann, Theo

    2008-01-01

    Since the approval of insulin in 1982, more than 120 recombinant drug substances have been approved and become available as extremely valuable therapeutic options. Exact copying of the most common human form is no longer a value per se, as challenges, primarily related to the pharmacokinetics of artificial recombinant drugs, can be overcome by diverging from the original. However, relatively minor changes in manufacturing or packaging may impact safety of therapeutic proteins. A major achievement is the development of recombinant proteins capable of entering a cell. Such drugs open up completely new opportunities by targeting intracellular mechanisms or by substituting intracellularly operating enzymes. Concerns that protein variants would cause an intolerable immune response turned out to be exaggerated. Although most recombinant drugs provoke some immune response, they are still well tolerated. This knowledge might result in a change in attitude towards antibody formation, i.e., neutralizing antibody activity (in vitro) may be overcome by dosing consistently on the basis of antibody titers and not only on body weight. As with other drugs, efficacy and safety of therapeutic proteins have to be demonstrated in clinical studies, and superiority over available products has to be proven instead of just claimed. PMID:18041103

  16. [Plant-Producers Of Recombinant Cytokines (Review)].

    PubMed

    Burlakovskii, M S; Yemel'yanov, V V; Lutova, L A

    2016-01-01

    Cytokines are a family of signaling polypeptides involved in cell-cell interactions in the process of the immune response, as well as in the regulation of a number of normal physiological functions. Cytokines are used in medicine for the treatment of cancer, immune disorders, viral infections, and other socially significant diseases, but the extent of their use is limited by the high production cost of the active agent. The development of this area of pharmacology is associated with the success of genetic engineering, which allows the production of significant amounts of protein by transgenic organisms. The review discusses the latest advances in the production of various cytokines with the use of genetically modified plants. PMID:27266244

  17. Protein Crystal Recombinant Human Insulin

    NASA Technical Reports Server (NTRS)

    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.

  18. Strand invasion promoted by recombination protein of coliphage

    NASA Astrophysics Data System (ADS)

    Rybalchenko, Nataliya; Golub, Efim I.; Bi, Baoyuan; Radding, Charles M.

    2004-12-01

    Studies of phage in vivo have indicated that its own recombination enzymes, protein and exonuclease, are capable of catalyzing two dissimilar pathways of homologous recombination that are widely distributed in nature: single-strand annealing and strand invasion. The former is an enzymatic splicing of overlapping ends of broken homologous DNA molecules, whereas the latter is characterized by the formation of a three-stranded synaptic intermediate and subsequent strand exchange. Previous studies in vitro have shown that protein has annealing activity, and that exonuclease, acting on branched substrates, can produce a perfect splice that requires only ligation for completion. The present study shows that protein can initiate strand invasion in vitro, as evidenced both by the formation of displacement loops (D-loops) in superhelical DNA and by strand exchange between colinear single-stranded and double-stranded molecules. Thus, protein can catalyze steps that are central to both strand annealing and strand invasion pathways of recombination. These observations add protein to a set of diverse proteins that appear to promote recognition of homology by a unitary mechanism governed by the intrinsic dynamic properties of base pairs in DNA. genetic recombination | phage λ

  19. Optimizing the yield of recombinant pharmaceutical proteins in plants.

    PubMed

    Twyman, Richard M; Schillberg, Stefan; Fischer, Rainer

    2013-01-01

    The production of recombinant pharmaceutical proteins in plants is entering a new phase with the recent approval of recombinant glucocerebrosidase produced in carrot cells and the successful production of clinical-grade proteins in diverse plant-based production platforms. In the long journey from concept to product, the field of molecular farming has faced technical and economic hurdles, many reflecting the initially limited productivity of plants compared to established platforms such as mammalian cells. This challenge has been met by innovative research aiming to increase recombinant protein yields and maximize the economic benefits of plants. Research has focused on increasing the intrinsic yield capability of plants by optimizing expression construct design, and also on novel strategies to avoid epigenetic silencing and environmental effects on protein accumulation. In this article, we discuss the diverse approaches that have been used to increase the productivity of plant-based platforms for the production of recombinant pharmaceutical proteins and consider future opportunities to maximize the potential of plants and increase their competitiveness outside niche markets. PMID:23394567

  20. Recombinant protein scaffolds for tissue engineering.

    PubMed

    Werkmeister, Jerome A; Ramshaw, John A M

    2012-02-01

    New biological materials for tissue engineering are now being developed using common genetic engineering capabilities to clone and express a variety of genetic elements that allow cost-effective purification and scaffold fabrication from these recombinant proteins, peptides or from chimeric combinations of these. The field is limitless as long as the gene sequences are known. The utility is dependent on the ease, product yield and adaptability of these protein products to the biomedical field. The development of recombinant proteins as scaffolds, while still an emerging technology with respect to commercial products, is scientifically superior to current use of natural materials or synthetic polymer scaffolds, in terms of designing specific structures with desired degrees of biological complexities and motifs. In the field of tissue engineering, next generation scaffolds will be the key to directing appropriate tissue regeneration. The initial period of biodegradable synthetic scaffolds that provided shape and mechanical integrity, but no biological information, is phasing out. The era of protein scaffolds offers distinct advantages, particularly with the combination of powerful tools of molecular biology. These include, for example, the production of human proteins of uniform quality that are free of infectious agents and the ability to make suitable quantities of proteins that are found in low quantity or are hard to isolate from tissue. For the particular needs of tissue engineering scaffolds, fibrous proteins like collagens, elastin, silks and combinations of these offer further advantages of natural well-defined structural scaffolds as well as endless possibilities of controlling functionality by genetic manipulation. PMID:22262725

  1. Incidence of Lettuce mosaic virus in lettuce and its detection by polyclonal antibodies produced against recombinant coat protein expressed in Escherichia coli.

    PubMed

    Sharma, Prachi; Sharma, Susheel; Singh, Jasvir; Saha, Swati; Baranwal, V K

    2016-04-01

    Lettuce mosaic virus (LMV), a member of the genus Potyvirus of family Potyviridae, causes mosaic disease in lettuce has recently been identified in India. The virus is seed borne and secondary infection occurs through aphids. To ensure virus freedom in seeds it is important to develop diagnostic tools, for serological methods the production of polyclonal antibodies is a prerequisite. The coat protein (CP) gene of LMV was amplified, cloned and expressed using pET-28a vector in Escherichia coli BL21DE3 competent cells. The LMV CP was expressed as a fusion protein containing a fragment of the E. coli His tag. The LMV CP/His protein reacted positively with a commercial antiserum against LMV in an immunoblot assay. Polyclonal antibodies purified from serum of rabbits immunized with the fusion protein gave positive results when LMV infected lettuce (Lactuca sativa) was tested at 1:1000 dilution in PTA-ELISA. These were used for specific detection of LMV in screening lettuce accessions. The efficacy of the raised polyclonal antiserum was high and it can be utilized in quarantine and clean seed production. PMID:26850143

  2. Recombinant protein expression plasmids optimized for industrial E. coli fermentation and plant systems produce biologically active human insulin-like growth factor-1 in transgenic rice and tobacco plants.

    PubMed

    Panahi, Mitra; Alli, Zaman; Cheng, Xiongying; Belbaraka, Loubaba; Belgoudi, Jaafar; Sardana, Ravinder; Phipps, Jenny; Altosaar, Illimar

    2004-06-01

    Human insulin-like growth factor-1 (hIGF-1) is a growth factor with clinical significance in medicine. The therapeutic potential of recombinant hIGF-1 (rthIGF-1) stems from the fact that hIGF-1 resembles insulin in many aspects of physiology. The expression of hIGF-1 in transgenic tobacco and rice plants using different expression cassettes is reported here. In the present study, two coding sequences were tested, one with the original human sequence, but partially optimized for expression in E. coli and the other with a plant-codon-optimized sequence that was expected to give a higher level of expression in plant systems. Three different hIGF-1 recombinant expression constructs were generated. All expression constructs utilized the maize ubiquitin 1 promoter with or without a signal sequence. Analyses conducted using a hIGF-1 specific ELISA kit showed all transgenic plants produced hIGF-1 and the accumulated hIGF-1 increased from the E. coli codon bias to higher levels when the hIGF-1 coding sequence was codon-optimized to match that of the maize zeamatin protein--the most transcribed gene in maize endosperm suspension cells. Further analyses that compared the functionality of the bacterial signal peptide Lam B in plants showed that this leader peptide led to lower expression levels when compared to transgenic plants that did not contain this sequence. This indicated that this expression construct was functional without removal of the bacterial signal sequence. The maize ubiquitin 1 promoter was found to be more active in rice plants than tobacco plants indicating that in this case, there was a class preference that was biased towards a monocot host. Biological analyses conducted using protein extracts from transgenic plants showed that the rthIGF-1 was effective in stimulating the in vitro growth and proliferation of human SH-SY5Y neuroblastoma cells. This indicated that the plant-produced rthIGF-1 was stable and biologically active. As some plants have been

  3. Inhibition of Human MCF-7 Breast Cancer Cells and HT-29 Colon Cancer Cells by Rice-Produced Recombinant Human Insulin-Like Growth Binding Protein-3 (rhIGFBP-3)

    PubMed Central

    Liu, Lizhong; Liu, Qiaoquan; Lan, Linlin; Tong, Peter C. Y.; Sun, Samuel S. M.

    2013-01-01

    Background Insulin-like growth factor binding protein-3 (IGFBP-3) is a multifunctional molecule which is closely related to cell growth, apoptosis, angiogenesis, metabolism and senescence. It combines with insulin-like growth factor-I (IGF-I) to form a complex (IGF-I/IGFBP-3) that can treat growth hormone insensitivity syndrome (GHIS) and reduce insulin requirement in patients with diabetes. IGFBP-3 alone has been shown to have anti-proliferation effect on numerous cancer cells. Methodology/Principal Findings We reported here an expression method to produce functional recombinant human IGFBP-3 (rhIGFBP-3) in transgenic rice grains. Protein sorting sequences, signal peptide and endoplasmic reticulum retention tetrapeptide (KDEL) were included in constructs for enhancing rhIGFBP-3 expression. Western blot analysis showed that only the constructs with signal peptide were successfully expressed in transgenic rice grains. Both rhIGFBP-3 proteins, with or without KDEL sorting sequence inhibited the growth of MCF-7 human breast cancer cells (65.76 ± 1.72% vs 45.00 ± 0.86%, p < 0.05; 50.84 ± 1.97% vs 45.00 ± 0.86%, p < 0.01 respectively) and HT-29 colon cancer cells (65.14 ±3.84% vs 18.01 ± 13.81%, p < 0.05 and 54.7 ± 9.44% vs 18.01 ± 13.81%, p < 0.05 respectively) when compared with wild type rice. Conclusion/Significance These findings demonstrated the feasibility of producing biological active rhIGFBP-3 in rice using a transgenic approach, which will definitely encourage more research on the therapeutic use of hIGFBP-3 in future. PMID:24143239

  4. Lipopolysaccharide induced conversion of recombinant prion protein

    PubMed Central

    Saleem, Fozia; Bjorndahl, Trent C; Ladner, Carol L; Perez-Pineiro, Rolando; Ametaj, Burim N; Wishart, David S

    2014-01-01

    The conformational conversion of the cellular prion protein (PrPC) to the β-rich infectious isoform PrPSc is considered a critical and central feature in prion pathology. Although PrPSc is the critical component of the infectious agent, as proposed in the “protein-only” prion hypothesis, cellular components have been identified as important cofactors in triggering and enhancing the conversion of PrPC to proteinase K resistant PrPSc. A number of in vitro systems using various chemical and/or physical agents such as guanidine hydrochloride, urea, SDS, high temperature, and low pH, have been developed that cause PrPC conversion, their amplification, and amyloid fibril formation often under non-physiological conditions. In our ongoing efforts to look for endogenous and exogenous chemical mediators that might initiate, influence, or result in the natural conversion of PrPC to PrPSc, we discovered that lipopolysaccharide (LPS), a component of gram-negative bacterial membranes interacts with recombinant prion proteins and induces conversion to an isoform richer in β sheet at near physiological conditions as long as the LPS concentration remains above the critical micelle concentration (CMC). More significant was the LPS mediated conversion that was observed even at sub-molar ratios of LPS to recombinant ShPrP (90–232). PMID:24819168

  5. Systems biology of recombinant protein production using Bacillus megaterium.

    PubMed

    Biedendieck, Rebekka; Borgmeier, Claudia; Bunk, Boyke; Stammen, Simon; Scherling, Christian; Meinhardt, Friedhelm; Wittmann, Christoph; Jahn, Dieter

    2011-01-01

    The Gram-negative bacterium Escherichia coli is the most widely used production host for recombinant proteins in both academia and industry. The Gram-positive bacterium Bacillus megaterium represents an increasingly used alternative for high yield intra- and extracellular protein synthesis. During the past two decades, multiple tools including gene expression plasmids and production strains have been developed. Introduction of free replicating and integrative plasmids into B. megaterium is possible via protoplasts transformation or transconjugation. Using His(6)- and StrepII affinity tags, the intra- or extracellular produced proteins can easily be purified in one-step procedures. Different gene expression systems based on the xylose controlled promoter P(xylA) and various phage RNA polymerase (T7, SP6, K1E) driven systems enable B. megaterium to produce up to 1.25g of recombinant protein per liter. Biomass concentrations of up to 80g/l can be achieved by high cell density cultivations in bioreactors. Gene knockouts and gene replacements in B. megaterium are possible via an optimized gene disruption system. For a safe application in industry, sporulation and protease-deficient as well as UV-sensitive mutants are available. With the help of the recently published B. megaterium genome sequence, it is possible to characterize bottle necks in the protein production process via systems biology approaches based on transcriptome, proteome, metabolome, and fluxome data. The bioinformatical platform (Megabac, http://www.megabac.tu-bs.de) integrates obtained theoretical and experimental data. PMID:21943898

  6. Vaccination against Anthrax with Attenuated Recombinant Strains of Bacillus anthracis That Produce Protective Antigen

    PubMed Central

    Barnard, John P.; Friedlander, Arthur M.

    1999-01-01

    The protective efficacy of several live, recombinant anthrax vaccines given in a single-dose regimen was assessed with Hartley guinea pigs. These live vaccines were created by transforming ΔANR and ΔSterne, two nonencapsulated, nontoxinogenic strains of Bacillus anthracis, with four different recombinant plasmids that express the anthrax protective antigen (PA) protein to various degrees. This enabled us to assess the effect of the chromosomal background of the strain, as well as the amount of PA produced, on protective efficacy. There were no significant strain-related effects on PA production in vitro, plasmid stability in vivo, survival of the immunizing strain in the host, or protective efficacy of the immunizing infection. The protective efficacy of the live, recombinant anthrax vaccine strains correlated with the anti-PA antibody titers they elicited in vivo and the level of PA they produced in vitro. PMID:9916059

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

    DOEpatents

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

    1996-01-01

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

  8. Comparative Evaluation of Recombinant Protein Production in Different Biofactories: The Green Perspective

    PubMed Central

    Capaldi, Stefano

    2014-01-01

    In recent years, the production of recombinant pharmaceutical proteins in heterologous systems has increased significantly. Most applications involve complex proteins and glycoproteins that are difficult to produce, thus promoting the development and improvement of a wide range of production platforms. No individual system is optimal for the production of all recombinant proteins, so the diversity of platforms based on plants offers a significant advantage. Here, we discuss the production of four recombinant pharmaceutical proteins using different platforms, highlighting from these examples the unique advantages of plant-based systems over traditional fermenter-based expression platforms. PMID:24745008

  9. Human recombinant type I collagen produced in plants.

    PubMed

    Shoseyov, Oded; Posen, Yehudit; Grynspan, Frida

    2013-07-01

    As a central element of the extracellular matrix, collagen is intimately involved in tissue development, remodeling, and repair and confers high tensile strength to tissues. Numerous medical applications, particularly, wound healing, cell therapy, bone reconstruction, and cosmetic technologies, rely on its supportive and healing qualities. Its synthesis and assembly require a multitude of genes and post-translational modifications, where even minor deviations can be deleterious or even fatal. Historically, collagen was always extracted from animal and human cadaver sources, but bare risk of contamination and allergenicity and was subjected to harsh purification conditions resulting in irreversible modifications impeding its biofunctionality. In parallel, the highly complex and stringent post-translational processing of collagen, prerequisite of its viability and proper functioning, sets significant limitations on recombinant expression systems. A tobacco plant expression platform has been recruited to effectively express human collagen, along with three modifying enzymes, critical to collagen maturation. The plant extracted recombinant human collagen type I forms thermally stable helical structures, fibrillates, and demonstrates bioactivity resembling that of native collagen. Deployment of the highly versatile plant-based biofactory can be leveraged toward mass, rapid, and low-cost production of a wide variety of recombinant proteins. As in the case of collagen, proper planning can bypass plant-related limitations, to yield products structurally and functionally identical to their native counterparts. PMID:23252967

  10. The advances and perspectives of recombinant protein production in the silk gland of silkworm Bombyx mori.

    PubMed

    Xu, Hanfu

    2014-10-01

    The silk gland of silkworm Bombyx mori, is one of the most important organs that has been fully studied and utilized so far. It contributes finest silk fibers to humankind. The silk gland has excellent ability of synthesizing silk proteins and is a kind tool to produce some useful recombinant proteins, which can be widely used in the biological, biotechnical and pharmaceutical application fields. It's a very active area to express recombinant proteins using the silk gland as a bioreactor, and great progress has been achieved recently. This review recapitulates the progress of producing recombinant proteins and silk-based biomaterials in the silk gland of silkworm in addition to the construction of expression systems. Current challenges and future trends in the production of valuable recombinant proteins using transgenic silkworms are also discussed. PMID:25113390

  11. ELISA for brucellosis detection based on three Brucella recombinant proteins.

    PubMed

    Thepsuriyanont, Pikun; Intarapuk, Apiradee; Chanket, Panita; Tunyong, Wittawat; Kalambaheti, Thareerat

    2014-01-01

    Control of brucellosis among farm animals, wildlife and humans require reliable diagnosis. Rose Bengal serological test (RBT) is based on lipopolysaccharide antigen of Brucella, which may cross react with other gram-negative bacteria and produce false positive result. Immunoreactive proteins, such as outer-membrane protein BP26, ribosome recycling factor protein CP24 and Brucella lumazine synthase (BLS), previously reported to be recognized by infected sheep sera, were selected for production of recombinant proteins for use in an ELISA in order to investigate immune response among goats and cows, in comparison with commercial RBT. Cut-off value for ELISA was based on the immune response of in vitro fertilized goats and cows. Goats positive for Brucella culture or by RBT were ELISA positive for either IgG or IgM against at least one recombinant protein. For animals with negative RBT, animals with positive ELISA could be detected, and 61.6% possessed ELISA values as high as in infected animals. Thus, this ELISA procedure is proposed as an alternative to RBT for screening of brucellosis in farm animals. PMID:24964662

  12. Multivalent Recombinant Protein Vaccine against Coccidioidomycosis

    PubMed Central

    Tarcha, Eric J.; Basrur, Venkatesha; Hung, Chiung-Yu; Gardner, Malcolm J.; Cole, Garry T.

    2006-01-01

    Coccidioidomycosis is a human respiratory disease that is endemic to the southwestern United States and is caused by inhalation of the spores of a desert soilborne fungus. Efforts to develop a vaccine against this disease have focused on identification of T-cell-reactive antigens derived from the parasitic cell wall which can stimulate protective immunity against Coccidioides posadasii infection in mice. We previously described a productive immunoproteomic/bioinformatic approach to the discovery of vaccine candidates which makes use of the translated genome of C. posadasii and a computer-based method of scanning deduced sequences of seroreactive proteins for epitopes that are predicted to bind to human major histocompatibility (MHC) class II-restricted molecules. In this study we identified a set of putative cell wall proteins predicted to contain multiple, promiscuous MHC II binding epitopes. Three of these were expressed by Escherichia coli, combined in a vaccine, and tested for protective efficacy in C57BL/6 mice. Approximately 90% of the mice survived beyond 90 days after intranasal challenge, and the majority cleared the pathogen. We suggest that the multicomponent vaccine stimulates a broader range of T-cell clones than the single recombinant protein vaccines and thereby may be capable of inducing protection in an immunologically heterogeneous human population. PMID:16988258

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

    DOEpatents

    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.

  14. Integrated continuous production of recombinant therapeutic proteins.

    PubMed

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

    2012-12-01

    In the current environment of diverse product pipelines, rapidly fluctuating market demands and growing competition from biosimilars, biotechnology companies are increasingly driven to develop innovative solutions for highly flexible and cost-effective manufacturing. To address these challenging demands, integrated continuous processing, comprised of high-density perfusion cell culture and a directly coupled continuous capture step, can be used as a universal biomanufacturing platform. This study reports the first successful demonstration of the integration of a perfusion bioreactor and a four-column periodic counter-current chromatography (PCC) system for the continuous capture of candidate protein therapeutics. Two examples are presented: (1) a monoclonal antibody (model of a stable protein) and (2) a recombinant human enzyme (model of a highly complex, less stable protein). In both cases, high-density perfusion CHO cell cultures were operated at a quasi-steady state of 50-60 × 10(6) cells/mL for more than 60 days, achieving volumetric productivities much higher than current perfusion or fed-batch processes. The directly integrated and automated PCC system ran uninterrupted for 30 days without indications of time-based performance decline. The product quality observed for the continuous capture process was comparable to that for a batch-column operation. Furthermore, the integration of perfusion cell culture and PCC led to a dramatic decrease in the equipment footprint and elimination of several non-value-added unit operations, such as clarification and intermediate hold steps. These findings demonstrate the potential of integrated continuous bioprocessing as a universal platform for the manufacture of various kinds of therapeutic proteins. PMID:22729761

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

    PubMed

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

    2011-12-01

    Over the last few years microalgae have gained increasing interest as a natural source of valuable compounds and as bioreactors for recombinant protein production. Natural high-value compounds including pigments, long-chain polyunsaturated fatty acids, and polysaccharides, which have a wide range of applications in the food, feed, cosmetics, and pharmaceutical industries, are currently produced with nontransgenic microalgae. However, transgenic microalgae can be used as bioreactors for the production of therapeutic and industrially relevant recombinant proteins. This technology shows great promise to simplify the production process and significantly decrease the production costs. To date, a variety of recombinant proteins have been produced experimentally from the nuclear or chloroplast genome of transgenic Chlamydomonas reinhardtii. These include monoclonal antibodies, vaccines, hormones, pharmaceutical proteins, and others. In this review, we outline recent progress in the production of recombinant proteins with transgenic microalgae as bioreactors, methods for genetic transformation of microalgae, and strategies for highly efficient expression of heterologous genes. In particular, we highlight the importance of maximizing the value of transgenic microalgae through producing recombinant proteins together with recovery of natural high-value compounds. Finally, we outline some important issues that need to be addressed before commercial-scale production of high-value recombinant proteins and compounds from transgenic microalgae can be realized. PMID:21882013

  16. Dynamics of unfolded protein response in recombinant CHO cells.

    PubMed

    Prashad, Kamal; Mehra, Sarika

    2015-03-01

    Genes in the protein secretion pathway have been targeted to increase productivity of monoclonal antibodies in Chinese hamster ovary cells. The results have been highly variable depending on the cell type and the relative amount of recombinant and target proteins. This paper presents a comprehensive study encompassing major components of the protein processing pathway in the endoplasmic reticulum (ER) to elucidate its role in recombinant cells. mRNA profiles of all major ER chaperones and unfolded protein response (UPR) pathway genes are measured at a series of time points in a high-producing cell line under the dynamic environment of a batch culture. An initial increase in IgG heavy chain mRNA levels correlates with an increase in productivity. We observe a parallel increase in the expression levels of majority of chaperones. The chaperone levels continue to increase until the end of the batch culture. In contrast, calreticulin and ERO1-L alpha, two of the lowest expressed genes exhibit transient time profiles, with peak induction on day 3. In response to increased ER stress, both the GCN2/PKR-like ER kinase and inositol-requiring enzyme-1alpha (Ire1α) signalling branch of the UPR are upregulated. Interestingly, spliced X-Box binding protein 1 (XBP1s) transcription factor from Ire1α pathway is detected from the beginning of the batch culture. Comparison with the expression levels in a low producer, show much lower induction at the end of the exponential growth phase. Thus, the unfolded protein response strongly correlates with the magnitude and timing of stress in the course of the batch culture. PMID:24504562

  17. Data Mining for Expressivity of Recombinant Protein Expression

    NASA Astrophysics Data System (ADS)

    Kira, Satoshi; Isoai, Atsushi; Yamamura, Masayuki

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

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

    PubMed

    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. PMID:26805756

  19. Production of antigens in Chlamydomonas reinhardtii: green microalgae as a novel source of recombinant proteins.

    PubMed

    Fuhrmann, Markus

    2004-01-01

    Recombinant small-scale proteins are produced in a number of systems, from bacteria like Escherichia coli, through lower eukaryotes like baker's yeast, up to mammalian cell cultures. However, the need for safe and cheap sources of large amounts of recombinant proteins for different purposes, including material sciences, diagnostics, and, of course, medical therapy, has forced the development of alternative production systems. Green microalgae are cheap and easily grown and offer a high protein content, which would seem to make them ideal hosts for the large-scale sustainable production of recombinant proteins in the future. In selected species, recombinant DNA can be introduced into the genomes of the nucleus, the chloroplast, and even the mitochondria, and thus the system offers both prokaryotic (chloroplast, mitochondria) and eukaryotic translation systems for a tailored expression of virtually any protein. PMID:14959830

  20. Recombinant Salmonella typhimurium outer membrane protein A is recognized by synovial fluid CD8 cells and stimulates synovial fluid mononuclear cells to produce interleukin (IL)-17/IL-23 in patients with reactive arthritis and undifferentiated spondyloarthropathy.

    PubMed

    Chaurasia, S; Shasany, A K; Aggarwal, A; Misra, R

    2016-08-01

    In developing countries, one-third of patients with reactive arthritis (ReA) and undifferentiated spondyloarthropathy (uSpA) are triggered by Salmonella typhimurium. Synovial fluid mononuclear cells (SFMCs) of patients with ReA and uSpA proliferate to low molecular weight fractions (lmwf) of outer membrane proteins (Omp) of S. typhimurium. To characterize further the immunity of Omp of Salmonella, cellular immune response to two recombinant proteins of lmwf, OmpA and OmpD of S. typhimurium (rOmpA/D-sal) was assessed in 30 patients with ReA/uSpA. Using flow cytometry, 17 of 30 patients' SF CD8(+) T cells showed significant intracellular interferon (IFN)-γ to Omp crude lysate of S. typhimurium. Of these 17, 11 showed significantly more CD8(+) CD69(+) IFN-γ T cells to rOmpA-sal, whereas only four showed reactivity to rOmpD-sal. The mean stimulation index was significantly greater in rOmpA-sal than rOmpD-sal [3·0 (1·5-6·5) versus 1·5 (1·0-2·75), P < 0·005]. Similarly, using enzyme-linked immunospot (ELISPOT) in these 17 patients, the mean spots of IFN-γ-producing SFMCs were significantly greater in rOmpA-sal than rOmpD-sal [44·9 (3·5-130·7) versus 19·25 (6-41), P < 0·05]. SFMCs stimulated by rOmpA-sal produced significantly more proinflammatory cytokines than rOmpD-sal: IFN-γ [1·44 (0·39-20·42) versus 0·72 (0·048-9·15) ng/ml, P < 0·05], interleukin (IL)-17 [28·60 (6·15-510·86) versus 11·84 (6·83-252·62) pg/ml, P < 0·05], IL-23 [70·19 (15-1161·16) versus 28·25 (> 15-241·52) pg/ml, P < 0·05] and IL-6 [59·78 (2·03-273·36) versus 10·17 (0·004-190·19) ng/ml, P < 0·05]. The rOmpA-sal-specific CD8(+) T cell response correlated with duration of current synovitis (r = 0·53, P < 0·05). Thus, OmpA of S. typhimurium is a target of SF CD8(+) T cells and drives SFMC to produce increased cytokines of the IL-17/IL-23 axis which contribute to the pathogenesis of Salmonella-triggered ReA. PMID:27060348

  1. Recombinant bovine herpesvirus-1 expressing p23 protein of Cryptosporidium parvum induces neutralizing antibodies in rabbits.

    PubMed

    Takashima, Yasuhiro; Xuan, Xuenan; Kimata, Isao; Iseki, Motohiro; Kodama, Yoshikatsu; Nagane, Noriko; Nagasawa, Hideyuki; Matsumoto, Yasunobu; Mikami, Takeshi; Otsuka, Haruki

    2003-04-01

    In order to develop a vaccine against cryptosporidiosis in cattle, we constructed a recombinant bovine herpesvirus-1 (BHV-1) expressing an immunodominant surface protein, p23, of Cryptosporidium parvum sporozoites. In the recombinant virus, the p23 gene under the control of a CAG promoter and a gene coding for an enhanced green fluorescent protein were integrated into the gG gene of BHV-1. Despite a low frequency of homologous recombination, cloning of the recombinants was easy because of the specific fluorescence of the plaques formed by recombinants. These plaques were among the plaques of the nonfluorescent parental virus. All clones selected for fluorescence also contained the p23 gene. In MDBK cells infected with the recombinant BHV-1, the antibody against the p23 protein recognized the p23 protein as an approximately 23-kDa specific band in Western blotting analysis. Rabbits immunized with the recombinant produced IgG against the p23 protein. It was also demonstrated that the sera of immunized rabbits reduced infection of C. parvum sporozoites in HCT-8 cells. The serum of an immunized rabbit reduced infection compared with the normal rabbit serum control. These results indicate that the recombinant BHV-1 induces neutralizing antibodies in rabbits. PMID:12760641

  2. Human recombinant neutralizing antibodies against hantaan virus G2 protein.

    PubMed

    Koch, Joachim; Liang, Mifang; Queitsch, Iris; Kraus, Annette A; Bautz, Ekkehard K F

    2003-03-30

    Old world hantaviruses, causing hemorrhagic fever with renal syndrome (HFRS), still present a public health problem in Asia and Eastern Europe. The majority of cases has been recorded in China. The aim of our study was to generate human recombinant neutralizing antibodies to a hantavirus by phage display technology. To preserve the structural identity of viral protein, the panning procedure was performed on native Hantaan (HTN) (76-118) virus propagated in Vero-E6 cells. In total, five complete human recombinant IgG antibodies were produced in a baculovirus expression system. All of them were able to completely neutralize HTN, and Seoul (SEO) virus in a plaque reduction neutralization test (PRNT). Three of these antibodies could also completely neutralize Dobrava (DOB) virus but not Puumala (PUU) virus. All antibodies bind to Hantaan virus G2 protein localized in the virus envelope. The sequence areas within the HTN (76-118)-G2 protein detected by five selected antibodies were mapped using peptide scans. Two partial epitopes, 916-KVMATIDSF-924 and 954-LVTKDIDFD-963, were recognized, which presumably are of paramount importance for docking of the virus to host cell receptors. A consensus motif 916-KVXATIXSF-924 could be identified by mutational analysis. The neutralizing antibodies to the most widely distributed hantaviruses causing HFRS might be promising candidates for the development of an agent for prevention and treatment of HFRS in patients. PMID:12706090

  3. Chemical Polysialylation of Recombinant Human Proteins.

    PubMed

    Smirnov, Ivan V; Vorobiev, Ivan I; Belogurov, Alexey A; Genkin, Dmitry D; Deyev, Sergey M; Gabibov, Alexander G

    2015-01-01

    Design of drug with prolonged therapeutic action is one of the rapid developing fields of modern medical science and required implementation of different methods of protein chemistry and molecular biology. There are several therapeutic proteins needing increasing of their stability, pharmacokinetic, and pharmacodynamics parameters. To make long-live DNA-encoded drug PEGylation was proposed. Alternatively polysialic (colominic) acid, extracted from the cell wall of E. coli, fractionated to the desired size by anion-exchange chromatography and chemically activated to the amine-reactive aldehyde form, may be chemically attached to the polypeptide chain. Conjugates of proteins and polysialic acid generally resemble properties of protein-PEG conjugates, but possess significant negative net charge and are thought to be fully degradable after endocytosis due to the presence of intracellular enzymes, hydrolyzing the polysialic acid. Complete biodegradation of the polysialic acid moiety makes this kind of conjugates preferable for creation of drugs, intended for chronic use. Here, we describe two different protocols of chemical polysialylation. First protocol was employed for the CHO-derived human butyrylcholinesterase with optimized for recovery of specific enzyme activity. Polysialic acid moieties are attached at various lysine residues. Another protocol was developed for high-yield conjugation of human insulin; major conjugation point is the N-terminal residue of the insulin's light chain. These methods may allow to produce polysialylated conjugates of various proteins or polypeptides with reasonable yield and without significant loss of functional activity. PMID:26082236

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

    DOEpatents

    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.

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

    DOEpatents

    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.

  6. Metal binding proteins, recombinant host cells and methods

    DOEpatents

    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.

  7. Recombinant human N-acetylgalactosamine-6-sulfate sulfatase (GALNS) produced in the methylotrophic yeast Pichia pastoris

    PubMed Central

    Rodríguez-López, Alexander; Alméciga-Díaz, Carlos J.; Sánchez, Jhonnathan; Moreno, Jefferson; Beltran, Laura; Díaz, Dennis; Pardo, Andrea; Ramírez, Aura María; Espejo-Mojica, Angela J.; Pimentel, Luisa; Barrera, Luis A.

    2016-01-01

    Mucopolysaccharidosis IV A (MPS IV A, Morquio A disease) is a lysosomal storage disease (LSD) produced by mutations on N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Recently an enzyme replacement therapy (ERT) for this disease was approved using a recombinant enzyme produced in CHO cells. Previously, we reported the production of an active GALNS enzyme in Escherichia coli that showed similar stability properties to that of a recombinant mammalian enzyme though it was not taken-up by culture cells. In this study, we showed the production of the human recombinant GALNS in the methylotrophic yeast Pichia pastoris GS115 (prGALNS). We observed that removal of native signal peptide and co-expression with human formylglycine-generating enzyme (SUMF1) allowed an improvement of 4.5-fold in the specific GALNS activity. prGALNS enzyme showed a high stability at 4 °C, while the activity was markedly reduced at 37 and 45 °C. It was noteworthy that prGALNS was taken-up by HEK293 cells and human skin fibroblasts in a dose-dependent manner through a process potentially mediated by an endocytic pathway, without any additional protein or host modification. The results show the potential of P. pastoris in the production of a human recombinant GALNS for the development of an ERT for Morquio A. PMID:27378276

  8. Recombinant human N-acetylgalactosamine-6-sulfate sulfatase (GALNS) produced in the methylotrophic yeast Pichia pastoris.

    PubMed

    Rodríguez-López, Alexander; Alméciga-Díaz, Carlos J; Sánchez, Jhonnathan; Moreno, Jefferson; Beltran, Laura; Díaz, Dennis; Pardo, Andrea; Ramírez, Aura María; Espejo-Mojica, Angela J; Pimentel, Luisa; Barrera, Luis A

    2016-01-01

    Mucopolysaccharidosis IV A (MPS IV A, Morquio A disease) is a lysosomal storage disease (LSD) produced by mutations on N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Recently an enzyme replacement therapy (ERT) for this disease was approved using a recombinant enzyme produced in CHO cells. Previously, we reported the production of an active GALNS enzyme in Escherichia coli that showed similar stability properties to that of a recombinant mammalian enzyme though it was not taken-up by culture cells. In this study, we showed the production of the human recombinant GALNS in the methylotrophic yeast Pichia pastoris GS115 (prGALNS). We observed that removal of native signal peptide and co-expression with human formylglycine-generating enzyme (SUMF1) allowed an improvement of 4.5-fold in the specific GALNS activity. prGALNS enzyme showed a high stability at 4 °C, while the activity was markedly reduced at 37 and 45 °C. It was noteworthy that prGALNS was taken-up by HEK293 cells and human skin fibroblasts in a dose-dependent manner through a process potentially mediated by an endocytic pathway, without any additional protein or host modification. The results show the potential of P. pastoris in the production of a human recombinant GALNS for the development of an ERT for Morquio A. PMID:27378276

  9. Production and secretion of recombinant proteins in Dictyostelium discoideum.

    PubMed

    Dittrich, W; Williams, K L; Slade, M B

    1994-06-01

    We have expressed useful amounts of three recombinant proteins in a new eukaryotic host/vector system. The cellular slime mold Dictyostelium discoideum efficiently secreted two recombinant products, a soluble form of the normally cell surface associated D. discoideum glycoprotein (PsA) and the heterologous protein glutathione-S-transferase (GST) from Schistosoma japonicum, while the enzyme beta-glucuronidase (GUS) from Escherichia coli was cell associated. Up to 20mg/l of recombinant PsA and 1mg/l of GST were obtained after purification from a standard, peptone based growth medium. The secretion signal peptide was correctly cleaved from the recombinant GST- and PsA-proteins and the expression of recombinant PsA was shown to be stable for at least one hundred generations in the absence of selection. PMID:7764951

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

    PubMed

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

    2012-01-01

    Microbial biocatalysts are used in a wide range of industries to produce large scale quantities of proteins, amino acids, and commodity chemicals. While the majority of these processes use glucose or other low-cost sugars as the substrate, Bacillus methanolicus is one example of a biocatalyst that has shown sustained growth on methanol as a carbon source at elevated temperature (50-53°C optimum) resulting in reduced feed and utility costs. Specifically, the complete chemical process enabled by this approach takes methane from natural gas, and following a low-cost conversion to methanol, can be used for the production of high value products. In this study, production of recombinant green fluorescent protein (GFPuv) by B. methanolicus is explored. A plasmid was constructed that incorporates the methanol dehydrogenase (mdh) promoter of B. methanolicus MGA3 together with the GFPuv gene. The plasmid, pNW33N, was shown to be effective for expression in other Bacillus strains, although not previously in B. methanolicus. A published electroporation protocol for transformation of B. methanolicus was modified to result in expression of GFP using plasmid pNW33N-mdh-GFPuv (pNmG). Transformation was confirmed by both agarose gel electrophoresis and by observation of green fluorescence under UV light exposure. The mass yield of cells and protein were measured in shake flask experiments. The optimum concentration of methanol for protein production was found to be at 200 mM. Higher concentrations than 200 mM resulted in slightly higher biomass production but lower amounts of recombinant protein. PMID:22275315

  11. Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

    PubMed Central

    Weidner, Maria; Taupp, Marcus; Hallam, Steven J.

    2010-01-01

    Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and easy to use expression system. As a single-celled microorganism P. pastoris is easy to manipulate and grows rapidly on inexpensive media at high cell densities. Being a eukaryote, P. pastoris is able to perform many of the post-translational modifications performed by higher eukaryotic cells and the obtained recombinant proteins undergo protein folding, proteolytic processing, disulfide bond formation and glycosylation [1]. As a methylotrophic yeast P. pastoris is capable of metabolizing methanol as its sole carbon source. The strong promoter for alcohol oxidase, AOX1, is tightly regulated and induced by methanol and it is used for the expression of the gene of interest. Accordingly, the expression of the foreign protein can be induced by adding methanol to the growth medium [2; 3]. Another important advantage is the secretion of the recombinant protein into the growth medium, using a signal sequence to target the foreign protein to the secretory pathway of P. pastoris. With only low levels of endogenous protein secreted to the media by the yeast itself and no added proteins to the media, a heterologous protein builds the majority of the total protein in the medium and facilitates following protein purification steps [3; 4]. The vector used here (pPICZαA) contains the AOX1 promoter for tightly regulated, methanol-induced expression of the gene of interest; the α-factor secretion signal for secretion of the recombinant protein, a Zeocin resistance gene for selection in both E. coli and Pichia and a C-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis) tag for detection and purification of a recombinant protein. We also show western blot analysis of the recombinant protein using the specific Anti-myc-HRP antibody recognizing the c-myc epitope on the parent vector. PMID:20186119

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

    PubMed Central

    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 recombinant protein in the intracellular compartment is critical, at this would lessen any competition in this region between the endogenous seed proteins and the introduced foreign protein. The production yields of several recombinant proteins have been greatly increased by this strategy. PMID:23563599

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

    PubMed

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

    2015-12-10

    Production of recombinant proteins with microalgae represents an alternative platform over plant- or bacterial-based expression systems for certain target proteins. Secretion of recombinant proteins allows accumulation of the target product physically separate from the valuable algal biomass. To date, there has been little investigation into the dynamics of recombinant protein secretion from microalgal hosts-the culture parameters that encourage secreted product accumulation and stability, while encouraging biomass production. In this work, the efficiency of recombinant protein production was optimized by adjusting cultivation parameters for a strain of Chlamydomonas reinhardtii previously engineered to secrete a functional recombinant Lolium perenne ice binding protein (LpIBP), which has applications as a frozen food texturing and cryopreservation additive, into its culture medium. Three media and several cultivation styles were investigated for effects on secreted LpIBP titres and culture growth. A combination of acetate and carbon dioxide feeding with illumination resulted in the highest overall biomass and recombinant protein titres up to 10mgL(-1) in the culture medium. Pure photoautotrophic production was possible using two media types, with recombinant protein accumulation in all cultivations correlating to culture cell density. Two different cultivation systems were used for scale-up to 10L cultivations, one of which produced yields of secreted recombinant protein up to 12mgL(-1) within six cultivation days. Functional ice recrystallization inhibition (IRI) of the LpIBP from total concentrated extracellular protein extracts was demonstrated in a sucrose solution used as a simplified ice cream model. IRI lasted up to 7 days, demonstrating the potential of secreted products from microalgae for use as food additives. PMID:25975624

  14. Optimising yeast as a host for recombinant protein production (review).

    PubMed

    Bonander, Nicklas; Bill, Roslyn M

    2012-01-01

    Having access to suitably stable, functional recombinant protein samples underpins diverse academic and industrial research efforts to understand the workings of the cell in health and disease. Synthesising a protein in recombinant host cells typically allows the isolation of the pure protein in quantities much higher than those found in the protein's native source. Yeast is a popular host as it is a eukaryote with similar synthetic machinery to the native human source cells of many proteins of interest, while also being quick, easy, and cheap to grow and process. Even in these cells the production of some proteins can be plagued by low functional yields. We have identified molecular mechanisms and culture parameters underpinning high yields and have consolidated our findings to engineer improved yeast cell factories. In this chapter, we provide an overview of the opportunities available to improve yeast as a host system for recombinant protein production. PMID:22454109

  15. Challenges and opportunities in the purification of recombinant tagged proteins.

    PubMed

    Pina, Ana Sofia; Lowe, Christopher R; Roque, Ana Cecília A

    2014-01-01

    The purification of recombinant proteins by affinity chromatography is one of the most efficient strategies due to the high recovery yields and purity achieved. However, this is dependent on the availability of specific affinity adsorbents for each particular target protein. The diversity of proteins to be purified augments the complexity and number of specific affinity adsorbents needed, and therefore generic platforms for the purification of recombinant proteins are appealing strategies. This justifies why genetically encoded affinity tags became so popular for recombinant protein purification, as these systems only require specific ligands for the capture of the fusion protein through a pre-defined affinity tag tail. There is a wide range of available affinity pairs "tag-ligand" combining biological or structural affinity ligands with the respective binding tags. This review gives a general overview of the well-established "tag-ligand" systems available for fusion protein purification and also explores current unconventional strategies under development. PMID:24334194

  16. Baculovirus Coinfection Strategy for Improved Galactosylation of Recombinant Glycoprotein Produced by Insect Cell Culture

    NASA Astrophysics Data System (ADS)

    Ney, Yap Wei; Rahman, Badarulhisam Abdul; Aziz, Azila Abdul

    Baculovirus Expression Vector System (BEVS) is widely used for the production of recombinant glycoproteins, but it is not ideal for pharmaceutical glycoprotein production due to incomplete glycosylation. The factors that ensure successful glycosylation are the presence of sufficient amount of glycosyltransferases, sugar nucleotides as the substrate donor and the recombinant protein as the substrate acceptor. In this study, we analyzed the galactosylation process by the introduction of ß-1,4galactosyltransferase (ß-1,4GalT) as the glycosyltransferase of interest and uridine-5`-diphosphogalactose (UDP-Gal) as the substrate donor. Recombinant human transferrin (rhTf) as a model protein was used as the substrate acceptor. Insect cell lines have been reported to produce a small amount of ß-1,4GalT and thus insufficient for effective galactosylation. In this study, we developed a method to produce galactosylated rhTf and optimized the expression of rhTf with better N-glycan quality. Recombinant ß-1,4GalT was introduced during protein expression by the coinfection of the BEVS with baculovirus carrying bovine ß-1,4GalT. To evaluate the extent of galactosylation by the coinfection strategy, a binding assay was established. In this binding assay, glycoprotein acceptor was absorbed onto ELISA plate surface. A lectin known as Ricinus communis agglutinin-I (RCA-I) labeled with peroxidase, was added and allowed to recognize Gal ß1>4GlcNAc group on the N-glycan of the glycoprotein, followed by appropriate color reaction measurements. Coexpression between rhTf and ß-1,4GalT did not show encouraging results due to the reduction of UDP-Gal upon baculovirus infection. This interesting finding suggested that the introduction of ß-1,4GalT alone was not sufficient for successful galactosylation. Alternatively, post harvest glycosylation method strategy seems to be a promising technique in the improvement of glycoprotein quality.

  17. Extraction and downstream processing of plant-derived recombinant proteins.

    PubMed

    Buyel, J F; Twyman, R M; Fischer, R

    2015-11-01

    glycans, the ability to scale up production rapidly for emergency responses and the ability to produce commodity recombinant proteins on an agricultural scale. PMID:25922318

  18. [Processing and Modification of Recombinant Spider Silk Proteins].

    PubMed

    Liu, Bin; Wang, Tao; Liu, Xiaobing; Luo, Yongen

    2015-08-01

    Due to its special sequence structure, spider silk protein has unique physical and chemical properties, mechanical properties and excellent biological properties. With the expansion of the application value of spider silk in many fields as a functional material, progress has been made in the studies on the expression of recombinant spider silk proteins through many host systems by gene recombinant techniques. Recombinant spider silk proteins can be processed into high performance fibers, and a wide range of nonfibrous morphologies. Moreover, for their excellent biocompatibility and low immune response they are ideal for biomedical applications. Here we review the process and mechanism of preparation in vitro, chemistry and genetic engineering modification on recombinant spider silk protein. PMID:26710473

  19. Clinical development of plant-produced recombinant pharmaceuticals: vaccines, antibodies and beyond.

    PubMed

    Yusibov, Vidadi; Streatfield, Stephen J; Kushnir, Natasha

    2011-03-01

    In the last few years, plants have become an increasingly attractive platform for recombinant protein production. This builds on two decades of research, starting with transgenic approaches to develop oral vaccines in which antigens or therapeutics can be delivered in processed plant biomass, and progressing to transient expression approaches whereby high yields of purified targets are administered parenterally. The advantages of plant-based expression systems include high scalability, low upstream costs, biocontainment, lack of human or animal pathogens, and ability to produce target proteins with desired structures and biological functions. Using transgenic and transient expression in whole plants or plant cell culture, a variety of recombinant subunit vaccine candidates, therapeutic proteins, including monoclonal antibodies, and dietary proteins have been produced. Some of these products have been tested in early phase clinical trials, and show safety and efficacy. Among those are mucosal vaccines for diarrheal diseases, hepatitis B and rabies; injectable vaccines for non-Hodgkin's lymphoma, H1N1 and H5N1 strains of influenza A virus, and Newcastle disease in poultry; and topical antibodies for the treatment of dental caries and HIV. As lead plant-based products have entered clinical trials, there has been increased emphasis on manufacturing under current Good Manufacturing Practice (cGMP) guidelines, and the preparation and presentation to the relevant government agencies of regulatory packages. PMID:21346417

  20. Recombinant human bone morphogenetic protein induces bone formation.

    PubMed Central

    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

    1990-01-01

    We 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 micrograms 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. Images PMID:2315314

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

    PubMed

    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. PMID:26614293

  2. Clinical trial to evaluate safety and immunogenicity of an oral inactivated enterotoxigenic Escherichia coli prototype vaccine containing CFA/I overexpressing bacteria and recombinantly produced LTB/CTB hybrid protein.

    PubMed

    Lundgren, A; Leach, S; Tobias, J; Carlin, N; Gustafsson, B; Jertborn, M; Bourgeois, L; Walker, R; Holmgren, J; Svennerholm, A-M

    2013-02-01

    We have developed a new oral vaccine against enterotoxigenic Escherichia coli (ETEC) diarrhea containing killed recombinant E. coli bacteria expressing increased levels of ETEC colonization factors (CFs) and a recombinant protein (LCTBA), i.e. a hybrid between the binding subunits of E. coli heat labile toxin (LTB) and cholera toxin (CTB). We describe a randomized, comparator controlled, double-blind phase I trial in 60 adult Swedish volunteers of a prototype of this vaccine. The safety and immunogenicity of the prototype vaccine, containing LCTBA and an E. coli strain overexpressing the colonization factor CFA/I, was compared to a previously developed oral ETEC vaccine, consisting of CTB and inactivated wild type ETEC bacteria expressing CFA/I (reference vaccine). Groups of volunteers were given two oral doses of either the prototype or the reference vaccine; the prototype vaccine was administered at the same or a fourfold higher dosage than the reference vaccine. The prototype vaccine was found to be safe and equally well-tolerated as the reference vaccine at either dosage tested. The prototype vaccine induced mucosal IgA (fecal secretory IgA and intestine-derived IgA antibody secreting cell) responses to both LTB and CFA/I, as well as serum IgA and IgG antibody responses to LTB. Immunization with LCTBA resulted in about twofold higher mucosal and systemic IgA responses against LTB than a comparable dose of CTB. The higher dose of the prototype vaccine induced significantly higher fecal and systemic IgA responses to LTB and fecal IgA responses to CFA/I than the reference vaccine. These results demonstrate that CF over-expression and inclusion of the LCTBA hybrid protein in an oral inactivated ETEC vaccine does not change the safety profile when compared to a previous generation of such a vaccine and that the prototype vaccine induces significant dose dependent mucosal immune responses against CFA/I and LTB. PMID:23306362

  3. Recombinant production and film properties of full-length hornet silk proteins.

    PubMed

    Kambe, Yusuke; Sutherland, Tara D; Kameda, Tsunenori

    2014-08-01

    Full-length versions of the four main components of silk cocoons of Vespa simillima hornets, Vssilk1-4, were produced as recombinant proteins in Escherichia coli. In shake flasks, the recombinant Vssilk proteins yielded 160-330mg recombinant proteinl(-1). Films generated from solutions of single Vssilk proteins had a secondary structure similar to that of films generated from native hornet silk. The films made from individual recombinant hornet silk proteins had similar or enhanced mechanical performance compared with films generated from native hornet silk, possibly reflecting the homogeneity of the recombinant proteins. The pH-dependent changes in zeta (ζ) potential of each Vssilk film were measured, and isoelectric points (pI) of Vssilk1-4 were determined as 8.9, 9.1, 5.0 and 4.2, respectively. The pI of native hornet silk, a combination of the four Vssilk proteins, was 4.7, a value similar to that of Bombyx mori silkworm silk. Films generated from Vssilk1 and 2 had net positive charge under physiological conditions and showed significantly higher cell adhesion activity. It is proposed that recombinant hornet silk is a valuable new material with potential for cell culture applications. PMID:24862540

  4. Recombinant Protein Production and Insect Cell Culture and Process

    NASA Technical Reports Server (NTRS)

    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.

  5. Recombinant protein production and insect cell culture and process

    NASA Technical Reports Server (NTRS)

    Spaulding, Glenn (Inventor); Prewett, Tacey (Inventor); Goodwin, Thomas (Inventor); Francis, Karen (Inventor); Andrews, Angela (Inventor); Oconnor, Kim (Inventor)

    1993-01-01

    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 the cultured insect cells as host for a virus encoding the described polypeptide such as baculovirus. The insect cells can also be a host for viral production.

  6. Recombinant Mycobacterium bovis bacillus Calmette-Guérin expressing Ag85B-IL-7 fusion protein enhances IL-17A-producing innate γδ T cells.

    PubMed

    Hatano, Shinya; Tamura, Toshiki; Umemura, Masayuki; Matsuzaki, Goro; Ohara, Naoya; Yoshikai, Yasunobu

    2016-05-11

    Interleukin 7 (IL-7) has an important function in the development and maintenance of IL-17A+ γδ T cells. We here constructed a recombinant Mycobacterium bovis bacillus Calmette-Guérin expressing antigen 85B (Ag85B)-IL-7 fusion protein (rBCG-Ag85B-IL-7). The Ag85B-IL-7 fusion protein and IL-7 were detected in the bacterial lysate of rBCG-Ag85B-IL-7. rBCG-Ag85B-IL-7 was the same in number as control rBCG expressing Ag85B (rBCG-Ag85B) in the lung at the early stage after intravenous inoculation, whereas the numbers of IL-17A+ γδ T cells and Ag-specific Th1 cells were significantly higher in the lungs of mice inoculated with rBCG-Ag85B-IL-7 than those inoculated with rBCG-Ag85B. The Ag-specific Th1 cell response was impaired in mice lacking IL-17A+ γδ T cells after inoculation with rBCG-Ag85B-IL-7. Thus, rBCG-Ag85B-IL-7 increases the pool size of IL-17A+ γδ T cells, which subsequently augment the Th1 response to mycobacterial infection. PMID:27079930

  7. Expression and Characterization of Recombinant Serratia liquefaciens Nucleases Produced with Baculovirus-mediated Silkworm Expression System.

    PubMed

    Iiyama, Kazuhiro; Lee, Jae Man; Tatsuke, Tuneyuki; Mon, Hiroaki; Kusakabe, Takahiro

    2016-06-01

    Baculovirus-Bombyx mori protein expression system has mainly been used for translation of eukaryotic proteins. In contrast, information pertaining to bacterial protein expression using this system is not sufficient. Therefore, recombinant nucleases from Serratia liquefaciens (rSlNucAs) were expressed in a Baculovirus-B. mori protein expression system. rSlNucAs containing the native signal peptide (rSlNucA-NSP) or silkworm 30-K signal peptide (rSlNucA-30K) at the NH2-terminus were constructed to enable secretion into the extracellular fraction. Both rSlNucA-30K and rSlNucA-NSP were successfully secreted into hemolymph of B. mori larvae. Affinity-purified rSlNucAs showed high nuclease activity. Optimum pH was 7.5 and half of maximum activity was maintained between pH 7.0 and 9.5. Optimum temperature was 35 °C. rSlNucAs showed sufficient activity in twofold-diluted radioimmunoprecipitation assay buffer and undiluted, mild lysis buffer. Genomic DNA of Escherichia coli was efficiently digested by rSlNucAs in the bacterial lysate. The results in this study suggest that rSlNucAs expressed by the Baculovirus-B. mori protein expression system will be a useful tool in molecular biology. Functional recombinant protein of bacteria was produced by Baculovirus-B. mori protein expression system. This system may be highly suitable for bacterial extracellular protein secreted via Sec pathway. PMID:27059494

  8. Overexpression and Enzymatic Assessment of Antigenic Fragments of Hyaluronidase Recombinant Protein From Streptococcus pyogenes

    PubMed Central

    Sadoogh Abbasian, Shabnam; Ghaznavi Rad, Ehsanollah; Akbari, Neda; Zolfaghari, Mohammad Reza; pakzad, Iraj; Abtahi, Hamid

    2014-01-01

    Background: Hyaluronidase catalyzes the hydrolysis of hyaluronan polymers to N-acetyl-D-glucosamine and D-glucuronic acid. This enzyme is a dimer of identical subunits. Hyaluronidase has different pharmaceutical and medical applications. Previously, we produced a recombinant hyaluronidase antigenic fragment of Streptococcus pyogenes. Objectives: This study aimed to improve the protein production and purity of hyaluronidase recombinant protein from S. pyogenes. In addition, the enzymatic activity of this protein was investigated. Materials and Methods: The expression of hyaluronidase antigenic fragments was optimized using IPTG concentration, time of induction, temperature, culture, and absorbance of 0.6-0.8-1 at 600 nm. Afterwards, the expressed proteins were purified and the enzymatic activity was assessed by turbid metric method. Results: Data indicated that maximum protein is produced in OD = 0.8, 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG), 37ºC, NB 1.5x, without glucose, incubated for overnight. The enzymatic activity of the recombinant protein was similar to the commercial form of hyaluronidase. Conclusions: The results showed that an antigenic fragment of the recombinant hyaluronidase protein from S. pyogenes has a considerable enzymatic activity. It can be suggested to use it for medical purposes. In addition, applications of bioinformatics software would facilitate the production of a smaller protein with same antigenic properties and enzymatic activity. PMID:25789122

  9. Purification of recombinant protein by cold-coacervation of fusion constructs incorporating resilin-inspired polypeptides.

    PubMed

    Lyons, Russell E; Elvin, Christopher M; Taylor, Karin; Lekieffre, Nicolas; Ramshaw, John A M

    2012-12-01

    Polypeptides containing between 4 and 32 repeats of a resilin-inspired sequence AQTPSSYGAP, derived from the mosquito Anopheles gambiae, have been used as tags on recombinant fusion proteins. These repeating polypeptides were inspired by the repeating structures that are found in resilins and sequence-related proteins from various insects. Unexpectedly, an aqueous solution of a recombinant resilin protein displays an upper critical solution temperature (cold-coacervation) when held on ice, leading to a separation into a protein rich phase, typically exceeding 200 mg/mL, and a protein-poor phase. We show that purification of recombinant proteins by cold-coacervation can be performed when engineered as a fusion partner to a resilin-inspired repeat sequence. In this study, we demonstrate the process by the recombinant expression and purification of enhanced Green fluorescent protein (EGFP) in E. coli. This facile purification system can produce high purity, concentrated protein solutions without the need for affinity chromatography or other time-consuming or expensive purification steps, and that it can be used with other bulk purification steps such as low concentration ammonium sulfate precipitation. Protein purification by cold-coacervation also minimizes the exposure of the target protein to enhanced proteolysis at higher temperature. PMID:22627880

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

    PubMed Central

    Rosano, Germán L.; Ceccarelli, Eduardo A.

    2014-01-01

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

  11. Optimizing transient recombinant protein expression in mammalian cells.

    PubMed

    Hopkins, Ralph F; Wall, Vanessa E; Esposito, Dominic

    2012-01-01

    Transient gene expression (TGE) in mammalian cells has become a routine process for expressing recombinant proteins in cell lines such as human embryonic kidney 293 and Chinese hamster ovary cells. The rapidly increasing need for recombinant proteins requires further improvements in TGE technology. While a great deal of focus has been directed toward optimizing the secretion of antibodies and other naturally secreted targets, much less work has been done on ways to improve cytoplasmic expression in mammalian cells. The benefits to protein production in mammalian cells, particularly for eukaryotic proteins, should be very significant - glycosylation and other posttranslational modifications will likely be native or near-native, solubility and protein folding would likely improve overexpression in heterologous hosts, and expression of proteins in their proper intracellular compartments is much more likely to occur. Improvements in this area have been slow, however, due to limited development of the cell culture processes needed for low-cost, higher-throughput expression in mammalian cells, and the relatively low diversity of DNA vectors for protein production in these systems. Here, we describe how the use of recombinational cloning, coupled with improvements in transfection protocols which increase speed and lower cost, can be combined to make mammalian cells much more amenable for routine recombinant protein expression. PMID:21987258

  12. Production of recombinant protein in Escherichia coli cultured in extract from waste product alga, Ulva lactuca.

    PubMed

    Rechtin, Tammy M; Hurst, Matthew; Potts, Tom; Hestekin, Jamie; Beitle, Robert; McLaughlin, John; May, Peter

    2014-01-01

    This study examined the potential for waste product alga, Ulva lactuca, to serve as a media component for recombinant protein production in Escherichia coli. To facilitate this investigation, U. lactuca harvested from Jamaica Bay was dried, and nutrients acid extracted for use as a growth media. The E. coli cell line BL21(DE3) was used to assess the effects on growth and production of recombinant green fluorescent protein (GFP). This study showed that media composed of acid extracts without further nutrient addition maintained E. coli growth and recombinant protein production. Extracts made from dried algae lots less than six-months-old were able to produce two-fold more GFP protein than traditional Lysogeny Broth media. PMID:24799463

  13. Generation of a Chinese Hamster Ovary Cell Line Producing Recombinant Human Glucocerebrosidase

    PubMed Central

    Novo, Juliana Branco; Morganti, Ligia; Moro, Ana Maria; Paes Leme, Adriana Franco; Serrano, Solange Maria de Toledo; Raw, Isaias; Ho, Paulo Lee

    2012-01-01

    Impaired activity of the lysosomal enzyme glucocerebrosidase (GCR) results in the inherited metabolic disorder known as Gaucher disease. Current treatment consists of enzyme replacement therapy by administration of exogenous GCR. Although effective, it is exceptionally expensive, and patients worldwide have a limited access to this medicine. In Brazil, the public healthcare system provides the drug free of charge for all Gaucher's patients, which reaches the order of $ 84 million per year. However, the production of GCR by public institutions in Brazil would reduce significantly the therapy costs. Here, we describe a robust protocol for the generation of a cell line producing recombinant human GCR. The protein was expressed in CHO-DXB11 (dhfr−) cells after stable transfection and gene amplification with methotrexate. As expected, glycosylated GCR was detected by immunoblotting assay both as cell-associated (~64 and 59 kDa) and secreted (63–69 kDa) form. Analysis of subclones allowed the selection of stable CHO cells producing a secreted functional enzyme, with a calculated productivity of 5.14 pg/cell/day for the highest producer. Although being laborious, traditional methods of screening high-producing recombinant cells may represent a valuable alternative to generate expensive biopharmaceuticals in countries with limited resources. PMID:23091360

  14. Biomimetic production of silk-like recombinant squid sucker ring teeth proteins.

    PubMed

    Ding, Dawei; Guerette, Paul A; Hoon, Shawn; Kong, Kiat Whye; Cornvik, Tobias; Nilsson, Martina; Kumar, Akshita; Lescar, Julien; Miserez, Ali

    2014-09-01

    The sucker ring teeth (SRT) of Humboldt squid exhibit mechanical properties that rival those of robust engineered synthetic polymers. Remarkably, these properties are achieved without a mineral phase or covalent cross-links. Instead, SRT are exclusively made of silk-like proteins called "suckerins", which assemble into nanoconfined β-sheet reinforced supramolecular networks. In this study, three streamlined strategies for full-length recombinant suckerin protein production and purification were developed. Recombinant suckerin exhibited high solubility and colloidal stability in aqueous-based solvents. In addition, the colloidal suspensions exhibited a concentration-dependent conformational switch, from random coil to β-sheet enriched structures. Our results demonstrate that recombinant suckerin can be produced in a facile manner in E. coli and processed from mild aqueous solutions into materials enriched in β-sheets. We suggest that recombinant suckerin-based materials offer potential for a range of biomedical and engineering applications. PMID:25068184

  15. Tying up the loose ends: circular permutation decreases the proteolytic susceptibility of recombinant proteins.

    PubMed

    Whitehead, Timothy A; Bergeron, Lisa M; Clark, Douglas S

    2009-10-01

    Recombinant proteins often suffer from poor expression because of proteolysis. Existing genetic engineering or fermentation strategies work for only a subset of cases where higher recombinant protein expression is needed. In this paper, we describe the use of circular permutation, wherein the original termini of a protein are concatenated and new termini are generated elsewhere with the sequence, as a general protein engineering strategy to produce full-length, active recombinant protein. We show that a circularly permuted variant of the thermosome (Group II chaperonin) from Methanocaldococcus jannaschii exhibited reduced proteolysis and increased expression in three different strains of Escherichia coli. Circular permutation of a different protein, TEM-1 beta-lactamase, by a similar method increased the expression lifetime of the protein in the periplasm of E. coli. Both circularly permuted proteins maintained activity near their wild-type counterparts and design criteria for selecting the sites for circular permutation are discussed. It is expected that this method will find broad utility for enhanced expression of recombinant proteins when proteolysis is a factor. PMID:19622546

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

    PubMed

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

    2014-01-01

    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. PMID:25523647

  17. Recent advances in recombinant protein expression by Corynebacterium, Brevibacterium, and Streptomyces: from transcription and translation regulation to secretion pathway selection.

    PubMed

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

    2013-11-01

    Gram-positive bacteria are widely used to produce recombinant proteins, amino acids, organic acids, higher alcohols, and polymers. Many proteins have been expressed in Gram-positive hosts such as Corynebacterium, Brevibacterium, and Streptomyces. The favorable and advantageous characteristics (e.g., high secretion capacity and efficient production of metabolic products) of these species have increased the biotechnological applications of bacteria. However, owing to multiplicity from genes encoding the proteins and expression hosts, the expression of recombinant proteins is limited in Gram-positive bacteria. Because there is a very recent review about protein expression in Bacillus subtilis, here we summarize recent strategies for efficient expression of recombinant proteins in the other three typical Gram-positive bacteria (Corynebacterium, Brevibacterium, and Streptomyces) and discuss future prospects. We hope that this review will contribute to the development of recombinant protein expression in Corynebacterium, Brevibacterium, and Streptomyces. PMID:24068337

  18. Synthesizing a Cellulase like Chimeric Protein by Recombinant Molecular Biology Techniques

    PubMed Central

    Banerjee, Hirendra Nath; Krauss, Christopher; Smith, Valerie; Mahaffey, Kelly; Boston, Ava

    2016-01-01

    In order to meet the Renewable Fuels Standard demands for 30 billion gallons of biofuels by the end of 2020, new technologies for generation of cellulosic ethanol must be exploited. Breaking down cellulose by cellulase enzyme is very important for this purpose but this is not thermostable and degrades at higher temperatures in bioreactors. Towards creation of a more ecologically friendly method of rendering bioethanol from cellulosic waste, we attempted to produce recombinant higher temperature resistant cellulases for use in bioreactors. The project involved molecular cloning of genes for cellulose-degrading enzymes based on bacterial source, expressing the recombinant proteins in E. coli and optimizing enzymatic activity. We were able to generate in vitro bacterial expression systems to produce recombinant His-tag purified protein which showed cellulase like activity. PMID:27468362

  19. Influence of protein formulation and carrier solution on asymmetrical flow field-flow fractionation: a case study of the plant-produced recombinant anthrax protective antigen pp-PA83.

    PubMed

    Palais, Caroline; Chichester, Jessica A; Manceva, Slobodanka; Yusibov, Vidadi; Arvinte, Tudor

    2015-02-01

    Asymmetrical flow field-flow fractionation (afFFF) was used to investigate the properties of a plant-produced anthrax toxin protective antigen, pp-PA83. The afFFF fractogram consisted of two main peaks with molar masses similar to the molecular mass of pp-PA83 monomer. afFFF carrier solutions strongly influenced the ratio and the intensity of the two main peaks. These differences indicate that conformation changes in the pp-PA83 molecule occurred during the afFFF analysis. Similar fractograms were obtained for different pp-PA83 formulations when the afFFF carrier solution and the protein formulation were the same (or very similar). The data show that in specific cases, afFFF could be used to study protein conformation and document the importance of studying the influence of the carrier solution on afFFF. PMID:25417936

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

    PubMed

    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. Biotechnol. Bioeng. 2016;113: 961-969. © 2015 Wiley Periodicals, Inc. PMID:26480251

  1. Potential of fragment recombination for rational design of proteins.

    PubMed

    Eisenbeis, Simone; Proffitt, William; Coles, Murray; Truffault, Vincent; Shanmugaratnam, Sooruban; Meiler, Jens; Höcker, Birte

    2012-03-01

    It is hypothesized that protein domains evolved from smaller intrinsically stable subunits via combinatorial assembly. Illegitimate recombination of fragments that encode protein subunits could have quickly led to diversification of protein folds and their functionality. This evolutionary concept presents an attractive strategy to protein engineering, e.g., to create new scaffolds for enzyme design. We previously combined structurally similar parts from two ancient protein folds, the (βα)(8)-barrel and the flavodoxin-like fold. The resulting "hopeful monster" differed significantly from the intended (βα)(8)-barrel fold by an extra β-strand in the core. In this study, we ask what modifications are necessary to form the intended structure and what potential this approach has for the rational design of functional proteins. Guided by computational design, we optimized the interface between the fragments with five targeted mutations yielding a stable, monomeric protein whose predicted structure was verified experimentally. We further tested binding of a phosphorylated compound and detected that some affinity was already present due to an intact phosphate-binding site provided by one fragment. The affinity could be improved quickly to the level of natural proteins by introducing two additional mutations. The study illustrates the potential of recombining protein fragments with unique properties to design new and functional proteins, offering both a possible pathway of protein evolution and a protocol to rapidly engineer proteins for new applications. PMID:22329686

  2. Proteomic differences in recombinant CHO cells producing two similar antibody fragments.

    PubMed

    Sommeregger, Wolfgang; Mayrhofer, Patrick; Steinfellner, Willibald; Reinhart, David; Henry, Michael; Clynes, Martin; Meleady, Paula; Kunert, Renate

    2016-09-01

    Chinese hamster ovary (CHO) cells are the most commonly used mammalian hosts for the production of biopharmaceuticals. To overcome unfavorable features of CHO cells, a lot of effort is put into cell engineering to improve phenotype. "Omics" studies investigating elevated growth rate and specific productivities as well as extracellular stimulus have already revealed many interesting engineering targets. However, it remains largely unknown how physicochemical properties of the recombinant product itself influence the host cell. In this study, we used quantitative label-free LC-MS proteomic analyses to investigate product-specific proteome differences in CHO cells producing two similar antibody fragments. We established recombinant CHO cells producing the two antibodies, 3D6 and 2F5, both as single-chain Fv-Fc homodimeric antibody fragments (scFv-Fc). We applied three different vector strategies for transgene delivery (i.e., plasmid, bacterial artificial chromosome, recombinase-mediated cassette exchange), selected two best performing clones from transgene variants and transgene delivery methods and investigated three consecutively passaged cell samples by label-free proteomic analysis. LC-MS-MS profiles were compared in several sample combinations to gain insights into different aspects of proteomic changes caused by overexpression of two different heterologous proteins. This study suggests that not only the levels of specific product secretion but the product itself has a large impact on the proteome of the cell. Biotechnol. Bioeng. 2016;113: 1902-1912. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID:26913574

  3. Expression and Characterization of Recombinant Campylobacter jejuni Chemotactic Proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Expression and Characterization of Recombinant Campylobacter jejuni Chemotactic Proteins Hung-Yueh Yeh*, Kelli L. Hiett, John E. Line, Brian B. Oakley and Bruce S. Seal, Poultry Microbiological Safety Research Unit, Richard B. Russell Agricultural Research Center, Agricultural Research Service, Uni...

  4. 21 CFR 878.4494 - Absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... produced by recombinant DNA technology. 878.4494 Section 878.4494 Food and Drugs FOOD AND DRUG... recombinant DNA technology. (a) Identification. An absorbable poly(hydroxybutyrate) surgical suture is an... deoxyribonucleic acid (DNA) technology. The device is intended for use in general soft tissue approximation...

  5. 21 CFR 878.4494 - Absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... produced by recombinant DNA technology. 878.4494 Section 878.4494 Food and Drugs FOOD AND DRUG... recombinant DNA technology. (a) Identification. An absorbable poly(hydroxybutyrate) surgical suture is an... deoxyribonucleic acid (DNA) technology. The device is intended for use in general soft tissue approximation...

  6. 21 CFR 878.4494 - Absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... produced by recombinant DNA technology. 878.4494 Section 878.4494 Food and Drugs FOOD AND DRUG... recombinant DNA technology. (a) Identification. An absorbable poly(hydroxybutyrate) surgical suture is an... deoxyribonucleic acid (DNA) technology. The device is intended for use in general soft tissue approximation...

  7. 21 CFR 878.4494 - Absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... produced by recombinant DNA technology. 878.4494 Section 878.4494 Food and Drugs FOOD AND DRUG... recombinant DNA technology. (a) Identification. An absorbable poly(hydroxybutyrate) surgical suture is an... deoxyribonucleic acid (DNA) technology. The device is intended for use in general soft tissue approximation...

  8. 21 CFR 878.4494 - Absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... produced by recombinant DNA technology. 878.4494 Section 878.4494 Food and Drugs FOOD AND DRUG... recombinant DNA technology. (a) Identification. An absorbable poly(hydroxybutyrate) surgical suture is an... deoxyribonucleic acid (DNA) technology. The device is intended for use in general soft tissue approximation...

  9. Acid extraction and purification of recombinant spider silk proteins.

    PubMed

    Mello, Charlene M; Soares, Jason W; Arcidiacono, Steven; Butler, Michelle M

    2004-01-01

    A procedure has been developed for the isolation of recombinant spider silk proteins based upon their unique stability and solubilization characteristics. Three recombinant silk proteins, (SpI)7, NcDS, and [(SpI)4/(SpII)1]4, were purified by extraction with organic acids followed by affinity or ion exchange chromatography resulting in 90-95% pure silk solutions. The protein yield of NcDS (15 mg/L culture) and (SpI)7 (35 mg/L) increased 4- and 5-fold, respectively, from previously reported values presumably due to a more complete solubilization of the expressed recombinant protein. [(SpI)4/(SpII)1]4, a hybrid protein based on the repeat sequences of spidroin I and spidroin II, had a yield of 12.4 mg/L. This method is an effective, reproducible technique that has broad applicability for a variety of silk proteins as well as other acid stable biopolymers. PMID:15360297

  10. Leptospirosis serodiagnosis by ELISA based on recombinant outer membrane protein.

    PubMed

    Chalayon, Piyanart; Chanket, Phanita; Boonchawalit, Toungporn; Chattanadee, Siriporn; Srimanote, Potjanee; Kalambaheti, Thareerat

    2011-05-01

    The outer membrane protein LipL21, LipL32, LipL41 and Loa22 of Leptospira interrogans serovar Copenhageni were previously revealed by immunoproteomic analysis, using sera from acute phase infection in a guinea pig. The full-length DNA of each protein was then cloned from the same serovar and expressed in pRSET vector. The obtained molecular weight (MW) of recombinant proteins rLipL21, rLipL32 and rLoa22 were slightly higher than the MW predicted from nucleotide sequences of each inserted gene, while only the N-terminal half of rLipL41 was obtained. Mice antiserum raised against each purified recombinant protein could react with the whole cell lysate of leptospiral serovars, implying that leptospiral native proteins shared a common epitope with recombinant protein. Serodiagnosis using recombinant protein antigen based on indirect ELISA procedure was developed in this study. The optimization of the ELISA components lead to determination of optical density (OD) from a single serum-dilution of 1:1000 in the leptospirosis patients group and normal healthy control group. The cut off OD values for both IgG and IgM class were investigated, and based on this fixed dilution only the IgG class could be used for differential diagnosis of patients and normal individuals. Compared with the MAT assay, ELISA assay utilizing both rLipL32 and rLoa22 as antigen, gave high accuracy and could thus be useful as a confirmative serology test. PMID:21353274

  11. Murine immune responses to a Plasmodium vivax-derived chimeric recombinant protein expressed in Brassica napus

    PubMed Central

    2011-01-01

    Background To develop a plant-based vaccine against Plasmodium vivax, two P. vivax candidate proteins were chosen. First, the merozoite surface protein-1 (MSP-1), a major asexual blood stage antigen that is currently considered a strong vaccine candidate. Second, the circumsporozoite protein (CSP), a component of sporozoites that contains a B-cell epitope. Methods A synthetic chimeric recombinant 516 bp gene encoding containing PvMSP-1, a Pro-Gly linker motif, and PvCSP was synthesized; the gene, named MLC, encoded a total of 172 amino acids. The recombinant gene was modified with regard to codon usage to optimize gene expression in Brassica napus. The Ti plasmid inducible gene transfer system was used for MLC chimeric recombinant gene expression in B. napus. Gene expression was confirmed by polymerase chain reaction (PCR), beta-glucuronidase reporter gene (GUS) assay, and Western blot. Results The MLC chimeric recombinant protein expressed in B. napus had a molecular weight of approximately 25 kDa. It exhibited a clinical sensitivity of 84.21% (n = 38) and a clinical specificity of 100% (n = 24) as assessed by enzyme-linked immunosorbent assay (ELISA). Oral immunization of BALB/c mice with MLC chimeric recombinant protein successfully induced antigen-specific IgG1 production. Additionally, the Th1-related cytokines IL-12 (p40), TNF, and IFN-γ were significantly increased in the spleens of the BALB/c mice. Conclusions The chimeric MLC recombinant protein produced in B. napus has potential as both as an antigen for diagnosis and as a valuable vaccine candidate for oral immunization against vivax malaria. PMID:21529346

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

    PubMed

    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. PMID:27322762

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

    PubMed

    Martínez-Alonso, Mónica; Villaverde, Antonio; Ferrer-Miralles, Neus

    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

  14. Spider silk fibers spun from soluble recombinant silk produced in mammalian cells.

    PubMed

    Lazaris, Anthoula; Arcidiacono, Steven; Huang, Yue; Zhou, Jiang-Feng; Duguay, Francois; Chretien, Nathalie; Welsh, Elizabeth A; Soares, Jason W; Karatzas, Costas N

    2002-01-18

    Spider silks are protein-based "biopolymer" filaments or threads secreted by specialized epithelial cells as concentrated soluble precursors of highly repetitive primary sequences. Spider dragline silk is a flexible, lightweight fiber of extraordinary strength and toughness comparable to that of synthetic high-performance fibers. We sought to "biomimic" the process of spider silk production by expressing in mammalian cells the dragline silk genes (ADF-3/MaSpII and MaSpI) of two spider species. We produced soluble recombinant (rc)-dragline silk proteins with molecular masses of 60 to 140 kilodaltons. We demonstrated the wet spinning of silk monofilaments spun from a concentrated aqueous solution of soluble rc-spider silk protein (ADF-3; 60 kilodaltons) under modest shear and coagulation conditions. The spun fibers were water insoluble with a fine diameter (10 to 40 micrometers) and exhibited toughness and modulus values comparable to those of native dragline silks but with lower tenacity. Dope solutions with rc-silk protein concentrations >20% and postspinning draw were necessary to achieve improved mechanical properties of the spun fibers. Fiber properties correlated with finer fiber diameter and increased birefringence. PMID:11799236

  15. Environment Control to Improve Recombinant Protein Yields in Plants Based on Agrobacterium-Mediated Transient Gene Expression.

    PubMed

    Fujiuchi, Naomichi; Matoba, Nobuyuki; Matsuda, Ryo

    2016-01-01

    Agrobacterium-mediated transient expression systems enable plants to produce a wide range of recombinant proteins on a rapid timescale. To achieve economically feasible upstream production and downstream processing, two yield parameters should be considered: (1) recombinant protein content per unit biomass and (2) recombinant protein productivity per unit area-time at the end of the upstream production. Because environmental factors in the upstream production have impacts on these parameters, environment control is important to maximize the recombinant protein yield. In this review, we summarize the effects of pre- and postinoculation environmental factors in the upstream production on the yield parameters and discuss the basic concept of environment control for plant-based transient expression systems. Preinoculation environmental factors associated with planting density, light quality, and nutrient supply affect plant characteristics, such as biomass and morphology, which in turn affect recombinant protein content and productivity. Accordingly, environment control for such plant characteristics has significant implications to achieve a high yield. On the other hand, postinoculation environmental factors, such as temperature, light intensity, and humidity, have been shown to affect recombinant protein content. Considering that recombinant protein production in Agrobacterium-mediated transient expression systems is a result of a series of complex biological events starting from T-DNA transfer from Agrobacterium tumefaciens to protein biosynthesis and accumulation in leaf tissue, we propose that dynamic environment control during the postinoculation process, i.e., changing environmental conditions at an appropriate timing for each event, may be a promising approach to obtain a high yield. Detailed descriptions of plant growth conditions and careful examination of environmental effects will significantly contribute to our knowledge to stably obtain high recombinant

  16. Environment Control to Improve Recombinant Protein Yields in Plants Based on Agrobacterium-Mediated Transient Gene Expression

    PubMed Central

    Fujiuchi, Naomichi; Matoba, Nobuyuki; Matsuda, Ryo

    2016-01-01

    Agrobacterium-mediated transient expression systems enable plants to produce a wide range of recombinant proteins on a rapid timescale. To achieve economically feasible upstream production and downstream processing, two yield parameters should be considered: (1) recombinant protein content per unit biomass and (2) recombinant protein productivity per unit area–time at the end of the upstream production. Because environmental factors in the upstream production have impacts on these parameters, environment control is important to maximize the recombinant protein yield. In this review, we summarize the effects of pre- and postinoculation environmental factors in the upstream production on the yield parameters and discuss the basic concept of environment control for plant-based transient expression systems. Preinoculation environmental factors associated with planting density, light quality, and nutrient supply affect plant characteristics, such as biomass and morphology, which in turn affect recombinant protein content and productivity. Accordingly, environment control for such plant characteristics has significant implications to achieve a high yield. On the other hand, postinoculation environmental factors, such as temperature, light intensity, and humidity, have been shown to affect recombinant protein content. Considering that recombinant protein production in Agrobacterium-mediated transient expression systems is a result of a series of complex biological events starting from T-DNA transfer from Agrobacterium tumefaciens to protein biosynthesis and accumulation in leaf tissue, we propose that dynamic environment control during the postinoculation process, i.e., changing environmental conditions at an appropriate timing for each event, may be a promising approach to obtain a high yield. Detailed descriptions of plant growth conditions and careful examination of environmental effects will significantly contribute to our knowledge to stably obtain high recombinant

  17. The recombinant expression and activity detection of MAF-1 fusion protein

    PubMed Central

    Fu, Ping; Wu, Jianwei; Gao, Song; Guo, Guo; Zhang, Yong; Liu, Jian

    2015-01-01

    This study establishes the recombinant expression system of MAF-1 (Musca domestica antifungal peptide-1) and demonstrates the antifungal activity of the expression product and shows the relationship between biological activity and structure. The gene segments on mature peptide part of MAF-1 were cloned, based on the primers designed according to the cDNA sequence of MAF-1. We constructed the recombinant prokaryotic expression plasmid using prokaryotic expression vector (pET-28a(+)) and converted it to the competent cell of BL21(DE3) to gain recombinant MAF-1 fusion protein with His tag sequence through purifying affinity chromatographic column of Ni-NTA. To conduct the Western Blotting test, recombinant MAF-1 fusion protein was used to produce the polyclonal antibody of rat. The antifungal activity of the expression product was detected using Candida albicans (ATCC10231) as the indicator. The MAF-1 recombinant fusion protein was purified to exhibit obvious antifungal activity, which lays the foundation for the further study of MAF-1 biological activity, the relationship between structure and function, as well as control of gene expression. PMID:26423137

  18. Replication protein A is required for meiotic recombination in Saccharomyces cerevisiae.

    PubMed Central

    Soustelle, Christine; Vedel, Michèle; Kolodner, Richard; Nicolas, Alain

    2002-01-01

    In Saccharomyces cerevisiae, meiotic recombination is initiated by transient DNA double-stranded breaks (DSBs). These DSBs undergo a 5' --> 3' resection to produce 3' single-stranded DNA ends that serve to channel DSBs into the RAD52 recombinational repair pathway. In vitro studies strongly suggest that several proteins of this pathway--Rad51, Rad52, Rad54, Rad55, Rad57, and replication protein A (RPA)--play a role in the strand exchange reaction. Here, we report a study of the meiotic phenotypes conferred by two missense mutations affecting the largest subunit of RPA, which are localized in the protein interaction domain (rfa1-t11) and in the DNA-binding domain (rfa1-t48). We find that both mutant diploids exhibit reduced sporulation efficiency, very poor spore viability, and a 10- to 100-fold decrease in meiotic recombination. Physical analyses indicate that both mutants form normal levels of meiosis-specific DSBs and that the broken ends are processed into 3'-OH single-stranded tails, indicating that the RPA complex present in these rfa1 mutants is functional in the initial steps of meiotic recombination. However, the 5' ends of the broken fragments undergo extensive resection, similar to what is observed in rad51, rad52, rad55, and rad57 mutants, indicating that these RPA mutants are defective in the repair of the Spo11-dependent DSBs that initiate homologous recombination during meiosis. PMID:12072452

  19. Different expression systems for production of recombinant proteins in Saccharomyces cerevisiae.

    PubMed

    Liu, Zihe; Tyo, Keith E J; Martínez, José L; Petranovic, Dina; Nielsen, Jens

    2012-05-01

    Yeast Saccharomyces cerevisiae has become an attractive cell factory for production of commodity and speciality chemicals and proteins, such as industrial enzymes and pharmaceutical proteins. Here we evaluate most important expression factors for recombinant protein secretion: we chose two different proteins (insulin precursor (IP) and α-amylase), two different expression vectors (POTud plasmid and CPOTud plasmid) and two kinds of leader sequences (the glycosylated alpha factor leader and a synthetic leader with no glycosylation sites). We used IP and α-amylase as representatives of a simple protein and a multi-domain protein, as well as a non-glycosylated protein and a glycosylated protein, respectively. The genes coding for the two recombinant proteins were fused independently with two different leader sequences and were expressed using two different plasmid systems, resulting in eight different strains that were evaluated by batch fermentations. The secretion level (µmol/L) of IP was found to be higher than that of α-amylase for all expression systems and we also found larger variation in IP production for the different vectors. We also found that there is a change in protein production kinetics during the diauxic shift, that is, the IP was produced at higher rate during the glucose uptake phase, whereas amylase was produced at a higher rate in the ethanol uptake phase. For comparison, we also refer to data from another study, (Tyo et al. submitted) in which we used the p426GPD plasmid (standard vector using URA3 as marker gene and pGPD1 as expression promoter). For the IP there is more than 10-fold higher protein production with the CPOTud vector compared with the standard URA3-based vector, and this vector system therefore represent a valuable resource for future studies and optimization of recombinant protein production in yeast. PMID:22179756

  20. Recombinant conotoxin, TxVIA, produced in yeast has insecticidal activity.

    PubMed

    Bruce, C; Fitches, E C; Chougule, N; Bell, H A; Gatehouse, J A

    2011-07-01

    Conotoxins are a diverse collection of more than 50,000 peptides produced by predatory marine snails of the genus Conus in order to immobilize their prey. Many conotoxins modulate the activity of ion channels, and show high specificity to their targets; as a result, some have valuable pharmaceutical applications. However, obtaining active peptide is difficult and to date has only been achieved though natural collection, chemical synthesis, or the use of prokaryotic expression systems, which often have the disadvantage of requiring subsequent steps to correctly fold the peptide. This paper reports the production of a conotoxin, TxVIA from Conus textile, as a biologically active recombinant protein, using the yeast Pichia pastoris as expression host. The presence of the pro-peptide was found to be necessary for the expression of biologically active conotoxin. We also show that TxVIA is not, as previously reported, mollusc-specific, but also shows insecticidal activity when injected into lepidopteran (cabbage moth) and dipteran (house fly) larvae. In contrast, recombinant TxVIA was not found to be molluscicidal to the grey field slug Deroceras reticulatum. PMID:21640131

  1. Using ion exchange chromatography to purify a recombinantly expressed protein.

    PubMed

    Duong-Ly, Krisna C; Gabelli, Sandra B

    2014-01-01

    Ion exchange chromatography (IEX) separates molecules by their surface charge, a property that can vary vastly between different proteins. There are two types of IEX, cation exhange and anion exchange chromatography. The protocol that follows was designed by the authors for anion exchange chromatography of a recombinantly expressed protein having a pI of 4.9 and containing two cysteine residues and one tryptophan residue, using an FPLC system. Prior to anion exchange, the protein had been salted out using ammonium sulfate precipitation and partially purified via hydrophobic interaction chromatography (see Salting out of proteins using ammonium sulfate precipitation and Use and Application of Hydrophobic Interaction Chromatography for Protein Purification). Slight modifications to this protocol may be made to accommodate both the protein of interest and the availability of equipment. PMID:24674065

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

    PubMed

    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. PMID:27165321

  3. Immunodiagnosis of Ehrlichia canis Infection with Recombinant Proteins

    PubMed Central

    McBride, Jere W.; Corstvet, Richard E.; Breitschwerdt, Edward B.; Walker, David H.

    2001-01-01

    Ehrlichia canis causes a potentially fatal rickettsial disease of dogs that requires rapid and accurate diagnosis in order to initiate appropriate therapy leading to a favorable prognosis. We recently reported the cloning of two immunoreactive E. canis proteins, P28 and P140, that were applicable for serodiagnosis of the disease. In the present study we cloned a new immunoreactive E. canis surface protein gene of 1,170 bp, which encodes a protein with a predicted molecular mass of 42.6 kDa (P43). The P43 gene was not detected in E. chaffeensis DNA by Southern blot, and antisera against recombinant P43 (rP43) did not react with E. chaffeensis as detected by indirect fluorescent antibody (IFA) assay. Forty-two dogs exhibiting signs and/or hematologic abnormalities associated with canine ehrlichiosis were tested by IFA assay and by recombinant Western immunoblot. Among the 22 samples that were IFA positive for E. canis, 100% reacted with rP43, 96% reacted with rP28, and 96% reacted with rP140. The specificity of the recombinant proteins compared to the IFAs was 96% for rP28, 88% for P43 and 63% for P140. The results of this study demonstrate that the rP43 and rP28 are sensitive and reliable serodiagnostic antigens for E. canis infections. PMID:11136790

  4. Overexpression of microRNAs enhances recombinant protein production in Chinese hamster ovary cells.

    PubMed

    Loh, Wan Ping; Loo, Bernard; Zhou, Lihan; Zhang, Peiqing; Lee, Dong-Yup; Yang, Yuansheng; Lam, Kong Peng

    2014-09-01

    MicroRNAs (miRNAs) are short, non-coding RNAs that can negatively regulate expression of multiple genes at post-transcriptional levels. Using miRNAs to target multiple genes and pathways is a promising cell-engineering strategy to increase recombinant protein production in mammalian cells. Here, we identified miRs-17, -19b, -20a, and -92a to be differentially expressed between high- and low- monoclonal antibody-producing Chinese hamster ovary (CHO) cell clones using next-generation sequencing and quantitative real-time PCR. These miRNAs were stably overexpressed individually and in combination in a high-producing clone to assess their effects on CHO cell growth, recombinant protein productivity and product quality. Stably transfected pools demonstrated 24-34% increases in specific productivity (qP) and 21-31% increases in titer relative to the parental clone, without significant alterations in proliferation rates. The highest protein-producing clones isolated from these pools exhibited 130-140% increases in qP and titer compared to the parental clone, without major changes in product aggregation and N-glycosylation profile. From our clonal data, correlations between enhanced qP/titer and increased levels of miRs-17, -19b, and -92a were observed. Our results demonstrate the potential of miRs-17, -19b, and -92a as cell-engineering targets to increase recombinant protein production in mammalian cells. PMID:24819042

  5. Systems Biology of Recombinant Protein Production in Bacillus megaterium

    NASA Astrophysics Data System (ADS)

    Biedendieck, Rebekka; Bunk, Boyke; Fürch, Tobias; Franco-Lara, Ezequiel; Jahn, Martina; Jahn, Dieter

    Over the last two decades the Gram-positive bacterium Bacillus megaterium was systematically developed to a useful alternative protein production host. Multiple vector systems for high yield intra- and extracellular protein production were constructed. Strong inducible promoters were combined with DNA sequences for optimised ribosome binding sites, various leader peptides for protein export and N- as well as C-terminal affinity tags for affinity chromatographic purification of the desired protein. High cell density cultivation and recombinant protein production were successfully tested. For further system biology based control and optimisation of the production process the genomes of two B. megaterium strains were completely elucidated, DNA arrays designed, proteome, fluxome and metabolome analyses performed and all data integrated using the bioinformatics platform MEGABAC. Now, solid theoretical and experimental bases for primary modeling attempts of the production process are available.

  6. Recombinant Antibody Production in Arabidopsis Seeds Triggers an Unfolded Protein Response1[W][OA

    PubMed Central

    De Wilde, Kirsten; De Buck, Sylvie; Vanneste, Kevin; Depicker, Ann

    2013-01-01

    Among the many plant-based production systems that are being tested for molecular farming, seeds are very attractive, as they provide a stable environment in which the accumulating recombinant proteins can be stored. However, it is not known exactly how high production levels of recombinant antibodies influence the endogenous transcriptome and proteome of the developing seed. To address this question, we studied the transcriptomic status in developing Arabidopsis (Arabidopsis thaliana) seeds 13 d post anthesis of three transgenic lines, producing varying levels of recombinant VHH or single-chain Fv antibody fragments fused to the human immunoglobulin G1-derived Fc fragment under the control of the β-PHASEOLIN seed-specific promoter. Using genome-wide Tiling arrays, we demonstrated that only a small proportion of the transcriptome was significantly changed in each of the lines compared with the wild type. Strikingly, in all three lines, we found a large overlap of up-regulated genes corresponding to protein folding, glycosylation/modification, translocation, vesicle transport, and protein degradation, suggestive of a state of cellular stress called the unfolded protein response. Moreover, the gene up-regulation amplitude was similar in all three lines. We hypothesize that the production of recombinant antibodies in the endoplasmic reticulum triggers endoplasmic reticulum stress, causing a disturbance of the normal cellular homeostasis. PMID:23188806

  7. Identification, recombinant production and structural characterization of four silk proteins from the Asiatic honeybee Apis cerana.

    PubMed

    Shi, Jiahai; Lua, Shixiong; Du, Ning; Liu, Xiangyang; Song, Jianxing

    2008-06-01

    Unlike silkworm and spider silks assembled from very large and repetitive fibrous proteins, the bee and ant silks were recently demonstrated to consist of four small and non-repetitive coiled-coil proteins. The design principle for this silk family remains largely unknown and so far no structural study is available on them in solution. The present study aimed to identify, express and characterize the Asiatic honeybee silk proteins using DLS, CD and NMR spectroscopy. Consequently, (1) four silk proteins are identified, with approximately 6, 10, 9 and 8% variations, respectively, from their European honeybee homologs. Strikingly, their recombinant forms can be produced in Escherichia coil with yields of 10-60 mg/l. (2) Despite containing approximately 65% coiled-coil sequences, four proteins have very low alpha-helix (9-27%) but unusually high random coil (45-56%) contents. Surprisingly, beta-sheet is also detected in four silk proteins (26-35%), implying the possible presence of beta-sheet in the bee and ant silks. (3) Four proteins lacking of the tight tertiary packing appear capable of interacting with each other weakly but this interaction triggers no significant formation of the tight tertiary packing. The study not only implies the promising potential to produce recombinant honeybee silk proteins for the development of various biomaterials; but also provides the first structural insight into the molecular mechanism underlying the formation of the coiled-coil silks. PMID:18394700

  8. Functional insights into recombinant TROSPA protein from Ixodes ricinus.

    PubMed

    Figlerowicz, Marek; Urbanowicz, Anna; Lewandowski, Dominik; Jodynis-Liebert, Jadwiga; Sadowski, Czeslaw

    2013-01-01

    Lyme disease (also called borreliosis) is a prevalent chronic disease transmitted by ticks and caused by Borrelia burgdorferi s. l. spirochete. At least one tick protein, namely TROSPA from I. scapularis, commonly occurring in the USA, was shown to be required for colonization of the vector by bacteria. Located in the tick gut, TROSPA interacts with the spirochete outer surface protein A (OspA) and initiates the tick colonization. Ixodes ricinus is a primary vector involved in B. burgdorferi s. l. transmission in most European countries. In this study, we characterized the capacities of recombinant TROSPA protein from I. ricinus to interact with OspA from different Borrelia species and to induce an immune response in animals. We also showed that the N-terminal part of TROSPA (a putative transmembrane domain) is not involved in the interaction with OspA and that reduction of the total negative charge on the TROSPA protein impaired TROSPA-OspA binding. In general, the data presented in this paper indicate that recombinant TROSPA protein retains the capacity to form a complex with OspA and induces a significant level of IgG in orally immunized rats. Thus, I. ricinus TROSPA may be considered a good candidate component for an animal vaccine against Borrelia. PMID:24204685

  9. An Efficient Genome-Wide Fusion Partner Screening System for Secretion of Recombinant Proteins in Yeast

    PubMed Central

    Bae, Jung-Hoon; Hyun Sung, Bong; Kim, Hyun-Jin; Park, Soon-Ho; Lim, Kwang-Mook; Kim, Mi-Jin; Lee, Cho-Ryong; Sohn, Jung-Hoon

    2015-01-01

    To produce rarely secreted recombinant proteins in the yeast Saccharomyces cerevisiae, we developed a novel genome-wide optimal translational fusion partner (TFP) screening system that involves recruitment of an optimal secretion signal and fusion partner. A TFP library was constructed from a genomic and truncated cDNA library by using the invertase-based signal sequence trap technique. The efficiency of the system was demonstrated using two rarely secreted proteins, human interleukin (hIL)-2 and hIL-32. Optimal TFPs for secretion of hIL-2 and hIL-32 were easily selected, yielding secretion of these proteins up to hundreds of mg/L. Moreover, numerous uncovered yeast secretion signals and fusion partners were identified, leading to efficient secretion of various recombinant proteins. Selected TFPs were found to be useful for the hypersecretion of other recombinant proteins at yields of up to several g/L. This screening technique could provide new methods for the production of various types of difficult-to-express proteins. PMID:26195161

  10. Liposomes containing recombinant E protein vaccine against duck Tembusu virus in ducks.

    PubMed

    Ma, Tengfei; Liu, Yongxia; Cheng, Jia; Liu, Yanhan; Fan, Wentao; Cheng, Ziqiang; Niu, Xudong; Liu, Jianzhu

    2016-04-27

    To obtain an effective vaccine candidate against duck Tembusu viral (DTMUV) disease which causes egg-drop and great economical loss in the Chinese duck industry, liposome vaccines containing recombinant E protein were prepared and assessed in this study. The recombinant plasmid (PET28a-E) was constructed and transformed into BL21 (DE3) cells to produce E proteins. The recombinant E proteins were purified and entrapped by liposomes through reverse-phase evaporation. Eighty-four cherry valley ducks were randomly divided into seven groups and inoculated intramuscularly at one- or seven-day-old with liposomes-E protein or Freund's adjuvant-E protein vaccine. Blood samples were collected from the first week to the tenth week for serum antibody, plasma for viremia, as well as oropharyngeal and cloacal swabs for virus shedding analyses after being challenged with a 10(2.4) 50% tissue culture infective dose (TCID50) of duck Tembusu virus. Results showed that serum antibody level of the liposomes vaccine was higher than the Freund's adjuvant vaccine, and inoculating twice was superior to once; furthermore, the viremia and virus shedding tests also proved that the liposomes vaccine can provide complete protection against DTMUV challenge. These results demonstrated that the liposomes-E protein vaccine could be used as a potential candidate vaccine to prevent DTMUV infection in ducks. PMID:27016654

  11. Cryptocaryon irritans recombinant proteins as potential antigens for sero-surveillance of cryptocaryonosis.

    PubMed

    Lokanathan, Y; Mohd-Adnan, A; Kua, B-C; Nathan, S

    2016-09-01

    Cryptocaryonosis is a major problem for mariculture, and the absence of suitable sero-surveillance tools for the detection of cryptocaryonosis makes it difficult to screen Cryptocaryon irritans-infected fish, particularly asymptomatic fish. In this study, we proposed a serum-based assay using selected C. irritans proteins to screen infected and asymptomatic fish. Eight highly expressed genes were chosen from an earlier study on C. irritans expressed sequence tags and ciliate glutamine codons were converted to universal glutamine codons. The chemically synthesized C. irritans genes were then expressed in an Escherichia coli expression host under optimized conditions. Five C. irritans proteins were successfully expressed in E. coli and purified by affinity chromatography. These proteins were used as antigens in an enzyme-linked immunosorbent assay (ELISA) to screen sera from experimentally immunized fish and naturally infected fish. Sera from both categories of fish reacted equally well with the expressed C. irritans recombinant proteins as well as with sonicated theronts. This study demonstrated the utility of producing ciliate recombinant proteins in a heterologous expression host. An ELISA was successfully developed to diagnose infected and asymptomatic fish using the recombinant proteins as antigens. PMID:27086498

  12. Tagging recombinant proteins to enhance solubility and aid purification.

    PubMed

    Walls, Dermot; Loughran, Sinéad T

    2011-01-01

    Protein fusion technology has enormously facilitated the efficient production and purification of individual recombinant proteins. The use of genetically engineered affinity and solubility-enhancing polypeptide "tags" has increased greatly in recent years and there now exists a considerable repertoire of these that can be used to solve issues related to the expression, stability, solubility, folding, and purification of their fusion partner. In the case of large-scale proteomic studies, the development of purification procedures tailored to individual proteins is not practicable, and affinity tags have therefore become indispensable tools for structural and functional proteomic initiatives that involve the expression of many proteins in parallel. Here, the rationale and applications of a range of established and more recently developed solubility-enhancing and affinity tags are outlined. PMID:20978965

  13. Making recombinant proteins in filamentous fungi- are we expecting too much?

    PubMed

    Nevalainen, Helena; Peterson, Robyn

    2014-01-01

    Hosts used for the production of recombinant proteins are typically high-protein secreting mutant strains that have been selected for a specific purpose, such as efficient production of cellulose-degrading enzymes. Somewhat surprisingly, sequencing of the genomes of a series of mutant strains of the cellulolytic Trichoderma reesei, widely used as an expression host for recombinant gene products, has shed very little light on the nature of changes that boost high-level protein secretion. While it is generally agreed and shown that protein secretion in filamentous fungi occurs mainly through the hyphal tip, there is growing evidence that secretion of proteins also takes place in sub-apical regions. Attempts to increase correct folding and thereby the yields of heterologous proteins in fungal hosts by co-expression of cellular chaperones and foldases have resulted in variable success; underlying reasons have been explored mainly at the transcriptional level. The observed physiological changes in fungal strains experiencing increasing stress through protein overexpression under strong gene promoters also reflect the challenge the host organisms are experiencing. It is evident, that as with other eukaryotes, fungal endoplasmic reticulum is a highly dynamic structure. Considering the above, there is an emerging body of work exploring the use of weaker expression promoters to avoid undue stress. Filamentous fungi have been hailed as candidates for the production of pharmaceutically relevant proteins for therapeutic use. One of the biggest challenges in terms of fungally produced heterologous gene products is their mode of glycosylation; fungi lack the functionally important terminal sialylation of the glycans that occurs in mammalian cells. Finally, exploration of the metabolic pathways and fluxes together with the development of sophisticated fermentation protocols may result in new strategies to produce recombinant proteins in filamentous fungi. PMID:24578701

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

    PubMed

    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. A Gateway recombination herpesvirus cloning system with negative selection that produces vectorless progeny.

    PubMed

    Kunec, Dusan; van Haren, Sandra; Burgess, Shane C; Hanson, Larry A

    2009-01-01

    Crossover recombination based on the lambda phage integration/excision functions enables insertion of a gene of interest into a specific locus by a simple one-step in vitro recombination reaction. Recently, a highly efficient recombination system for targeted mutagenesis, which utilizes lambda phage crossover recombination cloning, has been described for a human herpesvirus 2 bacterial artificial chromosome (BAC). The disadvantages of the system are that it allows only neutral selection (loss of green fluorescent protein) of desired recombinants and that it regenerates herpesvirus progeny containing the BAC sequence inserted in the herpesvirus genome. In this study, the existing channel catfish herpesvirus (CCV) infectious clone (in the form of overlapping fragments) was modified to allow introduction of foreign genes by modified lambda phage crossover recombination cloning. This novel system enables negative and neutral selection and regenerates vectorless herpesvirus progeny. Construction of two CCV mutants expressing lacZ, one from the native CCV ORF5 promoter and the other from the immediate-early cytomegalovirus promoter, demonstrated the efficiency and reliability of this system. This novel cloning system enables rapid incorporation, direct delivery and high-level expression of foreign genes by a herpesvirus. This system has broad utility and could be used to facilitate development of recombinant viruses, viral vectors and better vaccines. PMID:18948138

  16. Characterization of recombinant human diamine oxidase (rhDAO) produced in Chinese Hamster Ovary (CHO) cells.

    PubMed

    Gludovacz, Elisabeth; Maresch, Daniel; Bonta, Maximilian; Szöllösi, Helen; Furtmüller, Paul G; Weik, Robert; Altmann, Friedrich; Limbeck, Andreas; Borth, Nicole; Jilma, Bernd; Boehm, Thomas

    2016-06-10

    Human diamine oxidase (hDAO) efficiently degrades polyamines and histamine. Reduced enzyme activities might cause complications during pregnancy and be involved in histamine intolerance. So far hDAO has been characterized after isolation from either native sources or the heterologous production in insect cells. Accessibility to human enzyme is limited and insect cells produce non-human glycosylation patterns that may alter its biochemical properties. We present the heterologous expression of hDAO in Chinese Hamster Ovary (CHO) cells and a three step purification protocol. Analysis of metal content using ICP-MS revealed that 93% of the active sites were occupied by copper. Topaquinone (TPQ) cofactor content was determined using phenylhydrazine titration. Ninety-four percent of DAO molecules contained TPQ and therefore the copper content at the active site was indirectly confirmed. Mass spectrometric analysis was conducted to verify sequence integrity of the protein and to assess the glycosylation profile. Electronic circular dichroism and UV-vis spectra data were used to characterize structural properties. The substrate preference and kinetic parameters were in accordance with previous publications. The establishment of a recombinant production system for hDAO enables us to generate decent amounts of protein with negligible impurities to address new scientific questions. PMID:27063138

  17. A recombinant Bacillus anthracis strain producing the Clostridium perfringens Ib component induces protection against iota toxins.

    PubMed Central

    Sirard, J C; Weber, M; Duflot, E; Popoff, M R; Mock, M

    1997-01-01

    The Bacillus anthracis toxinogenic Sterne strain is currently used as a live veterinary vaccine against anthrax. The capacity of a toxin-deficient derivative strain to produce a heterologous antigen by using the strong inducible promoter of the B. anthracis pag gene was investigated. The expression of the foreign gene ibp, encoding the Ib component of iota toxin from Clostridium perfringens, was analyzed. A pag-ibp fusion was introduced by allelic exchange into a toxin-deficient Sterne strain, thereby replacing the wild-type pag gene. This recombinant strain, called BAIB, was stable and secreted large quantities of Ib protein in induced culture conditions. Mice given injections of live BAIB spores developed an antibody response specific to the Ib protein. The pag-ibp fusion was therefore functional both in vitro and in vivo. Moreover, the immunized animals were protected against a challenge with C. perfringens iota toxin or with the homologous Clostridium spiroforme toxin. The protective immunity was mediated by neutralizing antibodies. In conclusion, B. anthracis is promising for the development of live veterinary vaccines. PMID:9169728

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

    PubMed Central

    Zhong, Nan; Loppnau, Peter; Seitova, Alma; Ravichandran, Mani; Fenner, Maria; Jain, Harshika; Bhattacharya, Anandi; Hutchinson, Ashley; Paduch, Marcin; Lu, Vincent; Olszewski, Michal; Kossiakoff, Anthony A.; Dowdell, Evan; Koide, Akiko; Koide, Shohei; Huang, Haiming; Nadeem, Vincent; Sidhu, Sachdev S.; Greenblatt, Jack F.; Marcon, Edyta; Arrowsmith, Cheryl H.; Edwards, Aled M.; Gräslund, Susanne

    2015-01-01

    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. PMID:26437229

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed Central

    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

  1. Liposomes containing recombinant gp85 protein vaccine against ALV-J in chickens.

    PubMed

    Zhang, Limei; Cai, Dongjie; Zhao, Xiaona; Cheng, Ziqiang; Guo, Huijun; Qi, Chunhua; Liu, Jianzhu; Xu, Ruixue; Zhao, Peng; Cui, Zhizhong

    2014-05-01

    To study the potential of liposome vaccines in the clinical prevention of ALV-J, the effect of recombinant gp85 protein of subgroup J avian leukosis virus (ALV-J) entrapped by liposomes in chickens against ALV-J infection was investigated in this paper. A recombinant plasmid (PET28a-gp85) containing the PET28a vector and gp85 gene was constructed and then expressed in Rosetta (DE3) cells with 0.5mM IPTG to produce recombinant gp85 proteins that could be entrapped by liposomes through reverse-phase evaporation. The chickens were inoculated intramuscularly either once or twice with the liposomes or with Freund's adjuvant emulsion containing recombinant gp85 protein. Sixty chickens were raised to one week old for the first inoculation and to three weeks old for the second inoculation. Chickens raised to five weeks old were challenged with a 10(2.4) 50% tissue culture infective dose (TCID50) of ALV-J. Blood samples were collected from each chicken at weekly intervals for serum antibody and viremia analyses. Changes in serum antibodies showed that positive serum antibodies (S/P value >0.6) could be induced in all groups regardless of the frequency of inoculation but improved significantly in the twice-inoculated groups. As well, high levels of antibodies emerged earlier in the Freund's adjuvant groups but persisted longer in the liposome groups. Detection of viremia indicated that the liposomes provide better protection against ALV-J than Freund's adjuvant emulsion and that this protection is directly influenced by serum antibody levels. Overall, this study reveals the potential of liposome vaccines containing recombinant gp85 protein in the clinical prevention of ALV-J. PMID:24625339

  2. Immunogenicity of recombinant Plasmodium falciparum SERA proteins in rodents.

    PubMed

    Barr, P J; Inselburg, J; Green, K M; Kansopon, J; Hahm, B K; Gibson, H L; Lee-Ng, C T; Bzik, D J; Li, W B; Bathurst, I C

    1991-03-01

    We have expressed defined regions of the serine-repeat antigen (SERA) of the Honduras-1 strain of Plasmodium falciparum in the yeast Saccharomyces cerevisiae. Amino-terminal domains of the natural SERA protein have been shown previously to be targets for parasite-inhibitory murine monoclonal antibodies. Two recombinant SERA antigens were selected for purification and immunological analysis. The first (SERA 1), corresponding to amino acids 24-285 of the natural SERA precursor, was expressed by the ubiquitin fusion method. This allowed for in vivo cleavage by endogenous yeast ubiquitin hydrolase, and subsequent isolation of the mature polypeptide. The second, larger protein (SERA N), encompassing amino acids 24-506, was expressed at only low levels using this system, but could be isolated in high yields when fused to human gamma-interferon (gamma-IFN). Each purified protein was used to immunize mice with either Freund's adjuvant or a muramyl tripeptide adjuvant that has been used in humans. Sera from immunized mice were shown to be capable of in vitro inhibition of invasion of erythrocytes by the Honduras-1 strain of P. falciparum. The results suggest that a recombinant SERA antigen may be an effective component of a candidate malaria vaccine. PMID:2052035

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

    PubMed

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

    2014-05-01

    Although several expression systems are currently available for the production of recombinant proteins, they still have some inherent disadvantages, thereby resulting in the desire to explore a novel expression system for producing recombinant proteins. Dunaliella salina (D. salina) has been exploited as a novel expression system for the field of genetic engineering because of its distinct advantages, including low production cost, fast growth, easy culture, ease of transgenic manipulation, and modified abilities of transcription and translation. Thus far, studies on D. salina host have made great development and significant progress. This paper presents a comprehensive summary of the achievements of D. salina host from the following aspects: the advantages of D. salina cells, transformation methods, cloning of D. salina genes, and expression of exogenous genes into D. salina. Furthermore, the authors identified the current main obstacles and future application prospects for the recombinant proteins produced by D. salina, which could be used as a basis for the future maturation of D. salina expression system. PMID:24643734

  4. Recombinant Human Peptidoglycan Recognition Proteins Reveal Antichlamydial Activity.

    PubMed

    Bobrovsky, Pavel; Manuvera, Valentin; Polina, Nadezhda; Podgorny, Oleg; Prusakov, Kirill; Govorun, Vadim; Lazarev, Vassili

    2016-07-01

    Peptidoglycan recognition proteins (PGLYRPs) are innate immune components that recognize the peptidoglycan and lipopolysaccharides of bacteria and exhibit antibacterial activity. Recently, the obligate intracellular parasite Chlamydia trachomatis was shown to have peptidoglycan. However, the antichlamydial activity of PGLYRPs has not yet been demonstrated. The aim of our study was to test whether PGLYRPs exhibit antibacterial activity against C. trachomatis Thus, we cloned the regions containing the human Pglyrp1, Pglyrp2, Pglyrp3, and Pglyrp4 genes for subsequent expression in human cell lines. We obtained stable HeLa cell lines that secrete recombinant human PGLYRPs into culture medium. We also generated purified recombinant PGLYRP1, -2, and -4 and confirmed their activities against Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacteria. Furthermore, we examined the activities of recombinant PGLYRPs against C. trachomatis and determined their MICs. We also observed a decrease in the infectious ability of chlamydial elementary bodies in the next generation after a single exposure to PGLYRPs. Finally, we demonstrated that PGLYRPs attach to C. trachomatis elementary bodies and activate the expression of the chlamydial two-component stress response system. Thus, PGLYRPs inhibit the development of chlamydial infection. PMID:27160295

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

    PubMed

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

    2016-01-01

    Corynebacterium glutamicum, which has been for long an industrial producer of various L-amino acids, nucleic acids, and vitamins, is now also regarded as a potential host for the secretory production of recombinant proteins. To harness its potential as an industrial platform for recombinant protein production, the development of an efficient secretion system is necessary. Particularly, regarding protein production in large-scale bioreactors, it would be appropriate to develop a secretory expression system that is specialized for high cell density cultivation conditions. Here we isolated a new signal peptide that mediates the efficient secretion of recombinant proteins under high cell density cultivation conditions. The secretome of C. glutamicum ATCC 13032 under high cell density cultivation conditions was initially investigated, and one major protein was identified as a hypothetical protein encoded by cg1514. Novel secretory production systems were then developed using the Cg1514 signal peptide and its own promoter. Efficient protein secretion was demonstrated using three protein models: endoxylanase, α-amylase, and camelid antibody fragment (VHH). For large-scale production, fed-batch cultivations were also conducted and high yields were successfully achieved--as high as 1.07 g/L (endoxylanase), 782.6 mg/L (α-amylase), and 1.57 g/L (VHH)--in the extracellular medium. From the culture media, all model proteins could be simply purified by one-step column chromatography with high purities and recovery yields. To the best of our knowledge, this is the first report of the development of an efficient secretory expression system by secretome analysis under high cell density cultivation conditions in C. glutamicum. PMID:26134574

  6. Construction and characterization of recombinant Vibrio cholerae strains producing inactive cholera toxin analogs.

    PubMed

    Häse, C C; Thai, L S; Boesman-Finkelstein, M; Mar, V L; Burnette, W N; Kaslow, H R; Stevens, L A; Moss, J; Finkelstein, R A

    1994-08-01

    The catalytic A subunit of cholera toxin (CT-A) is capable of ADP-ribosylating the guanine nucleotide-binding protein, which regulates cell adenylyl cyclase, leading to the life-threatening diarrhea of cholera. Amino acids involved in the enzymatic activity of CT-A have previously been identified. By means of site-directed mutagenesis, an analog of the CT-A subunit gene was created with codon substitutions for both Arg-7 and Glu-112, each of which has been shown to produce subunits lacking ADP-ribosyltransferase activity. The mutated gene fragment was exchanged for the wild-type copy in the previously cloned ctxAB operon from El Tor biotype, Ogawa serotype Vibrio cholerae strain 3083, which produces CT-2. Further, the zonula occludens toxin gene, zot, was inactivated by an insertional mutation to create the new plasmid construct pCT-2*. Additionally, a DNA fragment encoding the B subunit of CT-1 (CT produced by classical biotype, Inaba serotype V. cholerae strain 569B) was exchanged for the homologous part in pCT-2*, resulting in the creation of pCT-1*. These plasmid constructs were introduced into the CT-negative V. cholerae mutant strain JBK70 (E1 Tor biotype, Inaba serotype); CT-A-B+ derivatives CVD101 and CVD103 of classical biotype Ogawa and Inaba serotype strains 395 and 569B, respectively; El Tor biotype Inaba and Ogawa serotype strains C6706 and C7258, respectively, recently isolated in Peru; and O139 (synonym Bengal) strain SG25-1 from the current epidemic in India. Recombinant toxins (CT-1* and CT-2*), partially purified from culture supernatants of transformed JBK70, were shown to be inactive on mouse Y1 adrenal tumor cells and in an in vitro ADP-ribosyltransferase assay. CT-1* and CT-2* reacted with polyclonal and monoclonal antibodies against both A and B subunits of CT. The toxin analogs reacted with antibodies against CT-A and CT-B on cellulose acetate strips and in a GM1 enzyme-linked immunosorbent assay; they reacted appropriately with B

  7. Fibroblast adhesion to recombinant tropoelastin expressed as a protein A-fusion protein.

    PubMed Central

    Grosso, L E; Parks, W C; Wu, L J; Mecham, R P

    1991-01-01

    A bovine tropoelastin cDNA encoding exons 15-36 that includes the elastin-receptor binding site was expressed in Escherichia coli as a fusion protein with Protein A from Staphylococcus aureus. After isolation of the fusion protein by affinity chromatography on Ig-Sepharose, the tropoelastin domain was separated from plasmid-pR1T2T-encoded Protein A (Protein A') by CNBr cleavage. Cell-adhesion assays demonstrated specific adhesion to the recombinant tropoelastin. Furthermore, the data indicate that interactions involving the bovine elastin receptor mediate nuchalligament fibroblast adhesion to the recombinant protein. In agreement with earlier studies of fibroblast chemotaxis to bovine tropoelastin, nuchal-ligament fibroblast adhesion demonstrated developmental regulation of the elastin receptor. Images Fig. 2. Fig. 3. PMID:1996952

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

    PubMed

    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. PMID:26631378

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

    PubMed Central

    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

    2015-01-01

    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. PMID:26631378

  10. Accelerated protein engineering for chemical biotechnology via homologous recombination.

    PubMed

    Nordwald, Erik M; Garst, Andrew; Gill, Ryan T; Kaar, Joel L

    2013-12-01

    Protein engineering has traditionally relied on random mutagenesis strategies to generate diverse libraries, which require high-throughput screening or selection methods to identify rare variants. Alternatively, approaches to semi-rational library construction can be used to minimize the screening load and enhance the efficiency by which improved mutants may be identified. Such methods are typically limited to characterization of relatively few variants due to the difficulties in generating large rational libraries. New tools from synthetic biology, namely multiplexed DNA synthesis and homologous recombination, provide a promising avenue to rapidly construct large, rational libraries. These technologies also enable incorporation of synthetically encoded features that permit efficient characterization of the fitness of each mutant. Extension of these tools to protein library design could complement rational protein design cycles in an effort to more systematically search complex fitness landscapes. The highly parallelized nature with which such libraries can be generated also has the potential to expand directed protein evolution from single protein targets to protein networks whose concerted activities are required for the biological function of interest. PMID:23540421

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

    PubMed

    Jia, Baolei; Jeon, Che Ok

    2016-08-01

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

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

    PubMed Central

    2016-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  14. Production of recombinant oxytocin through sulfitolysis of inteincontaining fusion protein.

    PubMed

    Esipov, Roman S; Stepanenko, Vasily N; Chupova, Larisa A; Miroshnikov, Anatoly I

    2012-05-01

    An artificial gene consisting of seven copies of an oxytocinoyl-lysine encoding sequence arranged in a tandem was synthesized and inserted downstream of the SspDnaB intein gene in a pTWIN1 plasmid. The corresponding fusion protein Dnab-7oxy contained 16 cysteine residues and formed inclusion bodies when expressed in E. coli. The standard protocol involving solubilization of the fusion protein and its autocatalytic cleavage on a chitin resin was not effective because of a very low yield of the cleavage reaction. Attempts to perform a refolding of the intein part of the fusion protein in solution were also unsuccessful because of a high level of protein aggregation. Sulfitolysis of cysteine residues is known to increase a solubility of proteins and peptides. Therefore we suggested a one-step approach that combines solubilization of inclusion bodies and sulfitolysis of a hybrid protein. The fusion protein was completely reduced and solubilized in 8M urea at pH 9.0 in the presence of sodium sulfite and sodium tetrathionate. The sulfitized protein was loaded onto a chitin column, an efficient cleavage was induced by a pH shift from 9.0 to 6.5, and seven successively connected oxytocinoyl- lysine units were released. The heptamer was subjected to trypsinolysis yielding sulfitized monomers of oxytocinoyllysine. Oxytocinoyl-lysine was refolded as described previously and treated by carboxypeptidase B to form the oxytocinic acid. The target oxytocin amide was then synthesized via methyl ester intermediate. Using this approach 6 mg of recombinant oxytocin can be obtained from 1 g of biomass. PMID:22316308

  15. Profiling highly conserved microrna expression in recombinant IgG-producing and parental Chinese hamster ovary cells.

    PubMed

    Lin, Nan; Davis, Angela; Bahr, Scott; Borgschulte, Trissa; Achtien, Katherine; Kayser, Kevin

    2011-07-01

    MicroRNAs (miRNAs) play important roles in global gene regulation. Researchers in recombinant protein production have proposed miRNAs as biomarkers and cell engineering targets. However, miRNA expression remains understudied in Chinese Hamster Ovary cells, one of the most commonly used host cell systems for therapeutic protein production. To profile highly conserved miRNA expression, we used the miRCURY™ miRNA array for screening miRNAs in CHO cells. The selection criteria for further miRNA profiling included positive hybridization signals and experimentally validated predicted regulatory targets. On the basis of screening, we selected 16 miRNAs for quantitative RT-PCR profiling. We profiled miR expression in parental CHO DG44 and CHO K1 cell lines as well as four recombinant DG44-derived CHO lines producing a recombinant human IgG. We observed that miR-221 and miR-222 were significantly downregulated in all IgG-producing cell lines when compared with parental DG44, whereas miR-125b was significantly downregulated in one IgG-producing line. In another IgG-producing line, miR-19a was significantly upregulated. miRNA expression was also profiled in two of these lines that were amplified by stepwise increase of methotrexate. In both amplified cell lines, let-7b and miR-221 were significantly downregulated. In parental CHO K1, let-7b, miR-15b, and miR-17 were significantly downregulated when compared with DG44. The results reported here are the first steps toward profiling highly conserved miRNAs and studying the clonal difference in miRNA expression in CHO cells and may shed light on using miRNAs in cell engineering. PMID:21692195

  16. RecQ promotes toxic recombination in cells lacking recombination intermediate-removal proteins.

    PubMed

    Magner, Daniel B; Blankschien, Matthew D; Lee, Jennifer A; Pennington, Jeanine M; Lupski, James R; Rosenberg, Susan M

    2007-04-27

    The RecQ-helicase family is widespread, is highly conserved, and includes human orthologs that suppress genomic instability and cancer. In vivo, some RecQ homologs promote reduction of steady-state levels of bimolecular recombination intermediates (BRIs), which block chromosome segregation if not resolved. We find that, in vivo, E. coli RecQ can promote the opposite: the net accumulation of BRIs. We report that cells lacking Ruv and UvrD BRI-resolution and -prevention proteins die and display failed chromosome segregation attributable to accumulation of BRIs. Death and segregation failure require RecA and RecF strand exchange proteins. FISH data show that replication is completed during chromosome-segregation failure/death of ruv uvrD recA(Ts) cells. Surprisingly, RecQ (and RecJ) promotes this death. The data imply that RecQ promotes the net accumulation of BRIs in vivo, indicating a second paradigm for the in vivo effect of RecQ-like proteins. The E. coli RecQ paradigm may provide a useful model for some human RecQ homologs. PMID:17466628

  17. RecQ Promotes Toxic Recombination in Cells Lacking Recombination-Intermediate-Removal Proteins

    PubMed Central

    Magner, Daniel B.; Blankschien, Matthew D.; Lee, Jennifer A.; Pennington, Jeanine M.; Lupski, James R.; Rosenberg, Susan M.

    2010-01-01

    Summary The RecQ-helicase family is widespread, highly conserved, and includes human orthologues that suppress genomic instability and cancer. In vivo, some RecQ homologues promote reduction of steady-state levels of bimolecular recombination intermediates (BRIs), which block chromosome segregation if not resolved. We find that in vivo, E. coli RecQ can promote the opposite: the net accumulation of BRIs. We report that cells lacking Ruv and UvrD BRI-resolution and -prevention proteins die and display failed chromosome segregation attributable to accumulation of BRIs. Death and segregation failure require RecA and RecF strand-exchange proteins. FISH data show that replication is completed during chromosome-segregation failure/death of ruv uvrD recA(Ts) cells. Surprisingly, RecQ (and RecJ) promote this death. The data imply that RecQ promotes the net accumulation of BRIs in vivo, indicating a second paradigm for the in-vivo effect of RecQ-like proteins. The E. coli RecQ paradigm may provide a useful model for some human RecQ homologues. PMID:17466628

  18. Recombinant isotope labeled and selenium quantified proteins for absolute protein quantification.

    PubMed

    Zinn, Nico; Winter, Dominic; Lehmann, Wolf D

    2010-03-15

    A novel, widely applicable method for the production of absolutely quantified proteins is described, which can be used as internal standards for quantitative proteomic studies based on mass spectrometry. These standards are recombinant proteins containing an isotope label and selenomethionine. For recombinant protein expression, assembly of expression vectors fitted to cell-free protein synthesis was conducted using the gateway technology which offers fast access to a variety of genes via open reading frame libraries and an easy shuttling of genes between vectors. The proteins are generated by cell-free expression in a medium in which methionine is exchanged against selenomethionine and at least one amino acid is exchanged by a highly stable isotope labeled analogue. After protein synthesis and purification, selenium is used for absolute quantification by element mass spectrometry, while the heavy amino acids in the protein serve as reference in subsequent analyses by LC-ESI-MS or MALDI-MS. Accordingly, these standards are denominated RISQ (for recombinant isotope labeled and selenium quantified) proteins. In this study, a protein was generated containing Lys+6 ([(13)C(6)]-lysine) and Arg+10 ([(13)C(6),(15)N(4)]-arginine) so that each standard tryptic peptide contains a labeled amino acid. Apolipoprotein A1 was synthesized as RISQ protein, and its use as internal standard led to quantification of a reference material within the specified value. Owing to their cell-free expression, RISQ proteins do not contain posttranslational modifications. Thus, correct quantitative data by ESI- or MALDI-MS are restricted to quantifications based on peptides derived from unmodified regions of the analyte protein. Therefore, besides serving as internal standards, RISQ proteins stand out as new tools for quantitative analysis of covalent protein modifications. PMID:20163147

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

    PubMed Central

    Bashiri, Ghader; Baker, Edward N

    2015-01-01

    Protein production using recombinant DNA technology has a fundamental impact on our understanding of biology through providing proteins for structural and functional studies. Escherichia coli (E. coli) has been traditionally used as the default expression host to over-express and purify proteins from many different organisms. E. coli does, however, have known shortcomings for obtaining soluble, properly folded proteins suitable for downstream studies. These shortcomings are even more pronounced for the mycobacterial pathogen Mycobacterium tuberculosis, the bacterium that causes tuberculosis, with typically only one third of proteins expressed in E. coli produced as soluble proteins. Mycobacterium smegmatis (M. smegmatis) is a closely related and non-pathogenic species that has been successfully used as an expression host for production of proteins from various mycobacterial species. In this review, we describe the early attempts to produce mycobacterial proteins in alternative expression hosts and then focus on available expression systems in M. smegmatis. The advantages of using M. smegmatis as an expression host, its application in structural biology and some practical aspects of protein production are also discussed. M. smegmatis provides an effective expression platform for enhanced understanding of mycobacterial biology and pathogenesis and for developing novel and better therapeutics and diagnostics. PMID:25303009

  20. The production of recombinant dengue virus E protein using Escherichia coli and Pichia pastoris.

    PubMed

    Sugrue, R J; Cui, T; Xu, Q; Fu, J; Chan, Y C

    1997-12-01

    The dengue virus envelope protein was expressed as a GST fusion protein using E. coli and P. pastoris as expression hosts. In E. coli the recombinant E protein is expressed initially as a soluble 81 kDa GST fusion protein. Treatment of the fusion protein with thrombin released a 55 kDa protein, which is the expected size for correctly processed, non-glycosylated recombinant E protein. The antiserum from animals immunised with this recombinant E protein was found to specifically recognise the dengue virus E protein in virus-infected cells, thus demonstrating the immunogenic nature of the recombinant E protein. This expression system allowed production of up to 2 mg of purified recombinant E protein from a 1 1 bacterial culture. In contrast, expression of this GST fusion protein in P. pastoris is associated with extensive proteolytic degradation of the recombinant E protein. However, this proteolytic degradation was not observed in the truncated E protein sequences which were expressed. One of these recombinant fusion proteins, GST E401 was secreted into the culture medium at levels of up to 100 microg/l of growth medium. PMID:9504761

  1. Production of Recombinant Proteins in the Chloroplast of the Green Alga Chlamydomonas reinhardtii.

    PubMed

    Guzmán-Zapata, Daniel; Macedo-Osorio, Karla Soledad; Almaraz-Delgado, Alma Lorena; Durán-Figueroa, Noé; Badillo-Corona, Jesus Agustín

    2016-01-01

    Chloroplast transformation in the green algae Chlamydomonas reinhardtii can be used for the production of valuable recombinant proteins. Here, we describe chloroplast transformation of C. reinhardtii followed by protein detection. Genes of interest integrate stably by homologous recombination into the chloroplast genome following introduction by particle bombardment. Genes are inherited and expressed in lines recovered after selection in the presence of an antibiotic. Recombinant proteins can be detected by conventional techniques like immunoblotting and purified from liquid cultures. PMID:26614282

  2. The recombined cccDNA produced using minicircle technology mimicked HBV genome in structure and function closely.

    PubMed

    Guo, Xiaoyan; Chen, Ping; Hou, Xiaohu; Xu, Wenjuan; Wang, Dan; Wang, Tian-Yan; Zhang, Liping; Zheng, Gang; Gao, Zhi-Liang; He, Cheng-Yi; Zhou, Boping; Chen, Zhi-Ying

    2016-01-01

    HBV covalently closed circular DNA (cccDNA) is drug-resistant and responsible for viral persistence. To facilitate the development of anti-cccDNA drugs, we developed a minicircle DNA vector (MC)-based technology to produce large quantity of recombined cccDNA (rcccDNA) resembling closely to its wild-type counterpart both in structure and function. The rcccDNA differed to the wild-type cccDNA (wtcccDNA) only in that it carried an extra 36-bp DNA recombinant product attR upstream of the preC/C gene. Using a procedure similar to standard plasmid production, milligrams of rcccDNA can be generated in common laboratories conveniently. The rcccDNA demonstrated many essential biological features of wtcccDNA, including: (1) undergoing nucleation upon nucleus entry; (2) serving as template for production of all HBV RNAs and proteins; (3) deriving virions capable of infecting tree shrew, and subsequently producing viral mRNAs, proteins, rcccDNA and infectious virions. As an example to develop anti-cccDNA drugs, we used the Crispr/Cas9 system to provide clear-cut evidence that rcccDNA was cleaved by this DNA editing tool in vitro. In summary, we have developed a convenient technology to produce large quantity of rcccDNA as a surrogate of wtcccDNA for investigating HBV biology and developing treatment to eradicate this most wide-spreading virus. PMID:27174254

  3. The recombined cccDNA produced using minicircle technology mimicked HBV genome in structure and function closely

    PubMed Central

    Guo, Xiaoyan; Chen, Ping; Hou, Xiaohu; Xu, Wenjuan; Wang, Dan; Wang, Tian-yan; Zhang, Liping; Zheng, Gang; Gao, Zhi-liang; He, Cheng-Yi; Zhou, Boping; Chen, Zhi-Ying

    2016-01-01

    HBV covalently closed circular DNA (cccDNA) is drug-resistant and responsible for viral persistence. To facilitate the development of anti-cccDNA drugs, we developed a minicircle DNA vector (MC)-based technology to produce large quantity of recombined cccDNA (rcccDNA) resembling closely to its wild-type counterpart both in structure and function. The rcccDNA differed to the wild-type cccDNA (wtcccDNA) only in that it carried an extra 36-bp DNA recombinant product attR upstream of the preC/C gene. Using a procedure similar to standard plasmid production, milligrams of rcccDNA can be generated in common laboratories conveniently. The rcccDNA demonstrated many essential biological features of wtcccDNA, including: (1) undergoing nucleation upon nucleus entry; (2) serving as template for production of all HBV RNAs and proteins; (3) deriving virions capable of infecting tree shrew, and subsequently producing viral mRNAs, proteins, rcccDNA and infectious virions. As an example to develop anti-cccDNA drugs, we used the Crispr/Cas9 system to provide clear-cut evidence that rcccDNA was cleaved by this DNA editing tool in vitro. In summary, we have developed a convenient technology to produce large quantity of rcccDNA as a surrogate of wtcccDNA for investigating HBV biology and developing treatment to eradicate this most wide-spreading virus. PMID:27174254

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

    PubMed Central

    Wang, Kevin Yueju; Wang, Nan; Liu, Dehu

    2013-01-01

    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. PMID:24416067

  5. Cloning, expression, and antigenic characterization of recombinant protein of Mycoplasma gallisepticum expressed in Escherichia coli.

    PubMed

    Rocha, T S; Tramuta, C; Catania, S; Matucci, A; Giuffrida, M G; Baro, C; Profiti, M; Bertolotti, L; Rosati, S

    2015-04-01

    Mycoplasma gallisepticum (MG) is a member of the most important avian mycoplasmas, causing chronic respiratory disease in chickens and leading to important economic losses in the poultry industry. Recombinant technology represents a strategic approach used to achieve highly reliable and specific diagnostic tests in veterinary diseases control: in particular this aspect is crucial for confirming mycoplasma infection and for maintaining mycoplasma-free breeder flocks. In this study, we identified a component of the pyruvate dehydrogenase dihydrolipoamide acetyltransferase (i.e., E2) protein by 2-dimensional electrophoresis (2-DE), characterized it in immunoblotting assays, and analyzed its recombinant (r-E2) in a rec-ELISA test. For full-length protein expression in Escherichia coli (EC) a point mutation was introduced. A rabbit antiserum produced against r-E2 was tested in a Western Blot using different samples of Mycoplasma species. The results showed the applicability of site-directed mutagenesis, with a good yield of the r-E2 after purification. Also, anti-E2 serum reacted with all the tested MG strains showing no cross reaction with other mycoplasmas. The developed E2 ELISA test was capable of detecting MG antibodies in the sera examined. Those results demonstrate the antigenic stability of the E2 protein which could represent a recombinant antigen with potential diagnostic applications. PMID:25667423

  6. Secretion of human interleukin-2 fused with green fluorescent protein in recombinant Pichia pastoris.

    PubMed

    Cha, Hyung Joon; Dalal, Nimish N; Bentley, William E

    2005-07-01

    Methylotrophic yeast Pichia pastoris is convenient for the expression of eukaryotic foreign proteins owing to its potential for posttranslational modifications, protein folding, and facile culturing. In this work, human interleukin (hIL)-2 was successfully produced as a secreted fusion form in recombinant P. pastoris. By employing green fluorescent protein (GFP) as a monitoring fusion partner, clear identification of fusion protein expression and quantification of intracellular hIL-2 were possible even though there was no correlation between culture supernatant fluorescence and secreted hIL-2 owing to high media interference. Importantly, by the addition of casamino acids in basal medium, we were able to enhance threefold amount of secreted hIL-2, which was present both as a fusion and as a clipped fragment. PMID:16014994

  7. Identification, Characterization and Down-Regulation of Cysteine Protease Genes in Tobacco for Use in Recombinant Protein Production

    PubMed Central

    Duwadi, Kishor; Chen, Ling; Menassa, Rima; Dhaubhadel, Sangeeta

    2015-01-01

    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. PMID:26148064

  8. Immobilized palladium(II) ion affinity chromatography for recovery of recombinant proteins with peptide tags containing histidine and cysteine.

    PubMed

    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. PMID:25277090

  9. Ligand binding and protein relaxation in heme proteins: a room temperature analysis of NO geminate recombination.

    PubMed

    Petrich, J W; Lambry, J C; Kuczera, K; Karplus, M; Poyart, C; Martin, J L

    1991-04-23

    Ultrafast absorption spectroscopy is used to study heme-NO recombination at room temperature in aqueous buffer on time scales where the ligand cannot leave its cage environment. While a single barrier is observed for the cage recombination of NO with heme in the absence of globin, recombination in hemoglobin and myoglobin is nonexponential. Examination of hemoglobin with and without inositol hexaphosphate points to proximal constraints as important determinants of the geminate rebinding kinetics. Molecular dynamics simulations of myoglobin and heme-imidazole subsequent to ligand dissociation were used to investigate the transient behavior of the Fe-proximal histidine coordinate and its possible involvement in geminate recombination. The calculations, in the context of the absorption measurements, are used to formulate a distinction between nonexponential rebinding that results from multiple protein conformations (substates) present at equilibrium or from nonequilibrium relaxation of the protein triggered by a perturbation such as ligand dissociation. The importance of these two processes is expected to depend on the time scale of rebinding relative to equilibrium fluctuations and nonequilibrium relaxation. Since NO rebinding occurs on the picosecond time scale of the calculated myoglobin relaxation, a time-dependent barrier is likely to be an important factor in the observed nonexponential kinetics. The general implications of the present results for ligand binding in heme proteins and its time and temperature dependence are discussed. It appears likely that, at low temperatures, inhomogeneous protein populations play an important role and that as the temperature is raised, relaxation effects become significant as well. PMID:2018766

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

    PubMed

    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. PMID:23247902

  11. Over-producing soluble protein complex and validating protein-protein interaction through a new bacterial co-expression system.

    PubMed

    Zeng, Jumei; Zhang, Lei; Li, Yuqing; Wang, Yi; Wang, Mingchao; Duan, Xin; He, Zheng-Guo

    2010-01-01

    Many proteins exert their functions through a protein complex and protein-protein interactions. However, the study of these types of interactions is complicated when dealing with toxic or hydrophobic proteins. It is difficult to use the popular Escherichia coli host for their expression, as these proteins in all likelihood require a critical partner protein to ensure their proper folding and stability. In the present study, we have developed a novel co-expression vector, pHEX, which is compatible with, and thus can be partnered with, many commercially available E. coli vectors, such as pET, pGEX and pMAL. The pHEX contains the p15A origin of replication and a T7 promoter, which can over-produce a His-tagged recombinant protein. The new co-expression system was demonstrated to efficiently co-produce and co-purify heterodimeric protein complexes, for example PE25/PPE41 (Rv2430c/Rv2431c) and ESAT6/CFP10 (Rv3874/Rv3875), from the human pathogen Mycobacterium tuberculosis H37Rv. Furthermore, the system was also effectively used to characterize protein-protein interactions through convenient affinity tags. Using an in vivo pull-down assay, for the first time we have confirmed the presence of three pairs of PE/PPE-related novel protein interactions in this pathogen. In summary, a convenient and efficient co-expression vector system has been successfully developed. The new system should be applicable to any protein complex or any protein-protein interaction of interest in a wide range of biological organisms. PMID:19747546

  12. Model based engineering of Pichia pastoris central metabolism enhances recombinant protein production

    PubMed Central

    Nocon, Justyna; Steiger, Matthias G.; Pfeffer, Martin; Sohn, Seung Bum; Kim, Tae Yong; Maurer, Michael; Rußmayer, Hannes; Pflügl, Stefan; Ask, Magnus; Haberhauer-Troyer, Christina; Ortmayr, Karin; Hann, Stephan; Koellensperger, Gunda; Gasser, Brigitte; Lee, Sang Yup; Mattanovich, Diethard

    2014-01-01

    The production of recombinant proteins is frequently enhanced at the levels of transcription, codon usage, protein folding and secretion. Overproduction of heterologous proteins, however, also directly affects the primary metabolism of the producing cells. By incorporation of the production of a heterologous protein into a genome scale metabolic model of the yeast Pichia pastoris, the effects of overproduction were simulated and gene targets for deletion or overexpression for enhanced productivity were predicted. Overexpression targets were localized in the pentose phosphate pathway and the TCA cycle, while knockout targets were found in several branch points of glycolysis. Five out of 9 tested targets led to an enhanced production of cytosolic human superoxide dismutase (hSOD). Expression of bacterial β-glucuronidase could be enhanced as well by most of the same genetic modifications. Beneficial mutations were mainly related to reduction of the NADP/H pool and the deletion of fermentative pathways. Overexpression of the hSOD gene itself had a strong impact on intracellular fluxes, most of which changed in the same direction as predicted by the model. In vivo fluxes changed in the same direction as predicted to improve hSOD production. Genome scale metabolic modeling is shown to predict overexpression and deletion mutants which enhance recombinant protein production with high accuracy. PMID:24853352

  13. [Recombinant proteins containing amino acid sequences of two ectatomin chains].

    PubMed

    Esipov, R S; Gurevich, A I; Kaiushin, A L; Korosteleva, M D; Miroshnikov, A I; Shevchenko, L V; Pluzhnikov, K A; Grishin, E V

    1997-12-01

    Artificial genes for chains A and B of ectatomin, an Ectatomma tuberculatum ant toxin, were obtained by chemical and enzymic synthesis and cloned into new plasmid vectors. Expression plasmids with the genes of hybrid proteins were constructed containing human interleukin-3 or its terminal 63-mer fragment as well as chains A and B of ectatomin, which are linked via a region containing the cleavage site of specific protease, enterokinase (hybrid proteins IL3ETOXA, IL3ETOXB, ILETOXA, and ILETOXB). Escherichia coli producer strains providing a high yield of IL3ETOXA and IL3ETOXB proteins as inclusion bodies were obtained. PMID:9499370

  14. Recombinant Nox4 cytosolic domain produced by a cell or cell-free base systems exhibits constitutive diaphorase activity

    SciTech Connect

    Nguyen, Minh Vu Chuong; Zhang, Leilei; Lhomme, Stanislas; Mouz, Nicolas

    2012-03-16

    Highlights: Black-Right-Pointing-Pointer A comparison of two bacterial cell and cell-free protein expression systems is presented. Black-Right-Pointing-Pointer Soluble and active truncated Nox4 proteins are produced. Black-Right-Pointing-Pointer Nox4 has a constitutive diaphorase activity which is independent of cytosolic factors. Black-Right-Pointing-Pointer Isoform Nox4B is unable to initiate the first electronic transfer step. Black-Right-Pointing-Pointer Findings contribute to the understanding of the mechanism of Nox4 oxidase activity. -- Abstract: The membrane protein NADPH (nicotinamide adenine dinucleotide phosphate) oxidase Nox4 constitutively generates reactive oxygen species differing from other NADPH oxidases activity, particularly in Nox2 which needs a stimulus to be active. Although the precise mechanism of production of reactive oxygen species by Nox2 is well characterized, the electronic transfer throughout Nox4 remains unclear. Our study aims to investigate the initial electronic transfer step (diaphorase activity) of the cytosolic tail of Nox4. For this purpose, we developed two different approaches to produce soluble and active truncated Nox4 proteins. We synthesized soluble recombinant proteins either by in vitro translation or by bacteria induction. While proteins obtained by bacteria induction demonstrate an activity of 4.4 {+-} 1.7 nmol/min/nmol when measured against iodonitro tetrazolium chloride and 20.5 {+-} 2.8 nmol/min/nmol with cytochrome c, the soluble proteins produced by cell-free expression system exhibit a diaphorase activity with a turn-over of 26 {+-} 2.6 nmol/min/nmol when measured against iodonitro tetrazolium chloride and 48 {+-} 20.2 nmol/min/nmol with cytochrome c. Furthermore, the activity of the soluble proteins is constitutive and does not need any stimulus. We also show that the cytosolic tail of the isoform Nox4B lacking the first NADPH binding site is unable to demonstrate any diaphorase activity pointing out the

  15. Triatoma Virus Recombinant VP4 Protein Induces Membrane Permeability through Dynamic Pores

    PubMed Central

    Sánchez-Eugenia, Rubén; Goikolea, Julen; Gil-Cartón, David; Sánchez-Magraner, Lissete

    2015-01-01

    ABSTRACT In naked viruses, membrane breaching is a key step that must be performed for genome transfer into the target cells. Despite its importance, the mechanisms behind this process remain poorly understood. The small protein VP4, encoded by the genomes of most viruses of the order Picornavirales, has been shown to be involved in membrane alterations. Here we analyzed the permeabilization activity of the natively nonmyristoylated VP4 protein from triatoma virus (TrV), a virus belonging to the Dicistroviridae family within the Picornavirales order. The VP4 protein was produced as a C-terminal maltose binding protein (MBP) fusion to achieve its successful expression. This recombinant VP4 protein is able to produce membrane permeabilization in model membranes in a membrane composition-dependent manner. The induced permeability was also influenced by the pH, being greater at higher pH values. We demonstrate that the permeabilization activity elicited by the protein occurs through discrete pores that are inserted on the membrane. Sizing experiments using fluorescent dextrans, cryo-electron microscopy imaging, and other, additional techniques showed that recombinant VP4 forms heterogeneous proteolipidic pores rather than common proteinaceous channels. These results suggest that the VP4 protein may be involved in the membrane alterations required for genome transfer or cell entry steps during dicistrovirus infection. IMPORTANCE During viral infection, viruses need to overcome the membrane barrier in order to enter the cell and replicate their genome. In nonenveloped viruses membrane fusion is not possible, and hence, other mechanisms are implemented. Among other proteins, like the capsid-forming proteins and the proteins required for viral replication, several viruses of the order Picornaviridae contain a small protein called VP4 that has been shown to be involved in membrane alterations. Here we show that the triatoma virus VP4 protein is able to produce membrane

  16. Recombinant Sheep Pox Virus Proteins Elicit Neutralizing Antibodies.

    PubMed

    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

    2016-01-01

    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. PMID:27338444

  17. Recombinant Sheep Pox Virus Proteins Elicit Neutralizing Antibodies

    PubMed Central

    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.

    2016-01-01

    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. PMID:27338444

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

    PubMed

    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. PMID:11348724

  19. Residual Endotoxin Contaminations in Recombinant Proteins Are Sufficient to Activate Human CD1c+ Dendritic Cells

    PubMed Central

    Schwarz, Harald; Schmittner, Maria; Duschl, Albert; Horejs-Hoeck, Jutta

    2014-01-01

    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. PMID:25478795

  20. Optimizing Escherichia coli as a protein expression platform to produce Mycobacterium tuberculosis immunogenic proteins

    PubMed Central

    2013-01-01

    Background A number of valuable candidates as tuberculosis vaccine have been reported, some of which have already entered clinical trials. The new vaccines, especially subunit vaccines, need multiple administrations in order to maintain adequate life-long immune memory: this demands for high production levels and degree of purity. Results In this study, TB10.4, Ag85B and a TB10.4-Ag85B chimeric protein (here-after referred as full) - immunodominant antigens of Mycobacterium tuberculosis - were expressed in Escherichia coli and purified to homogeneity. The rational design of expression constructs and optimization of fermentation and purification conditions allowed a marked increase in solubility and yield of the recombinant antigens. Indeed, scaling up of the process guaranteed mass production of all these three antigens (2.5-25 mg of pure protein/L cultivation broth). Quality of produced soluble proteins was evaluated both by mass spectrometry to assess the purity of final preparations, and by circular dichroism spectroscopy to ascertain the protein conformation. Immunological tests of the different protein products demonstrated that when TB10.4 was fused to Ag85B, the chimeric protein was more immunoreactive than either of the immunogenic protein alone. Conclusions We reached the goal of purifying large quantities of soluble antigens effective in generating immunological response against M. tuberculosis by a robust, controlled, scalable and economically feasible production process. PMID:24252280

  1. Strain engineering to prevent norleucine incorporation during recombinant protein production in Escherichia coli.

    PubMed

    Veeravalli, Karthik; Laird, Michael W; Fedesco, Mark; Zhang, Yu; Yu, X Christopher

    2015-01-01

    Incorporation of norleucine in place of methionine residues during recombinant protein production in Escherichia coli is well known. Continuous feeding of methionine is commonly used in E. coli recombinant protein production processes to prevent norleucine incorporation. Although this strategy is effective in preventing norleucine incorporation, there are several disadvantages associated with continuous feeding. Continuous feeding increases the operational complexity and the overall cost of the fermentation process. In addition, the continuous feed leads to undesirable dilution of the fermentation medium possibly resulting in lower cell densities and recombinant protein yields. In this work, the genomes of three E. coli hosts were engineered by introducing chromosomal mutations that result in methionine overproduction in the cell. The recombinant protein purified from the fermentations using the methionine overproducing hosts had no norleucine incorporation. Furthermore, these studies demonstrated that the fermentations using one of the methionine overproducing hosts exhibited comparable fermentation performance as the control host in three different recombinant protein production processes. PMID:25315437

  2. Fluorescent labeling for clonal selection of Marc 145 cells secreting high levels of recombinant protein PBD-1.

    PubMed

    Huang, Hai-Jun; Peng, Xia; Deng, Bing; Huang, Cong; Li, Jie; Qian, Yun-Guo; Gao, Qi-Shuang; Xiang, Min; Lu, Shun; Chen, Zhi-Hua; Zhan, Cai-Yao; Zhou, Li; Tao, Bi-Fei; Liu, Jie; Tan, Ben-Zhong

    2016-03-01

    Despite the powerful impact gene expression markers like the green fluorescent protein (GFP) or enhanced GFP (EGFP) exert on linking the expression of recombinant protein for selection of high producers in recent years, there is still a strong incentive to develop more economical and efficient methods for isolating mammalian cell clones secreting high levels of recombinant proteins. Here we present a new method based on the co-expression of EGFP that allows clonal selection in standard 96-well cell culture plates. The genes encoding the EGFP protein and the related protein are linked by an internal ribosome entry site and thus are transcribed into the same mRNA in an independent translation process. Since both proteins arise from a common mRNA, the EGFP expression level correlates with the expression level of the therapeutic protein in each clone. By expressing recombinant porcine β-defensin 1 in Marc 145 cells, we demonstrate the robustness and performance of this technique. The method can be served as an alternative to identify high-producer clones with various cell sorting methods. PMID:25297006

  3. Creation of a producent, optimization of expression, and purification of recombinant Yersinia pseudotuberculosis L-asparaginase.

    PubMed

    Sidoruk, K V; Pokrovsky, V S; Borisova, A A; Omeljanuk, N M; Aleksandrova, S S; Pokrovskaya, M V; Gladilina, Ju A; Bogush, V G; Sokolov, N N

    2011-12-01

    Recombinant E. coli strain producing Y. pseudotuberculosis Q66CJ2 (YpA) L-asparaginase II was created. Gene ansB homologue encoding Y. pseudotuberculosis IP 32953 L-asparaginase precursor was synthesized. The gene was cloned in pBad24 expression vector and expressed in E. coli BL21 (DE3) strain. Optimal conditions for the producer strain culturing were selected. An effective method for isolation and purification of the enzyme by two-staged column chromatography was developed. PMID:22808465

  4. Strategies for successful recombinant expression of disulfide bond-dependent proteins in Escherichia coli

    PubMed Central

    de Marco, Ario

    2009-01-01

    Bacteria are simple and cost effective hosts for producing recombinant proteins. However, their physiological features may limit their use for obtaining in native form proteins of some specific structural classes, such as for instance polypeptides that undergo extensive post-translational modifications. To some extent, also the production of proteins that depending on disulfide bridges for their stability has been considered difficult in E. coli. Both eukaryotic and prokaryotic organisms keep their cytoplasm reduced and, consequently, disulfide bond formation is impaired in this subcellular compartment. Disulfide bridges can stabilize protein structure and are often present in high abundance in secreted proteins. In eukaryotic cells such bonds are formed in the oxidizing environment of endoplasmic reticulum during the export process. Bacteria do not possess a similar specialized subcellular compartment, but they have both export systems and enzymatic activities aimed at the formation and at the quality control of disulfide bonds in the oxidizing periplasm. This article reviews the available strategies for exploiting the physiological mechanisms of bactera to produce properly folded disulfide-bonded proteins. PMID:19442264

  5. Self-assembly studies of native and recombinant fibrous proteins

    NASA Astrophysics Data System (ADS)

    Wilson, Donna Lucille

    unmodified silk protein. A sequence block from the native primary structure of collagen IV, as well as sequences of selected collagen-modifying enzymes, were manipulated through recombinant DNA technology. Collagen IV is found primarily in the basement membrane of cells and typically characterized by a loose "chicken-mesh" network of individual molecules assembled via their end regions. (Abstract shortened by UMI.)

  6. A role for homologous recombination proteins in cell cycle regulation

    PubMed Central

    Kostyrko, Kaja; Bosshard, Sandra; Urban, Zuzanna; Mermod, Nicolas

    2015-01-01

    Eukaryotic cells respond to DNA breaks, especially double-stranded breaks (DSBs), by activating the DNA damage response (DDR), which encompasses DNA repair and cell cycle checkpoint signaling. The DNA damage signal is transmitted to the checkpoint machinery by a network of specialized DNA damage-recognizing and signal-transducing molecules. However, recent evidence suggests that DNA repair proteins themselves may also directly contribute to the checkpoint control. Here, we investigated the role of homologous recombination (HR) proteins in normal cell cycle regulation in the absence of exogenous DNA damage. For this purpose, we used Chinese Hamster Ovary (CHO) cells expressing the Fluorescent ubiquitination-based cell cycle indicators (Fucci). Systematic siRNA-mediated knockdown of HR genes in these cells demonstrated that the lack of several of these factors alters cell cycle distribution, albeit differentially. The knock-down of MDC1, Rad51 and Brca1 caused the cells to arrest in the G2 phase, suggesting that they may be required for the G2/M transition. In contrast, inhibition of the other HR factors, including several Rad51 paralogs and Rad50, led to the arrest in the G1/G0 phase. Moreover, reduced expression of Rad51B, Rad51C, CtIP and Rad50 induced entry into a quiescent G0-like phase. In conclusion, the lack of many HR factors may lead to cell cycle checkpoint activation, even in the absence of exogenous DNA damage, indicating that these proteins may play an essential role both in DNA repair and checkpoint signaling. PMID:26125600

  7. A Recombinant Multiepitope Protein for Hepatitis B Diagnosis

    PubMed Central

    de Souza, Marilen Queiroz; Galdino, Alexsandro Sobreira; dos Santos, José Carlos; Soares, Marcus Vinicius; de Nóbrega, Yanna C.; Álvares, Alice da Cunha Morales; de Freitas, Sonia Maria; Torres, Fernando Araripe Gonçalves; Felipe, Maria Sueli Soares

    2013-01-01

    Hepatitis B is a liver inflammation caused by hepatitis B virus (HBV) and can be diagnosed in clinical stage by hepatitis B core antibody from IgM class (anti-HBcIgM). Hepatitis B core antibody from IgG class (Anti-HBcIgG) appears quickly after IgM, reaching high titers in chronic hepatitis, and remains even after cure. Since hepatitis B core antibody (anti-HBc) is the first antibody identified and sometimes the only marker detected during the course of infection, it can be used both to indicate HBV acute infection (anti-HBc-IgM) and to identify individuals who have come into contact with the virus (anti-HBc-IgG). In this work we propose a recombinant hepatitis B core multiepitope antigen (rMEHB) to be used for diagnosis of hepatitis B. For this purpose, a synthetic gene coding for rMEHB was designed and cloned into vector pET21a with a 6xHis tag at the C-terminal. Time course induction in E. coli showed an induced protein with an apparent molecular mass of ~21 kDa. Protein purification was performed by a single step with affinity chromatography Ni-NTA. Circular dichroism spectroscopy indicated rMEHB as a thermal stable protein at pH 7.0 and 8.0. In these conditions rMEHB was successfully used to perform an enzyme linked immuno sorbent assay (ELISA) with positive and negative sera. PMID:24294596

  8. Recombinant aequorin and green fluorescent protein as valuable tools in the study of cell signalling.

    PubMed

    Chiesa, A; Rapizzi, E; Tosello, V; Pinton, P; de Virgilio, M; Fogarty, K E; Rizzuto, R

    2001-04-01

    Luminous proteins include primary light producers, such as aequorin, and secondary photoproteins that in some organisms red-shift light emission for better penetration in space. When expressed in heterologous systems, both types of proteins may act as versatile reporters capable of monitoring phenomena as diverse as calcium homoeostasis, protein sorting, gene expression, and so on. The Ca(2+)-sensitive photoprotein aequorin was targeted to defined intracellular locations (organelles, such as mitochondria, endoplasmic reticulum, sarcoplasmic reticulum, Golgi apparatus and nucleus, and cytoplasmic regions, such as the bulk cytosol and the subplasmalemmal rim), and was used to analyse Ca(2+) homoeostasis at the subcellular level. We will discuss this application, reviewing its advantages and disadvantages and the experimental procedure. The applications of green fluorescent protein (GFP) are even broader. Indeed, the ability to molecularly engineer and recombinantly express a strongly fluorescent probe has provided a powerful tool for investigating a wide variety of biological events in live cells (e.g. tracking of endogenous proteins, labelling of intracellular structures, analysing promoter activity etc.). More recently, the demonstration that, using appropriate mutants and/or fusion proteins, GFP fluorescence can become sensitive to physiological parameters or activities (ion concentration, protease activity, etc.) has further expanded its applications and made GFP the favourite probe of cell biologists. We will here present two applications in the field of cell signalling, i.e. the use of GFP chimaeras for studying the recruitment of protein kinase C isoforms and the activity of intracellular proteases. PMID:11256942

  9. Recombinant aequorin and green fluorescent protein as valuable tools in the study of cell signalling.

    PubMed Central

    Chiesa, A; Rapizzi, E; Tosello, V; Pinton, P; de Virgilio, M; Fogarty, K E; Rizzuto, R

    2001-01-01

    Luminous proteins include primary light producers, such as aequorin, and secondary photoproteins that in some organisms red-shift light emission for better penetration in space. When expressed in heterologous systems, both types of proteins may act as versatile reporters capable of monitoring phenomena as diverse as calcium homoeostasis, protein sorting, gene expression, and so on. The Ca(2+)-sensitive photoprotein aequorin was targeted to defined intracellular locations (organelles, such as mitochondria, endoplasmic reticulum, sarcoplasmic reticulum, Golgi apparatus and nucleus, and cytoplasmic regions, such as the bulk cytosol and the subplasmalemmal rim), and was used to analyse Ca(2+) homoeostasis at the subcellular level. We will discuss this application, reviewing its advantages and disadvantages and the experimental procedure. The applications of green fluorescent protein (GFP) are even broader. Indeed, the ability to molecularly engineer and recombinantly express a strongly fluorescent probe has provided a powerful tool for investigating a wide variety of biological events in live cells (e.g. tracking of endogenous proteins, labelling of intracellular structures, analysing promoter activity etc.). More recently, the demonstration that, using appropriate mutants and/or fusion proteins, GFP fluorescence can become sensitive to physiological parameters or activities (ion concentration, protease activity, etc.) has further expanded its applications and made GFP the favourite probe of cell biologists. We will here present two applications in the field of cell signalling, i.e. the use of GFP chimaeras for studying the recruitment of protein kinase C isoforms and the activity of intracellular proteases. PMID:11256942

  10. Sustained release emphasizing recombinant human bone morphogenetic protein-2.

    PubMed

    Hollinger; Uludag; Winn

    1998-05-01

    Bone homeostasis is a dynamic process involving a myriad of cells and substrates modulated by regulatory signals such as hormones, growth and differentiating factors. When this environment is damaged, the regenerative sequalae follows a programmed pattern, and the capacity for successful recovery is often dependent on the extent of the injury. Many bony deficits that are excessively traumatic will not result in complete recovery and require therapeutic intervention(s) such as autografting or grafting from banked bone. However, for numerous reasons, an unacceptably high rate of failure is associated with these conventional therapies. Thus, alternative approaches are under investigation. A class of osteogenic regulatory molecules, the bone morphogenetic proteins (BMPs), have been isolated, cloned and characterized as potent supplements to augment bone regeneration. Optimizing a therapeutic application for BMPs may be dependent upon localized sustained release which in kind relies on a safe and well characterized carrier system. This review will discuss the current status of BMPs in bone regeneration and specifically will present the potential for a clinical therapeutic role of recombinant human BMP-2 sustained release carrier systems. PMID:10837631

  11. Purification of recombinant proteins from mammalian cell culture using a generic double-affinity chromatography scheme.

    PubMed

    Cass, Brian; Pham, Phuong Lan; Kamen, Amine; Durocher, Yves

    2005-03-01

    Transient transfection of mammalian cells has proven to be a useful technique for the rapid production of recombinant proteins because of its ability to produce milligram quantities within 2 weeks following cloning of their corresponding cDNA. This rapid production also requires a fast and efficient purification scheme that can be applied generically, typically through the use of affinity tags such as the polyhistidine-tag for capture by immobilized metal-affinity chromatography (IMAC) or the Strep-tag II, which binds to the StrepTactin affinity ligand. However, one-step purification using either of these tags has disadvantages in terms of yield, elution conditions, and purity. Here, we show that the addition of both Strep-tag-II and (His)(8) to the C-terminal of r-proteins allows efficient purification by consecutive IMAC and StrepTactin affinity. This approach has been successfully demonstrated using the intracellular protein DsRed, as well as two secreted proteins, secreted alkaline phosphatase (SEAP) and vascular endothelial growth factor (VEGF), all produced by transient transfection of HEK293-EBNA1 cells in medium supplemented with bovine calf serum. All proteins were purified to >99% homogeneity with yields varying from 29 to 81%. PMID:15721774

  12. GENERATION OF RECOMBINANT BACULOVIRUS VIA LIPOSOME MEDIATED TRANSFECTION

    EPA Science Inventory

    Baculovirus expression vectors have become a popular method of producing recombinant proteins. Production of recombinant virus requires the transfection of both the native viral DNA and a transfer plasmid into insect cells where recombination takes place. While several methods of...

  13. Metabolic flux profiling of recombinant protein secreting Pichia pastoris growing on glucose:methanol mixtures

    PubMed Central

    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

  14. Display of recombinant proteins at the surface of lactic acid bacteria: strategies and applications.

    PubMed

    Michon, C; Langella, P; Eijsink, V G H; Mathiesen, G; Chatel, J M

    2016-01-01

    Lactic acid bacteria (LAB) are promising vectors of choice to deliver active molecules to mucosal tissues. They are recognized as safe by the World Health Organization and some strains have probiotic properties. The wide range of potential applications of LAB-driven mucosal delivery includes control of inflammatory bowel disease, vaccine delivery, and management of auto-immune diseases. Because of this potential, strategies for the display of proteins at the surface of LAB are gaining interest. To display a protein at the surface of LAB, a signal peptide and an anchor domain are necessary. The recombinant protein can be attached to the membrane layer, using a transmembrane anchor or a lipoprotein-anchor, or to the cell wall, by a covalent link using sortase mediated anchoring via the LPXTG motif, or by non-covalent liaisons employing binding domains such as LysM or WxL. Both the stability and functionality of the displayed proteins will be affected by the kind of anchor used. The most commonly surfaced exposed recombinant proteins produced in LAB are antigens and antibodies and the most commonly used LAB are lactococci and lactobacilli. Although it is not necessarily so that surface-display is the preferred localization in all cases, it has been shown that for certain applications, such as delivery of the human papillomavirus E7 antigen, surface-display elicits better biological responses, compared to cytosolic expression or secretion. Recent developments include the display of peptides and proteins targeting host cell receptors, for the purpose of enhancing the interactions between LAB and host. Surface-display technologies have other potential applications, such as degradation of biomass, which is of importance for some potential industrial applications of LAB. PMID:27142045

  15. Protein-protein interactions in a higher-order structure direct lambda site-specific recombination.

    PubMed

    Thompson, J F; de Vargas, L M; Skinner, S E; Landy, A

    1987-06-01

    The highly directional site-specific recombination of bacteriophage lambda is tightly regulated by the binding of three different proteins to a complex array of sites. The manner in which these reactions are both stimulated and inhibited by co-operative binding of proteins to specific sites on the P arm of attP and AttR has been elucidated by correlation of nuclease protection with recombination studies of both wild-type and mutant DNAs. In addition to co-operative forces, there is a specific competitive interaction that allows the protein-DNA complex to serve as a "biological switch". This switch does not depend upon the simple occlusion of DNA binding sites by neighboring proteins; but, rather, the outcome of this competition is dependent on long-range interactions that vary between the higher-order structures of attP and attR. These higher-order structures are dependent on cooperative interactions involving three proteins binding to five or more sites. PMID:2958633

  16. Glycosylation characterization of recombinant human erythropoietin produced in glycoengineered Pichia pastoris by mass spectrometry.

    PubMed

    Gong, Bing; Burnina, Irina; Stadheim, Terrance A; Li, Huijuan

    2013-12-01

    Glycosylation plays a critical role in the in vivo efficacy of both endogenous and recombinant erythropoietin (EPO). Using mass spectrometry, we characterized the N-/O-linked glycosylation of recombinant human EPO (rhEPO) produced in glycoengineered Pichia pastoris and compared with the glycosylation of Chinese hamster ovary (CHO) cell-derived rhEPO. While the three predicted N-linked glycosylation sites (Asn24, Asn38 and Asn83) showed complete site occupancy, Pichia- and CHO-derived rhEPO showed distinct differences in the glycan structures with the former containing sialylated bi-antennary glycoforms and the latter containing a mixture of sialylated bi-, tri- and tetra-antennary structures. Additionally, the N-linked glycans from Pichia-produced rhEPO were similar across all three sites. A low level of O-linked mannosylation was detected on Pichia-produced rhEPO at position Ser126, which is also the O-linked glycosylation site for endogenous human EPO and CHO-derived rhEPO. In summary, the mass spectrometric analyses revealed that rhEPO derived from glycoengineered Pichia has a highly uniform bi-antennary N-linked glycan composition and preserves the orthogonal O-linked glycosylation site present on endogenous human EPO and CHO-derived rhEPO. PMID:24338886

  17. Recombineering in Corynebacterium glutamicum combined with optical nanosensors: a general strategy for fast producer strain generation.

    PubMed

    Binder, Stephan; Siedler, Solvej; Marienhagen, Jan; Bott, Michael; Eggeling, Lothar

    2013-07-01

    Recombineering in bacteria is a powerful technique for genome reconstruction, but until now, it was not generally applicable for development of small-molecule producers because of the inconspicuous phenotype of most compounds of biotechnological relevance. Here, we establish recombineering for Corynebacterium glutamicum using RecT of prophage Rac and combine this with our recently developed nanosensor technology, which enables the detection and isolation of productive mutants at the single-cell level via fluorescence-activated cell sorting (FACS). We call this new technology RecFACS, which we use for genomic site-directed saturation mutagenesis without relying on pre-constructed libraries to directly isolate L-lysine-producing cells. A mixture of 19 different oligonucleotides was used targeting codon 81 in murE of the wild-type, at a locus where one single mutation is known to cause L-lysine production. Using RecFACS, productive mutants were screened and isolated. Sequencing revealed 12 different amino acid exchanges in the targeted murE codon, which caused different L-lysine production titers. Apart from introducing a rapid genome construction technology for C. glutamicum, the present work demonstrates that RecFACS is suitable to simply create producers as well as genetic diversity in one single step, thus establishing a new general concept in synthetic biology. PMID:23630315

  18. Better and faster: improvements and optimization for mammalian recombinant protein production.

    PubMed

    Almo, Steven C; Love, James D

    2014-06-01

    Thanks to numerous technological advances, the production of recombinant proteins in mammalian cell lines has become an increasingly routine task that is no longer viewed as a heroic enterprise. While production in prokaryotic or lower eukaryotic systems may be more rapid and economical, the advantages of producing large amounts of protein that closely resembles the native form is often advantageous and may be essential for the realization of functionally active material for biological studies or biopharmaceuticals. The correct folding, processing and post-translational modifications conferred by expression in a mammalian cell is relevant to all classes of proteins, including cytoplasmic, secreted or integral membrane proteins. Therefore considerable efforts have focused on the development of growth media, cell lines, transformation methods and selection techniques that enable the production of grams of functional protein in weeks, rather than months. This review will focus on a plethora of methods that are broadly applicable to the high yield production of any class of protein (cytoplasmic, secreted or integral membrane) from mammalian cells. PMID:24721463

  19. Better and faster: improvements and optimization for mammalian recombinant protein production

    PubMed Central

    Almo, Steven C.; Love, James D.

    2014-01-01

    Thanks to numerous technological advances, the production of recombinant proteins in mammalian cell lines has become an increasingly routine task that is no longer viewed as a heroic enterprise. While production in prokaryotic or lower eukaryotic systems may be more rapid and economical, the advantages of producing large amounts of protein that closely resembles the native form is often advantageous and may be essential for the realization of functionally active material for biological studies or biopharmaceuticals. The correct folding, processing and post-translational modifications conferred by expression in a mammalian cell is relevant to all classes of proteins, including cytoplasmic, secreted or integral membrane proteins. Therefore considerable efforts have focused on the development of growth media, cell lines, transformation methods and selection techniques that enable the production of grams of functional protein in weeks, rather than months. This review will focus on a plethora of methods that are broadly applicable to the high yield production of any class of protein (cytoplasmic, secreted or integral membrane) from mammalian cells. PMID:24721463

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

    PubMed

    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. PMID:26614285

  1. Western Blot Detection of Human Anti-Chikungunya Virus Antibody with Recombinant Envelope 2 Protein.

    PubMed

    Yang, Zhaoshou; Lee, Jihoo; Ahn, Hye-Jin; Chong, Chom-Kyu; Dias, Ronaldo F; Nam, Ho-Woo

    2016-04-01

    Chikungunya virus (CHIKV), a tropical pathogen, has re-emerged and has massive outbreaks abruptly all over the world. Containing many dominant epitopes, the envelope E2 protein of CHIKV has been explored for the vaccination or diagnosis. In the present study, the antigenicity of a recombinant expressed intrinsically disorder domain (IUD) of E2 was tested for the detection of the antibody against CHIKV through western blot method. The gene of the IUD of E2 was inserted into 2 different vectors and expressed as recombinant GST-E2 and recombinant MBP-E2 fusion protein, respectively. Two kinds of fusion proteins were tested with 30 CHIKV patient sera and 30 normal sera, respectively. Both proteins were detected by 25 patients sera (83.3%) and 1 normal serum (3.3%). This test showed a relatively high sensitivity and very high specificity of the recombinant E2 proteins to be used as diagnostic antigens against CHIKV infection. PMID:27180586

  2. Western Blot Detection of Human Anti-Chikungunya Virus Antibody with Recombinant Envelope 2 Protein

    PubMed Central

    Yang, Zhaoshou; Lee, Jihoo; Ahn, Hye-Jin; Chong, Chom-Kyu; Dias, Ronaldo F.; Nam, Ho-Woo

    2016-01-01

    Chikungunya virus (CHIKV), a tropical pathogen, has re-emerged and has massive outbreaks abruptly all over the world. Containing many dominant epitopes, the envelope E2 protein of CHIKV has been explored for the vaccination or diagnosis. In the present study, the antigenicity of a recombinant expressed intrinsically disorder domain (IUD) of E2 was tested for the detection of the antibody against CHIKV through western blot method. The gene of the IUD of E2 was inserted into 2 different vectors and expressed as recombinant GST-E2 and recombinant MBP-E2 fusion protein, respectively. Two kinds of fusion proteins were tested with 30 CHIKV patient sera and 30 normal sera, respectively. Both proteins were detected by 25 patients sera (83.3%) and 1 normal serum (3.3%). This test showed a relatively high sensitivity and very high specificity of the recombinant E2 proteins to be used as diagnostic antigens against CHIKV infection. PMID:27180586

  3. Production of tag-free recombinant fusion protein encompassing promiscuous T cell epitope of tetanus toxoid and dog zona pellucida glycoprotein-3 for contraceptive vaccine development.

    PubMed

    Gupta, Neha; Shrestha, Abhinav; Panda, Amulya Kumar; Gupta, Satish Kumar

    2013-07-01

    Affinity tags can interfere in various physicochemical properties and immunogenicity of the recombinant proteins. In the present study, tag-free recombinant fusion protein encompassing promiscuous T cell epitope of tetanus toxoid [TT; amino acid (aa) residues 830-844] followed by dilysine linker and dog zona pellucida glycoprotein-3 (ZP3; aa residues 23-348) (TT-KK-ZP3) was expressed in Escherichia coli. The recombinant protein, expressed as inclusion bodies (IBs), was purified by isolation of IBs, processed to remove host cell proteins, followed by solubilization and refolding. A specific 39 kDa protein including ZP3 was identified by SDS-PAGE. CD spectra showed the presence of α-helices and β-sheets, and fluorescent spectroscopy revealed emission maxima of 265 A.U. at 339 nm for refolded protein and showed red shift in the presence of 6 M guanidine hydrochloride. Immunization of inbred FvB/J female mice with purified recombinant TT-KK-ZP3 (25 μg/animal) led to generation of high antibody titers against the recombinant protein. The antibodies reacted specifically with ZP matrix surrounding mouse oocytes. Immunized mice showed significant reduction in fertility as compared to the control group. The studies described herein provide a simple method to produce and purify tag-free recombinant protein for the development of a contraceptive vaccine. PMID:23242635

  4. Recombinant Minimalist Spider Wrapping Silk Proteins Capable of Native-Like Fiber Formation

    PubMed Central

    Xu, Lingling; Rainey, Jan K.; Meng, Qing; Liu, Xiang-Qin

    2012-01-01

    Spider silks are desirable biomaterials characterized by high tensile strength, elasticity, and biocompatibility. Spiders produce different types of silks for different uses, although dragline silks have been the predominant focus of previous studies. Spider wrapping silk, made of the aciniform protein (AcSp1), has high toughness because of its combination of high elasticity and tensile strength. AcSp1 in Argiope trifasciata contains a 200-aa sequence motif that is repeated at least 14 times. Here, we produced in E. coli recombinant proteins consisting of only one to four of the 200-aa AcSp1 repeats, designated W1 to W4. We observed that purified W2, W3 and W4 proteins could be induced to form silk-like fibers by shear forces in a physiological buffer. The fibers formed by W4 were ∼3.4 µm in diameter and up to 10 cm long. They showed an average tensile strength of 115 MPa, elasticity of 37%, and toughness of 34 J cm−3. The smaller W2 protein formed fewer fibers and required a higher protein concentration to form fibers, whereas the smallest W1 protein did not form silk-like fibers, indicating that a minimum of two of the 200-aa repeats was required for fiber formation. Microscopic examinations revealed structural features indicating an assembly of the proteins into spherical structures, fibrils, and silk-like fibers. CD and Raman spectral analysis of protein secondary structures suggested a transition from predominantly α-helical in solution to increasingly β-sheet in fibers. PMID:23209681

  5. Yeast-produced recombinant virus-like particles of coxsackievirus A6 elicited protective antibodies in mice.

    PubMed

    Zhou, Yu; Shen, Chaoyun; Zhang, Chao; Zhang, Wei; Wang, Lili; Lan, Ke; Liu, Qingwei; Huang, Zhong

    2016-08-01

    Coxsackievirus A6 (CA6) has recently emerged as the predominant pathogen of hand, foot and mouth disease (HFMD), causing significant morbidity in children and adults. The increasing prevalence of CA6 infection and its associated disease burden underscore the need for effective CA6 vaccines. However, CA6 grows poorly in cultured cells, making it difficult to develop inactivated whole-virus or live attenuated vaccines. Here we report the development of a recombinant virus-like particle (VLP) based CA6 vaccine. CA6 VLPs were produced in Pichia pastoris yeast transformed with a vector encoding both P1 and 3CD proteins of CA6. Immunization with CA6 VLPs elicited CA6-specific serum antibodies in mice. Passive transfer of anti-VLP antisera protected recipient mice against lethal CA6 challenge. Collectively, these results demonstrate that CA6 VLPs represent a viable CA6 vaccine candidate which warrants further preclinical and clinical development. PMID:27315772

  6. Comparative Evaluation of Heterologous Production Systems for Recombinant Pulmonary Surfactant Protein D

    PubMed Central

    Salgado, Daniela; Fischer, Rainer; Schillberg, Stefan; Twyman, Richard M.; Rasche, Stefan

    2014-01-01

    Commercial surfactant products derived from animal lungs are used for the treatment of respiratory diseases in premature neonates. These products contain lipids and the hydrophobic surfactant proteins B and C, which help to lower the surface tension in the lungs. Surfactant products are less effective when pulmonary diseases involve inflammatory complications because two hydrophilic surfactant proteins (A and D) are lost during the extraction process, yet surfactant protein D (SP-D) is a component of the innate immune system that helps to reduce lung inflammation. The performance of surfactant products could, therefore, be improved by supplementing them with an additional source of SP-D. Recombinant SP-D (rSP-D) is produced in mammalian cells and bacteria (Escherichia coli), and also experimentally in the yeast Pichia pastoris. Mammalian cells produce full-size SP-D, but the yields are low and the cost of production is high. In contrast, bacteria produce a truncated form of SP-D, which is active in vitro and in vivo, and higher yields can be achieved at a lower cost. We compare the efficiency of production of rSP-D in terms of the total yields achieved in each system and the amount of SP-D needed to meet the global demand for the treatment of pulmonary diseases, using respiratory distress syndrome as a case study. PMID:25538707

  7. Immunogenicity and protective efficacy of recombinant S1 domain of the porcine epidemic diarrhea virus spike protein.

    PubMed

    Oh, Jongsuk; Lee, Kyung-Won; Choi, Hwan-Won; Lee, Changhee

    2014-11-01

    Porcine epidemic diarrhea virus (PEDV) is a highly contagious enteric pathogen of swine. Acute PEDV outbreaks have continually emerged in most swine-producing Asian countries and, recently, in the United States, causing significant economic losses in the pig industry. The spike (S) protein of PEDV is a type 1 transmembrane envelope glycoprotein and consists of the S1 and S2 domains, which are responsible for virus binding and fusion, respectively. Since the S1 domain is involved in a specific high-affinity interaction with the cellular receptor and induction of neutralizing antibody in the natural host, it is a primary target for the development of effective vaccines against PEDV. In this study, a codon-optimized PEDV S1 gene containing amino acid residues 25-738 was synthesized based on a multiple alignment of the S amino acid sequences of PEDV field isolates and used to establish a stable porcine cell line constitutively expressing the PEDV S1 protein. The purified recombinant S1 protein was found to mediate highly potent antibody responses in immunized rabbits. The antibodies strongly recognized the recombinant S1 protein from cell lysates and supernatants of S1-expressing cells, whereas they bound weakly to the authentic S protein of PEDV vaccine strain SM98-1. Furthermore, a serum neutralization test revealed that the rabbit antisera completely inhibit infection of the PEDV vaccine strain at a serum dilution of 1:16. We then tested the ability of vaccination with the recombinant S1 protein to protect piglets against PEDV. Late-term pregnant sows were inoculated intramuscularly with the purified S1 protein, and the outcome was investigated in passively immunized suckling piglets after a virulent PEDV challenge. The results showed that vaccination with S1 protein efficiently protected neonatal piglets against PEDV. Our data suggest that the recombinant S1 protein shows potential as an effective and safe subunit vaccine for PED prevention. PMID:25008896

  8. Skeletal ligament healing using the recombinant human amelogenin protein.

    PubMed

    Hanhan, Salem; Ejzenberg, Ayala; Goren, Koby; Saba, Faris; Suki, Yarden; Sharon, Shay; Shilo, Dekel; Waxman, Jacob; Spitzer, Elad; Shahar, Ron; Atkins, Ayelet; Liebergall, Meir; Blumenfeld, Anat; Deutsch, Dan; Haze, Amir

    2016-05-01

    Injuries to ligaments are common, painful and debilitating, causing joint instability and impaired protective proprioception sensation around the joint. Healing of torn ligaments usually fails to take place, and surgical replacement or reconstruction is required. Previously, we showed that in vivo application of the recombinant human amelogenin protein (rHAM(+)) resulted in enhanced healing of the tooth-supporting tissues. The aim of this study was to evaluate whether amelogenin might also enhance repair of skeletal ligaments. The rat knee medial collateral ligament (MCL) was chosen to prove the concept. Full thickness tear was created and various concentrations of rHAM(+), dissolved in propylene glycol alginate (PGA) carrier, were applied to the transected MCL. 12 weeks after transection, the mechanical properties, structure and composition of transected ligaments treated with 0.5 μg/μl rHAM(+) were similar to the normal un-transected ligaments, and were much stronger, stiffer and organized than control ligaments, treated with PGA only. Furthermore, the proprioceptive free nerve endings, in the 0.5 μg/μl rHAM(+) treated group, were parallel to the collagen fibres similar to their arrangement in normal ligament, while in the control ligaments the free nerve endings were entrapped in the scar tissue at different directions, not parallel to the axis of the force. Four days after transection, treatment with 0.5 μg/μl rHAM(+) increased the amount of cells expressing mesenchymal stem cell markers at the injured site. In conclusion application of rHAM(+) dose dependently induced mechanical, structural and sensory healing of torn skeletal ligament. Initially the process involved recruitment and proliferation of cells expressing mesenchymal stem cell markers. PMID:26917487

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

    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. PMID:19005652

  11. Continuous production of flexible fibers from transgenically produced honeybee silk proteins.

    PubMed

    Poole, Jacinta; Church, Jeffrey S; Woodhead, Andrea L; Huson, Mickey G; Sriskantha, Alagacone; Kyratzis, Ilias L; Sutherland, Tara D

    2013-10-01

    Flexible and solvent stable fibers are produced after concentrated recombinant honeybee protein solutions are extruded into a methanol bath, dried, drawn in aqueous methanol, then covalently cross-linked using dry heat. Proteins in solution are predominantly coiled coil. Significant levels of non-orientated ß-sheets form during drying or after coagulation in aqueous methanol. Drawing generally aligns the coiled coil component parallel with the fibre axis and ß-sheet component perpendicular to the fiber axis. The fibres are readily handled, stable in the strong protein denaturants, urea and guanidinium, and suitable for a range of applications such as weaving and knitting. PMID:23881528

  12. Recominant Pinoresino-Lariciresinol Reductase, Recombinant Dirigent Protein And Methods Of Use

    DOEpatents

    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. Measurement of the degree of polarization of the spectra from laser produced recombining Al plasmas.

    PubMed

    Kim, Jaehoon; Kim, Dong-Eon

    2002-07-01

    Using a polarization-resolved UV-visible spectrometer, the degree of polarization of the spectra from laser produced Al plasmas was measured. The polarization resolution was achieved by using either a dichroic polarizer or a calcite crystal. The degree of polarization of Al III 4s(2)S(1/2)-4p(2)P(o)(3/2) transition at 569.66 nm was measured at different positions from a target surface. The degree of polarization was observed to be 2.1+/-0.13% at a distance of 220 microm from the target and decreased as the distance from the target increased, vanishing at a distance of about 1.3 mm from the target. To avoid the possible error due to the shot-to-shot variation of the line intensity, a calcite crystal was used to simultaneously observe the two polarization components, obtaining a similar result. The electron temperature of about 3 eV and the density of 2 x 10(17) cm(-3) measured spectroscopically indicates that the plasma was in the recombining phase. This is a report on the observation of the polarization of a transition in a laser-produced recombining Al plasma. PMID:12241524

  14. A dual-functional E. coli vector for expressing recombinant protein with high solubility and antigen presentation ability.

    PubMed

    Chuang, Chin-Kai; Su, Yu-Show; Fan, Chiu-Tin; Lee, Wen-Chuan; Chen, Ming-Yu

    2009-05-01

    A dual-functional Escherichia coli expression vector capable of producing soluble recombinant proteins with high immunogenicity in animals is introduced. This vector expresses polypeptides fused to a PTD-J-domain peptide. The J-domain peptide is derived from murine Hsp40 by using optimized codons for E. coli. The association of the J-domain to the nucleotide binding domain of the DnaK chaperone increases the probability that the fused polypeptide will be folded by the DnaK and hence increases the solubility of the recombinant protein. The PTD-J-domain can also enhance the immunogenicity of the fused chicken IGF-I polypeptide as well as an oligo-peptide derived from haptoglobin in rodents, possibly via the association with either the extracellular or intracellular Hsp70 proteins. PMID:19162194

  15. Expression and functional characterization of the plant antimicrobial snakin-1 and defensin recombinant proteins.

    PubMed

    Kovalskaya, Natalia; Hammond, Rosemarie W

    2009-01-01

    In this study, for the first time, functionally active, recombinant, cysteine-rich plant proteins snakin-1 (SN1) and defensin (PTH1) were expressed and purified using a prokaryotic expression system. The overall level of antimicrobial activities of SN1 and PTH1 produced in Escherichia coli was commensurate with that of the same proteins previously obtained from plant tissues. Both proteins exhibited strong antibacterial activity against the phytopathogenic bacterium Clavibacter michiganensis subsp. sepedonicus (50% inhibitory concentration (IC(50)) 1.5-8 microM) and antifungal activity against the phytopathogenic fungi Colletotrichum coccoides and Botrytis cinerea (IC(50) 5-14 microM). Significantly weaker activity was observed against Pseudomonas syringae pv. syringae and Pseudomonas syringae pv. tabaci. A pronounced synergistic antimicrobial effect against P. syringae pv. syringae and an additive effect against P. syringae pv. tabaci occurred with a combination of SN1 and PTH1. Aggregation of C. michiganensis subsp. sepedonicus bacterial cells at all protein concentrations tested was observed with the combination of SN1 and PTH1 and with SN1 alone. Our results demonstrate the use of a cost effective prokaryotic expression system for generation and in vitro characterization of plant cysteine-rich proteins with potential antimicrobial activities against a wide range of phytopathogenic microorganisms in order to select the most effective agents for future in vivo studies. PMID:18824107

  16. Phylogenetic analysis of Puumala virus subtype Bavaria, characterization and diagnostic use of its recombinant nucleocapsid protein.

    PubMed

    Mertens, Marc; Kindler, Eveline; Emmerich, Petra; Esser, Jutta; Wagner-Wiening, Christiane; Wölfel, Roman; Petraityte-Burneikiene, Rasa; Schmidt-Chanasit, Jonas; Zvirbliene, Aurelija; Groschup, Martin H; Dobler, Gerhard; Pfeffer, Martin; Heckel, Gerald; Ulrich, Rainer G; Essbauer, Sandra S

    2011-10-01

    Puumala virus (PUUV) is the predominant hantavirus species in Germany causing large numbers of mild to moderate cases of haemorrhagic fever with renal syndrome (HFRS). During an outbreak in South-East Germany in 2004 a novel PUUV subtype designated Bavaria was identified as the causative agent of HFRS in humans [1]. Here we present a molecular characterization of this PUUV strain by investigating novel partial and almost entire nucleocapsid (N) protein-encoding small (S-) segment sequences and partial medium (M-) segment sequences from bank voles (Myodes glareolus) trapped in Lower Bavaria during 2004 and 2005. Phylogenetic analyses confirmed their classification as subtype Bavaria, which is further subdivided into four geographical clusters. The entire N protein, harbouring an amino-terminal hexahistidine tag, of the Bavarian strain was produced in yeast Saccharomyces cerevisiae and showed a slightly different reactivity with N-specific monoclonal antibodies, compared to the yeast-expressed N protein of the PUUV strain Vranica/Hällnäs. Endpoint titration of human sera from different parts of Germany and from Finland revealed only very slight differences in the diagnostic value of the different recombinant proteins. Based on the novel N antigen indirect and monoclonal antibody capture IgG-ELISAs were established. By using serum panels from Germany and Finland their validation demonstrated a high sensitivity and specificity. In summary, our investigations demonstrated the Bavarian PUUV strain to be genetically divergent from other PUUV strains and the potential of its N protein for diagnostic applications. PMID:21598005

  17. High expression of functional adenovirus DNA polymerase and precursor terminal protein using recombinant vaccinia virus.

    PubMed Central

    Stunnenberg, H G; Lange, H; Philipson, L; van Miltenburg, R T; van der Vliet, P C

    1988-01-01

    Initiation of Adenovirus (Ad) DNA replication occurs by a protein-priming mechanism in which the viral precursor terminal protein (pTP) and DNA polymerase (pol) as well as two nuclear DNA-binding proteins from uninfected HeLa cells are required. Biochemical studies on the pTP and DNA polymerase proteins separately have been hampered due to their low abundance and their presence as a pTP-pol complex in Ad infected cells. We have constructed a genomic sequence containing the large open reading frame from the Ad5 pol gene to which 9 basepairs from a putative exon were ligated. When inserted behind a modified late promoter of vaccinia virus the resulting recombinant virus produced enzymatically active 140 kDa Ad DNA polymerase. The same strategy was applied to express the 80 kDa pTP gene in a functional form. Both proteins were overexpressed at least 30-fold compared to extracts from Adenovirus infected cells and, when combined, were fully active for initiation in an in vitro Adenovirus DNA replication system. Images PMID:3362670

  18. Regioselective covalent immobilization of recombinant antibody-binding proteins A, G, and L for construction of antibody arrays.

    PubMed

    Seo, Jin-soo; Lee, Sungwon; Poulter, C Dale

    2013-06-19

    Immobilized antibodies are useful for the detection of antigens in highly sensitive microarray diagnostic applications. Arrays with the antibodies attached regioselectively in a uniform orientation are typically more sensitive than those with random orientations. Direct regioselective immobilization of antibodies on a solid support typically requires a modified form of the protein. We now report a general approach for the regioselective attachment of antibodies to a surface using truncated forms of antibody-binding proteins A, G, and L that retain the structural motifs required for antibody binding. The recombinant proteins have a C-terminal CVIX protein farnesyltransferase recognition motif that allows us to append a bioorthogonal azide or alkyne moiety and use the Cu(I)-catalyzed Huisgen cycloaddition to attach the binding proteins to a suitably modified glass surface. This approach offers several advantages. The recombinant antibody-binding proteins are produced in Escherichia coli, chemoselectively modified posttranslationally in the cell-free homogenate, and directly attached to the glass surface without the need for purification at any stage of the process. Complexes between immobilized recombinant proteins A, G, and L and their respective strongly bound antibodies were stable to repeated washing with PBST buffer at pH 7.2. However, the antibodies could be stripped from the slides by treatment with 0.1 M glycine·HCl buffer, pH 2.6, for 30 min and regenerated by shaking with PBS buffer, pH 7.2, at 4 °C overnight. The recombinant forms of proteins A, G, and L can be used separately or in combination to give glass surfaces capable of binding a wide variety of antibodies. PMID:23746333

  19. The lambda red proteins promote efficient recombination between diverged sequences: implications for bacteriophage genome mosaicism.

    PubMed

    Martinsohn, Jann T; Radman, Miroslav; Petit, Marie-Agnès

    2008-05-01

    Genome mosaicism in temperate bacterial viruses (bacteriophages) is so great that it obscures their phylogeny at the genome level. However, the precise molecular processes underlying this mosaicism are unknown. Illegitimate recombination has been proposed, but homeologous recombination could also be at play. To test this, we have measured the efficiency of homeologous recombination between diverged oxa gene pairs inserted into lambda. High yields of recombinants between 22% diverged genes have been obtained when the virus Red Gam pathway was active, and 100 fold less when the host Escherichia coli RecABCD pathway was active. The recombination editing proteins, MutS and UvrD, showed only marginal effects on lambda recombination. Thus, escape from host editing contributes to the high proficiency of virus recombination. Moreover, our bioinformatics study suggests that homeologous recombination between similar lambdoid viruses has created part of their mosaicism. We therefore propose that the remarkable propensity of the lambda-encoded Red and Gam proteins to recombine diverged DNA is effectively contributing to mosaicism, and more generally, that a correlation may exist between virus genome mosaicism and the presence of Red/Gam-like systems. PMID:18451987

  20. Constructing a recombinant hyaluronic acid biosynthesis operon and producing food-grade hyaluronic acid in Lactococcus lactis.

    PubMed

    Sheng, Juzheng; Ling, Peixue; Wang, Fengshan

    2015-02-01

    Hyaluronic acid (HA), a natural high molecular weight polysaccharide, is produced by Streptococcus zooepidemicus. However, Streptococcus has several drawbacks including its potential to produce exotoxins, so there is demand for an alternative HA source. Here, a recombinant HA biosynthesis operon, as well as the HA biosynthesis operon of S. zooepidemicus were introduced into L. lactis using the nisin-controlled expression system, respectively. HA was successfully synthesized by recombinant L. lactis. Furthermore, overexpression of the endogenous enzymes directing the synthesis of precursor sugars was effective at increasing HA production, and increasing the supply of UDP-activated monosaccharide donors aided synthesis of monodisperse HA polysaccharides. Besides GRAS host strain (L. lactis) and NICE system, the selecting marker (lacF gene) of the recombinant strain is also food grade. Therefore, HA produced by recombinant L. lactis overcomes the problems associated with Streptococcus and provides a source of food-grading HA appropriate for widespread biotechnological applications. PMID:25447786

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

    PubMed Central

    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

  2. Five RecA-like proteins of Schizosaccharomyces pombe are involved in meiotic recombination.

    PubMed Central

    Grishchuk, A L; Kohli, J

    2003-01-01

    The genome of Schizosaccharomyces pombe contains five genes that code for proteins with sequence similarity to the Escherichia coli recombination protein RecA: rad51+, rhp55+, rhp57+, rlp1+, and dmc1+. We analyzed the effect of deletion of each of these genes on meiotic recombination and viability of spores. Meiotic recombination levels were different from wild type in all recA-related mutants in several genetic intervals, suggesting that all five RecA homologs of S. pombe are required for normal levels of meiotic recombination. Spore viability was reduced in rad51, rhp55, and rhp57 mutants, but not in rlp1 and dmc1. It is argued that reduction of crossover is not the only cause for the observed reduction of spore viability. Analysis of double and triple mutants revealed that Rad51 and Dmc1 play major and partially overlapping roles in meiotic recombination, while Rhp55, Rhp57, and Rlp1 play accessory roles. Remarkably, deletion of Rlp1 decreases the frequency of intergenic recombination (crossovers), but increases intragenic recombination (gene conversion). On the basis of our results, we present a model for the involvement of five RecA-like proteins of S. pombe in meiotic recombination and discuss their respective roles. PMID:14668362

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

    PubMed

    Cantu-Bustos, J Enrique; Cano Del Villar, Kevin D; Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Zarate, Xristo

    2016-06-01

    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. PMID:27014739

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

    PubMed Central

    Cantu-Bustos, J. Enrique; Cano del Villar, Kevin D.; Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Zarate, Xristo

    2016-01-01

    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. PMID:27014739

  5. A Bacillus anthracis strain deleted for six proteases serves as an effective host for production of recombinant proteins.

    PubMed

    Pomerantsev, Andrei P; Pomerantseva, Olga M; Moayeri, Mahtab; Fattah, Rasem; Tallant, Cynthia; Leppla, Stephen H

    2011-11-01

    Bacillus anthracis produces a number of extracellular proteases that impact the integrity and yield of other proteins in the B. anthracis secretome. In this study we show that anthrolysin O (ALO) and the three anthrax toxin proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF), produced from the B. anthracis Ames 35 strain (pXO1⁺, pXO2⁻), are completely degraded at the onset of stationary phase due to the action of proteases. An improved Cre-loxP gene knockout system was used to sequentially delete the genes encoding six proteases (InhA1, InhA2, camelysin, TasA, NprB, and MmpZ). The role of each protease in degradation of the B. anthracis toxin components and ALO was demonstrated. Levels of the anthrax toxin components and ALO in the supernatant of the sporulation defective, pXO1⁺ A35HMS mutant strain deleted for the six proteases were significantly increased and remained stable over 24 h. A pXO1-free variant of this six-protease mutant strain, designated BH460, provides an improved host strain for the preparation of recombinant proteins. As an example, BH460 was used to produce recombinant EF, which previously has been difficult to obtain from B. anthracis. The EF protein produced from BH460 had the highest in vivo potency of any EF previously purified from B. anthracis or Escherichia coli hosts. BH460 is recommended as an effective host strain for recombinant protein production, typically yielding greater than 10mg pure protein per liter of culture. PMID:21827967

  6. Rational engineering of recombinant picornavirus capsids to produce safe, protective vaccine antigen.

    PubMed

    Porta, Claudine; Kotecha, Abhay; Burman, Alison; Jackson, Terry; Ren, Jingshan; Loureiro, Silvia; Jones, Ian M; Fry, Elizabeth E; Stuart, David I; Charleston, Bryan

    2013-03-01

    Foot-and-mouth disease remains a major plague of livestock and outbreaks are often economically catastrophic. Current inactivated virus vaccines require expensive high containment facilities for their production and maintenance of a cold-chain for their activity. We have addressed both of these major drawbacks. Firstly we have developed methods to efficiently express recombinant empty capsids. Expression constructs aimed at lowering the levels and activity of the viral protease required for the cleavage of the capsid protein precursor were used; this enabled the synthesis of empty A-serotype capsids in eukaryotic cells at levels potentially attractive to industry using both vaccinia virus and baculovirus driven expression. Secondly we have enhanced capsid stability by incorporating a rationally designed mutation, and shown by X-ray crystallography that stabilised and wild-type empty capsids have essentially the same structure as intact virus. Cattle vaccinated with recombinant capsids showed sustained virus neutralisation titres and protection from challenge 34 weeks after immunization. This approach to vaccine antigen production has several potential advantages over current technologies by reducing production costs, eliminating the risk of infectivity and enhancing the temperature stability of the product. Similar strategies that will optimize host cell viability during expression of a foreign toxic gene and/or improve capsid stability could allow the production of safe vaccines for other pathogenic picornaviruses of humans and animals. PMID:23544011

  7. Purification of human recombinant interleukin 1 receptor antagonist proteins upon Bacillus subtilis sporulation.

    PubMed

    Maurizi, G; Di Cioccio, V; Macchia, G; Bossù, P; Bizzarri, C; Visconti, U; Boraschi, D; Tagliabue, A; Ruggiero, P

    1997-03-01

    Human interleukin 1 receptor antagonist (IL-1ra) and IL-1ra mutants were constitutively expressed in recombinant Bacillus subtilis in endocellular and active form. In order to optimize the purification of the recombinant proteins, a new method has been developed. After bacterial growth in fermenter, release of recombinant protein was achieved by starvation-induced sporulation. The sporulation supernatant was recovered by centrifugation, filtered, and subjected sequentially to cation- and anion-exchange chromatography. Alternatively, the fermenter's contents were directly subjected to expanded bed adsorption on a Streamline cation-exchange column, thus avoiding the centrifugation and filtration steps. Up to 88 mg of biological active purified recombinant protein per liter of culture was obtained, with a 72-79% recovery and 98% purity, depending on the molecule. By using the method described here, it is possible to achieve a spontaneous release of recombinant proteins expressed endocellularly at high levels in B. subtilis without need of a cell breakage step. Thus, this method could allow purification of the endocellular recombinant protein as if it were secreted. Furthermore, when using the expanded bed adsorption, highly purified protein was obtained in only two steps after sporulation. Among the advantages of the method, one of the most relevant is the possibility of keeping the system closed up to completion of the first purification step. PMID:9056487

  8. The human potential of a recombinant pandemic influenza vaccine produced in tobacco plants

    PubMed Central

    Jul-Larsen, Åsne; Madhun, Abdullah S.; Brokstad, Karl A.; Montomoli, Emanuele; Yusibov, Vidadi; Cox, Rebecca J.

    2012-01-01

    Rapid production of influenza vaccine antigen is an important challenge when a new pandemic occurs. Production of recombinant antigens in plants is a quick, cost effective and up scalable new strategy for influenza vaccine production. In this study, we have characterized a recombinant influenza haemagglutinin antigen (HAC1) that was derived from the 2009 pandemic H1N1 (pdmH1N1) virus and expressed in tobacco plants. Volunteers vaccinated with the 2009 pdmH1N1 oil-in-water adjuvanted vaccine provided serum and lymphocyte samples that were used to study the immunogenic properties of the HAC1 antigen in vitro. By 7 d post vaccination, the vaccine fulfilled the licensing criteria for antibody responses to the HA detected by haemagglutination inhibition and single radial hemolysis. By ELISA and ELISPOT analysis we showed that HAC1 was recognized by specific serum antibodies and antibody secreting cells, respectively. We conducted a kinetic analysis and found a peak of serum HAC1 specific antibody response between day 14 and 21 post vaccination by ELISA. We also detected elevated production of IL-2 and IFNγ and low frequencies of CD4+ T cells producing single or multiple Th1 cytokines after stimulating PBMCs (peripheral blood mononuclear cells) with the HAC1 antigen in vitro. This indicates that the antigen can interact with T cells, although confirming that an effective adjuvant would be required to improve the T-cell stimulation of plant based vaccines. We conclude that the tobacco derived recombinant HAC1 antigen is a promising vaccine candidate recognized by both B and T cells. PMID:22634440

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

    PubMed

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

    2015-08-01

    Microalgae have potential as platforms for the synthesis of high-value recombinant proteins due to their many beneficial attributes including ease of cultivation, lack of pathogenic agents, and low-cost downstream processing. However, current recombinant protein levels are low compared to other microbial platforms and stable insertion of transgenes is available in only a few microalgal species. We have explored different strategies aimed at increasing growth rate and recombinant protein production in the Chlamydomonas reinhardtii chloroplast. A novel fluorescent protein (vivid Verde Fluorescent Protein, VFP) was expressed under the control of the native atpA promoter/5'UTR element. VFP levels were detected by western blotting, with increased protein levels observed when co-expressed with a gene encoding the Escherichia coli Spy chaperone. We used these transformant lines to study the effect of temperature, light and media on recombinant protein production and cell growth. VFP levels and fluorescence, assessed by flow cytometry, allowed a determination of improved cultivation conditions as 30°C under mixotrophic mode. These conditions were tested for the accumulation of an antimicrobial endolysin (Cpl-1) of potential commercial interest, observing that the outcome obtained for VFP could not be easily replicated for Cpl-1. This study suggests that recombinant protein expression is product-specific and needs to be optimized individually. PMID:26098300

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

    PubMed Central

    Baganz, Frank; Purton, Saul

    2015-01-01

    Abstract Microalgae have potential as platforms for the synthesis of high‐value recombinant proteins due to their many beneficial attributes including ease of cultivation, lack of pathogenic agents, and low‐cost downstream processing. However, current recombinant protein levels are low compared to other microbial platforms and stable insertion of transgenes is available in only a few microalgal species. We have explored different strategies aimed at increasing growth rate and recombinant protein production in the Chlamydomonas reinhardtii chloroplast. A novel fluorescent protein (vivid Verde Fluorescent Protein, VFP) was expressed under the control of the native atpA promoter/5'UTR element. VFP levels were detected by western blotting, with increased protein levels observed when co‐expressed with a gene encoding the Escherichia coli Spy chaperone. We used these transformant lines to study the effect of temperature, light and media on recombinant protein production and cell growth. VFP levels and fluorescence, assessed by flow cytometry, allowed a determination of improved cultivation conditions as 30°C under mixotrophic mode. These conditions were tested for the accumulation of an antimicrobial endolysin (Cpl‐1) of potential commercial interest, observing that the outcome obtained for VFP could not be easily replicated for Cpl‐1. This study suggests that recombinant protein expression is product‐specific and needs to be optimized individually. PMID:26098300