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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. 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).

  3. Quality assessment of recombinant proteins produced in plants.

    PubMed

    Medrano, Giuliana; Dolan, Maureen C; Condori, Jose; Radin, David N; Cramer, Carole L

    2012-01-01

    Plant-based expression technologies for recombinant proteins have begun to receive acceptance for pharmaceuticals and other commercial markets. Protein products derived from plants offer safer, more cost-effective, and less capital-intensive alternatives to traditional manufacturing systems using microbial fermentation or animal cell culture bioreactors. Moreover, plants are now known to be capable of expressing bioactive proteins from a diverse array of species including animals and humans. Methods development to assess the quality and performance of proteins manufactured in plants are essential to support the QA/QC demands as plant-produced protein products transition to the commercial marketplace. Within the pharmaceutical arena, process validation and acceptance criteria for biological products must comply with the Food and Drug Administration (FDA) and ICH Q6B guidelines in order to initiate the regulatory approval process. Detailed product specifications will also need to be developed and validated for plant-made proteins for the bioenergy, food, chemical synthesis, or research reagent markets.We have, therefore, developed assessment methods for important qualitative and quantitative parameters of the products and the manufacturing methods utilized in plant-based production systems. In this chapter, we describe a number of procedures to validate product identity and characteristics including mass analyses, antibody cross-reactivity, N-terminal sequencing, and bioactivity. We also address methods for routine assessment of yield, recovery, and purity. The methods presented are those developed for the synthesis and recovery of the avian cytokine, chicken interleukin-12 (ChIL-12), produced in the leaves of Nicotiana benthamiana. The ChIL-12 protein used as a model for this chapter includes a C-terminal histidine epitope (HIS-tag) and, thus, these methods may be directly applicable to other HIS-tagged proteins produced in plants. However, the overall strategy

  4. [Biosynthesis and isolation of a recombinant protein for producing genetically-engineered human proinsulin].

    PubMed

    Ivankin, A N; Mitaleva, S I; Nekliudov, A D

    1998-01-01

    Isolation of the recombinant protein from a genetically engineered Escherichia coli 1854 producer for further chemical enzymatic transformation into human insulin through proinsulin was studied. Under optimal conditions, the recombinant protein formation was more than 35% of the total cell proteins. Structures of the polypeptides obtained and purified chromatographically were confirmed by amino acid analysis. Human proinsulin was derived from the recombinant protein isolated.

  5. Controlled glycosylation of plant-produced recombinant proteins.

    PubMed

    Strasser, Richard; Altmann, Friedrich; Steinkellner, Herta

    2014-12-01

    Despite their recognized importance for therapeutic proteins, the production of structurally defined glycans is still a challenging issue. However, an increased understanding of glycosylation pathways, recent advances in analytical tools, and emerging technologies for subcellular targeting using chimeric glycosyltransferases are facilitating the rational design of new glycan biosynthetic pathways. Plants are particularly amenable to glyco-engineering approaches and thus they are increasingly being used for the production of recombinant proteins. Here we summarize the main achievements in the field of in planta glyco-engineering for the production of therapeutically relevant proteins. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Overview of the purification of recombinant proteins produced in Escherichia coli.

    PubMed

    Wingfield, Paul T

    2003-02-01

    The updated version of this unit presents an overview of recombinant protein purification with special emphasis on proteins expressed in E. coli. The first section deals with information pertinent to protein purification that can be derived from translation of the cDNA sequence. This is followed by a discussion of common problems associated with bacterial protein expression. A flow chart summarizes approaches for establishing solubility and localization of bacterially produced proteins. Purification strategies for both soluble and insoluble proteins are also reviewed. A section on glycoproteins produced in bacteria in the nonglycosylated state is included to emphasize that, although they may not be useful for in vivo studies, such proteins are well suited for structural studies. Finally, protein handling, scale and aims of purification, and specialized equipment needed for recombinant protein purification and characterization are discussed. The methodologies and approaches described here are essentially suitable for laboratory-scale operations.

  7. 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.

  8. 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

  9. 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.

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

    PubMed

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

    2017-01-01

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

  11. Efficient preservation in a silicon oxide matrix of Escherichia coli, producer of recombinant proteins.

    PubMed

    Desimone, Martín F; De Marzi, Mauricio C; Copello, Guillermo J; Fernández, Marisa M; Malchiodi, Emilio L; Diaz, Luis E

    2005-10-01

    The aim of this work was to study the use of silicon oxide matrices for the immobilization and preservation of recombinant-protein-producing bacteria. We immobilized Escherichia coli BL21 transformants containing different expression plasmids. One contained DNA coding for a T-cell receptor beta chain, which was expressed as inclusion bodies in the cytoplasm. The other two encoded bacterial superantigens Staphylococcal Enterotoxin G and Streptococcal Superantigen, which were expressed as soluble proteins in the periplasm. The properties of immobilization and storage stability in inorganic matrices prepared from two precursors, silicon dioxide and tetraethoxysilane, were studied. Immobilized E. coli was stored in sealed tubes at 4 and 20 degrees C and the number of viable cells and level of recombinant protein production were analyzed weekly. Different tests showed that the biochemical characteristics of immobilized E. coli remained intact. At both temperatures selected, we found that the number of bacteria in silicon dioxide-derived matrix was of the same order of magnitude (10(9) cfu ml(-1)) as before immobilization, for 2 months. After 2 weeks, cells immobilized in an alkoxide-derived matrix decreased to 10(4) cfu ml(-1) at 4 degrees C, and no viable cells were detected at 20 degrees C. We found that immobilized bacteria could be used as a starter to produce recombinant proteins with yields comparable to those obtained from glycerol stocks: 15 mg l(-1) for superantigens and 2 mg l(-1) for T-cell receptor beta chain. These results contribute to the development of methods for microbial cell preservation under field conditions.

  12. Haematococcus as a promising cell factory to produce recombinant pharmaceutical proteins.

    PubMed

    Saei, Amir Ata; Ghanbari, Parisa; Barzegari, Abolfazl

    2012-11-01

    The need for recombinant pharmaceutical proteins has urged scientists all over the world to search for better protein expression systems which have higher capabilities and flexibilities. Although a number of protein expression systems are now available, no system is ideal and different systems lack specific properties. Here, microalga Haematococcus is discussed as a new protein expression system which merits cheap growth medium, fast growth rate, ease of manipulation and scale-up, ease of transformation, potential of exploiting in bioreactors and ability to exert post-translational modifications to the proteins. This green single-cell plant has favorable biological and biotechnological features for production of remarkable yields of recombinant proteins with high functionality. In this review article, we highlight the favorable biotechnological characteristics of Haematococcus for lowering costs and facilitating scale-up of recombinant protein production along with its superior biological features for genetic engineering.

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

    PubMed

    Tagliavia, Marcello; Cuttitta, Angela

    2016-01-01

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

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

    PubMed

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

    2015-07-01

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

  15. Effective non-denaturing purification method for improving the solubility of recombinant actin-binding proteins produced by bacterial expression.

    PubMed

    Chung, Jeong Min; Lee, Sangmin; Jung, Hyun Suk

    2017-05-01

    Bacterial expression is commonly used to produce recombinant and truncated mutant eukaryotic proteins. However, heterologous protein expression may render synthesized proteins insoluble. The conventional method used to express a poorly soluble protein, which involves denaturation and refolding, is time-consuming and inefficient. There are several non-denaturing approaches that can increase the solubility of recombinant proteins that include using different bacterial cell strains, altering the time of induction, lowering the incubation temperature, and employing different detergents for purification. In this study, we compared several non-denaturing protocols to express and purify two insoluble 34 kDa actin-bundling protein mutants. The solubility of the mutant proteins was not affected by any of the approaches except for treatment with the detergent sarkosyl. These results indicate that sarkosyl can effectively improve the solubility of insoluble proteins during bacterial expression. Copyright © 2016. Published by Elsevier Inc.

  16. Challenges in electrochemical pre-purification of recombinant proteins from green plant tissues: sgfp produced in tobacco leaves.

    PubMed

    Robić, Goran

    2013-01-01

    The use of recombinant proteins has increased greatly in recent years, as have the number of techniques and materials used for their production and purification. The principal advantage of using plants as bioreactors is the cost of the recombinant protein production, which is about 1000-fold lower as in the case of using CHO cells commonly applied in industry today. Among the different types of "green" bioreactors being studied today, there is a general consensus among scientists that production in green plant tissues such as leaves is more feasible. However, the presence of chlorophyll and phenolic compounds in plant extracts, which can precipitate and denature the proteins besides damaging separation membranes and gels, makes this technology impracticable on a commercial scale. Electrochemically produced aluminium hydroxide gel can be used to adsorb these compounds, and pre-purify recombinant synthetic green fluorescent protein (sGFP) produced in Nicotiana benthamiana leaves. Removal efficiencies of 99.7% of chlorophyll, 88.5% of phenolic compounds, and 38.5% of native proteins from the N. benthamiana extracts were achieved without removing sGFP from the extracts. Since electrochemical preparation of aluminum hydroxide gel is a cost-effective technique, its use can substantially contribute to the development of future production platforms for recombinant proteins produced in green plant tissues of pharmaceutical and industrial interest.

  17. Production of specific IgY antibody to the recombinant FanC protein produced in Escherichia coli

    PubMed Central

    Nasiri, Khadijeh; Zibaee, Saeed; Nassiri, Mohammadreza; Tahmoorespur, Mojtaba; Haghparast, Alireza

    2016-01-01

    Objective(s): Enterotoxigenic Escherichia coli (ETEC) strains are one of the primary causes of diarrhea in newborn calves and in humans, pigs, and sheep. IgY technology has been identified as a promising alternative to generating a mass amount of specific antibody for use in immunotherapy and immunodiagnostics. The purpose of this study was to produce specific antibody by egg yolk antibody (IgY) to recombinant FanC protein from ETEC. Materials and Methods: FanC (K99) gene was amplified from ETEC by specific primers and polymerase chain reaction. The gene was cloned and subcloned into pTZ57R/T and pET32a (+) vectors, respectively. Recombinant vector was transferred into E. coli BL21 CodonPlus (DE3). Protein expression was investigated by 1 mM IPTG induction. Hens were immunized by the purified recombinant FanC protein. The activity and specificity of the IgY antibody were detected by dot-blotting, Western blotting, and indirect ELISA. Results: We obtained FanC specific IgYs by immunizing the hens with the recombinant FanC protein. The anti-FanC IgY showed binding specifically to the FanC protein of ETEC. Conclusion: The results emphasize that specific IgY against the recombinant FanC protein could be recommended as a candidate for passive immunization against ETEC infection in animals and humans. PMID:27746871

  18. Optimization of culturing conditions of recombined Escherichia coli to produce umami octopeptide-containing protein.

    PubMed

    Zhang, Yin; Wei, Xiong; Lu, Zhou; Pan, Zhongli; Gou, Xinhua; Venkitasamy, Chandrasekar; Guo, Siya; Zhao, Liming

    2017-07-15

    Using synthesized peptides to verify the taste of natural peptides was probably the leading cause for tasting disputes regarding umami peptides. A novel method was developed to prepare the natural peptide which could be used to verify the taste of umami peptide. A controversial octopeptide was selected and gene engineering was used to structure its Escherichia coli. expressing vector. A response surface method was adopted to optimize the expression conditions of the recombinant protein. The results of SDS-PAGE for the recombinant protein indicated that the recombinant expression system was successfully structured. The fitting results of the response surface experiment showed that the OD600 value was the key factor which influenced the expression of the recombinant protein. The optimal culturing process conditions predicted with the fitting model were an OD600 value of 0.5, an IPTG concentration of 0.6mM, a culturing temperature of 28.75°C and a culturing time of 5h.

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

    PubMed Central

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

    2010-01-01

    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

  20. The pEAQ vector series: the easy and quick way to produce recombinant proteins in plants.

    PubMed

    Peyret, Hadrien; Lomonossoff, George P

    2013-09-01

    The pEAQ vectors are a series of plasmids designed to allow easy and quick production of recombinant proteins in plants. Their main feature is the use of the Cowpea Mosaic Virus hypertranslational "CPMV-HT" expression system, which provides high yields of recombinant protein through extremely high translational efficiency without the need for viral replication. Since their creation, the pEAQ vectors have been used to produce a wide variety of proteins in plants. Viral proteins and Virus-Like Particles (VLPs) have been of particular interest, but other types of proteins including active enzymes have also been expressed. While the pEAQ vectors have mostly been used in a transient expression context, through agroinfiltration of leaves, they have also been shown to be suitable for the production of stably transformed lines of both cell cultures and whole plants. This paper looks back on the genesis of the pEAQ vectors and reviews their use so far.

  1. An optimized sericin-1 expression system for mass-producing recombinant proteins in the middle silk glands of transgenic silkworms.

    PubMed

    Wang, Feng; Xu, Hanfu; Yuan, Lin; Ma, Sanyuan; Wang, Yuancheng; Duan, Xiaoli; Duan, Jianping; Xiang, Zhonghuai; Xia, Qingyou

    2013-10-01

    The middle silk gland (MSG) of silkworm is thought to be a potential host for mass-producing valuable recombinant proteins. Transgenic MSG expression systems based on the usage of promoter of sericin1 gene (sericin-1 expression system) have been established to produce various recombinant proteins in MSG. However, further modifying the activity of the sericin-1 expression system to yield higher amounts of recombinant proteins is still necessary. In this study, we provide an alternative modification strategy to construct an efficient sericin-1 expression system by using the hr3 enhancer (hr3 CQ) from a Chongqing strain of the Bombyx mori nuclear polyhedrosis virus (BmNPV) and the 3'UTRs of the fibroin heavy chain (Fib-HPA), the fibroin light chain (Fib-LPA), and Sericin1 (Ser1PA) genes. We first analyzed the effects of these DNA elements on expression of luciferase, and found that the combination of hr3 CQ and Ser1PA was most effective to increase the activity of luciferase. Then, hr3 CQ and Ser1PA were used to modify the sericin1 expression system. Transgenic silkworms bearing these modified sericin1 expression vectors were generated by a piggyBac transposon mediated genetic transformation method. Our results showed that mRNA level of DsRed reporter gene in transgenic silkworms containing hr3 CQ and Ser1PA significantly increased by 9 fold to approximately 83 % of that of endogenous sericin1. As the results of that, the production of recombinant RFP increased by 16 fold to 9.5 % (w/w) of cocoon shell weight. We conclude that this modified sericin-1 expression system is efficient and will contribute to the MSG as host to mass produce valuable recombinant proteins.

  2. Immunodiagnosis of Prune dwarf virus using antiserum produced to its recombinant coat protein.

    PubMed

    Abou-Jawdah, Yusuf; Sobh, Hana; Cordahi, Nada; Kawtharani, Hadia; Nemer, George; Maxwell, Douglas P; Nakhla, Mark K

    2004-10-01

    Certification represents the first line of defense against fruit tree viruses. For certification or surveys dealing with large number of samples, ELISA is still considered the technique of choice and requires a continuous supply of good quality antibodies. Prune dwarf virus (PDV) is among the major viruses affecting stone fruits; it belongs to the genus Ilarvirus named so for its isometric labile particles. Recombinant DNA technology was investigated for production of PDV antiserum to avoid labile virus purification and virus maintenance problems. The PDV coat protein gene (CP) was cloned into a protein expression bacterial plasmid vector which allowed a good level of expression of up to 2mg native protein/L culture. The recombinant PDV CP was injected into rabbits and the crude antiserum was successfully used in indirect ELISA at dilutions of up to 1:5000 to detect PDV in infected leaf samples. Similar results were obtained in dot blot immunoassays (DBIA). The antibodies were used in double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and results were comparable to a reference commercial kit. The crude antiserum was efficiently used for coating ELISA plates, thereby reducing test costs.

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

    PubMed

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

    2010-09-01

    Over-expression of heme binding proteins in Escherichia 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.

  4. Prokaryotic High-Level Expression System in Producing Adhesin Recombinant Protein E of Nontypeable Haemophilus influenzae

    PubMed Central

    Tavakoli, Minoo; Bouzari, Saeed; Siadat, Seyed Davar; Najar Peerayeh, Shahin; Jafari, Anis

    2015-01-01

    Background: Adhesion protein E (PE) of Haemophilus influenzae is a 16 - 18 kDa protein with 160 amino acids which causes adhesion to epithelial cells and acts as a major factor in pathogenesis. Objectives: In this study, we performed cloning, expression and purification of PE as a candidate antigen for vaccine design upon further study. Materials and Methods: At first, the pe gene of NTHi ATCC 49766 strain (483 bp) was amplified by PCR. Then, to sequence the resulted amplicon, it was cloned into TA vector (pTZ57R/T). In the next step, the sequenced gene was sub-cloned in pBAD/gIII A vector and transformed into competent Escherichia coli TOP10. For overexpression, the recombinant bacteria were grown in broth medium containing arabinose and the recombinant protein was purified using metal affinity chromatography (Ni-nitrilotriacetic acid) (Ni-NTA agarose). Finally, the protein was detected using sodium dodecyl sulfate polyacrylamide gel electrophores (SDS-PAG) and confirmed by western blotting. Results: The cloned gene was confirmed by PCR, restriction digestion and sequencing. The sequenced gene was searched for homology in GenBank and 99% similarity was found to the already deposited genes in GenBank. Then we obtained PE using Ni-NTA agarose with up to 7 mg/mL concentration. Conclusions: The pe gene was successfully cloned and confirmed by sequencing. Finally, PE was obtained with high concentration. Due to high homology and similarity among the pe gene from NTHi ATCC 49766 and other NTHi strains in GenBank, we believe that the protein is a universal antigen to be used as a vaccine design candidate and further studies to evaluate its immunogenicity is underway. PMID:26034537

  5. Hydrodynamic behavior of shaking flasks used for producing a recombinant protein by filamentous bacteria

    NASA Astrophysics Data System (ADS)

    Cordova Aguilar, Maria Soledad; Garcia, Monica; Trujillo-Roldan, Mauricio Alberto; Ascanio, Gabriel; Zenit, Roberto; Soto, Enrique

    2012-11-01

    Shake flasks are widely used for culture research. The agitation rate is one of the factors that determines the mass transfer. However, it has not been studied in detail. In this work, a comparison of the hydrodynamic performance for conventional, baffled and coiled spring Erlenmeyer flasks is presented. The velocity fields for a horizontal plane were measured by means of a Particle Image Velocimetry (PIV) technique and high speed videos were recorded to observe the behavior of the interface as a function of the agitation rate. It was observed not only that there is a strong dependence between the geometry and the hydrodynamics, but also there is a good agreement with the results obtained previously by Gamboa et al., in 2011, with the evaluation of the influence of culture conditions of S. lividans on protein O-glycosylation. The turbulence intensity increases with shaken rate. However, for the baffled geometry, it was observed a decrease for a critical speed, which is related with the in-phase and out-phase regions. These results can be an explanation for the variations in protein productivity as a function of the flask geometry and the differences in aggregation morphology and the pattern of O-glycosylation of the recombinant protein.

  6. GATEWAY technology and E. coli recombinant system produce a properly folded and functional recombinant allergen of the lipid transfer protein of apple (Mal d 3).

    PubMed

    Borges, Jean-Philippe; Culerrier, Raphaël; Aldon, Didier; Barre, Annick; Benoist, Hervé; Saurel, Olivier; Milon, Alain; Didier, Alain; Rougé, Pierre

    2010-04-01

    The lipid transfer protein of apple fruit, Mal d 3, has been produced as a soluble recombinant protein in transformed Escherichia coli cells using the GATEWAY technology. Circular dichroism spectra showing the protein essentially consists of alpha-helices indicate that the rMal d 3 is properly folded. The (1)H NMR spectra also indicates a correct fold for the recombinant allergen. The reactivity of rMal d 3 towards IgE from apple allergic patients and in vitro degranulation activity measured on transformed rat basophil leukemia cells expressing the human Fc epsilon RI alpha-subunit of rMal d 3 is similar to those of the native allergen purified from apple fruits. The expression of active rMal d 3 in E. coli is readily feasible and offers an interesting alternative to the production of recombinant allergen in the yeast Pichia pastoris. This expression in E. coli open the way to the modification of Mal d 3 by site-directed mutagenesis for immunotherapy purposes. (c) 2009 Elsevier Inc. All rights reserved.

  7. Making recombinant extracellular matrix proteins.

    PubMed

    Ruggiero, Florence; Koch, Manuel

    2008-05-01

    A variety of approaches to understand extracellular matrix protein structure and function require production of recombinant proteins. Moreover, the expression of heterologous extracellular matrix proteins, in particular collagens, using the recombinant technology is of major interest to the biomedical industry. Although extracellular matrix proteins are large, modular and often multimeric, most of them have been successfully produced in various expression systems. This review provides important factors, including the design of the construct, the cloning strategies, the expression vectors, the transfection method and the host cell systems, to consider in choosing a reliable and cost-effective way to make recombinant extracellular matrix proteins. Advantages and drawbacks of each system have been appraised. Protocols that may ease efficient recombinant production of extracellular matrix are described. Emphasis is placed on the recombinant collagen production. Members of the collagen superfamily exhibit specific structural features and generally require complex post-translational modifications to retain full biological activity that make more arduous their recombinant production.

  8. 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.

  9. Construction of Recombinant Bacmid Containing M2e-Ctxb and Producing the Fusion Protein in Insect Cell Lines

    PubMed Central

    Mirzaei, Nima; Mokhtari Azad, Talat; Nategh, Rakhshandeh; Soleimanjahi, Hoorieh; Amirmozafari, Nour

    2014-01-01

    Background: Sequence variations in glycoproteins of influenza virus surface impel us to design new candidate vaccines yearly. Ectodomain of influenza M2 protein is a surface and highly conserved protein. M2e in influenza vaccines may eliminate the need for changing vaccine formulation every year. Objectives: In this study, a recombinant baculovirus containing M2e and cholera toxin subunit B fusion gene was generated with transposition process to express in large amounts in insect cell lines. Materials and Methods: M2e-ctxB fusion gene was created and cloned into pFastBac HT. The recombinant vector was transformed into DH10Bac cells to introduce the fusion gene into the bacmid DNA via a site-specific transposition process. The recombinant bacmid was then extracted from white colonies and further analyzed using PCR, DNA sequence analyzing, and indirect immunofluorescence assay. Results: PCR and DNA sequence analyzing results showed that the fusion gene was constructed as a single open reading frame and was successfully inserted into bacmid DNA. Moreover, indirect immunofluorescence results showed that the fusion gene was successfully expressed. Conclusions: Baculovirus expression vector system is valuable to produce M2e based influenza vaccines due to its simple utilization and ease of target gene manipulation. The expressed protein in such systems can improve the evaluating process of new vaccination strategies. PMID:24719728

  10. Recombinant anthrax toxin receptor-Fc fusion proteins produced in plants protect rabbits against inhalational anthrax.

    PubMed

    Wycoff, Keith L; Belle, Archana; Deppe, Dorothée; Schaefer, Leah; Maclean, James M; Haase, Simone; Trilling, Anke K; Liu, Shihui; Leppla, Stephen H; Geren, Isin N; Pawlik, Jennifer; Peterson, Johnny W

    2011-01-01

    Inhalational anthrax, a zoonotic disease caused by the inhalation of Bacillus anthracis spores, has a ∼50% fatality rate even when treated with antibiotics. Pathogenesis is dependent on the activity of two toxic noncovalent complexes: edema toxin (EdTx) and lethal toxin (LeTx). Protective antigen (PA), an essential component of both complexes, binds with high affinity to the major receptor mediating the lethality of anthrax toxin in vivo, capillary morphogenesis protein 2 (CMG2). Certain antibodies against PA have been shown to protect against anthrax in vivo. As an alternative to anti-PA antibodies, we produced a fusion of the extracellular domain of human CMG2 and human IgG Fc, using both transient and stable tobacco plant expression systems. Optimized expression led to the CMG2-Fc fusion protein being produced at high levels: 730 mg/kg fresh leaf weight in Nicotiana benthamiana and 65 mg/kg in N. tabacum. CMG2-Fc, purified from tobacco plants, fully protected rabbits against a lethal challenge with B. anthracis spores at a dose of 2 mg/kg body weight administered at the time of challenge. Treatment with CMG2-Fc did not interfere with the development of the animals' own immunity to anthrax, as treated animals that survived an initial challenge also survived a rechallenge 30 days later. The glycosylation of the Fc (or lack thereof) had no significant effect on the protective potency of CMG2-Fc in rabbits or on its serum half-life, which was about 5 days. Significantly, CMG2-Fc effectively neutralized, in vitro, LeTx-containing mutant forms of PA that were not neutralized by anti-PA monoclonal antibodies.

  11. 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

  12. Control of specific carbon dioxide production in a fed-batch culture producing recombinant protein using a soft sensor.

    PubMed

    Gustavsson, Robert; Lukasser, Cornelia; Mandenius, Carl-Fredrik

    2015-04-20

    The feeding of a fed-batch cultivation producing recombinant protein was controlled by a soft sensor set-up. It was assumed that the control approach could be based on the cell's production of carbon dioxide and that this parameter indicates the metabolic state occurring at induced protein expression. The soft sensor used the on-line signals from a carbon dioxide analyser and a near-infrared (NIR) probe for biomass to estimate the specific production rate qCO2. Control experiments were carried out with various qCO2 set-points where we observe that the feeding of nutrients to the culture could easily be controlled and resulted in a decreased variability compared to uncontrolled cultivations. We therefore suggest that this control approach could serve as an alternative to other commonly applied methods such as controlling the cell's overflow metabolism of acetate or the cell's specific growth rate. However, further studies of the internal metabolic fluxes of CO2 during protein expression would be recommended for a more precise characterization of the relationship between qCO2 and protein expression in order to fully interpret the control behaviour.

  13. Causes of proteolytic degradation of secreted recombinant proteins produced in methylotrophic yeast Pichia pastoris: case study with recombinant ovine interferon-tau.

    PubMed

    Sinha, Jayanta; Plantz, Bradley A; Inan, Mehmet; Meagher, Michael M

    2005-01-05

    It was observed that during fermentative production of recombinant ovine interferon-tau (r-oIFN-tau) in Pichia pastoris, a secreted recombinant protein, the protein was degraded increasingly after 48 h of induction and the rate of degradation increased towards the end of fermentation at 72 h, when the fermentation was stopped. Proteases, whose primary source was the vacuoles, was found in increasing levels in the cytoplasm and in the fermentation broth after 48 h of induction and reached maximal values when the batch was completed at 72 h. Protease levels at various cell fractions as well as in the culture supernatant were lower when glycerol was used as the carbon source instead of methanol. It can be concluded that methanol metabolism along with cell lysis towards the end of fermentation contributes to increased proteolytic activity and eventual degradation of recombinant protein.

  14. Validation of the manufacturing process used to produce long-acting recombinant factor IX Fc fusion protein.

    PubMed

    McCue, J; Osborne, D; Dumont, J; Peters, R; Mei, B; Pierce, G F; Kobayashi, K; Euwart, D

    2014-07-01

    Recombinant factor IX Fc (rFIXFc) fusion protein is the first of a new class of bioengineered long-acting factors approved for the treatment and prevention of bleeding episodes in haemophilia B. The aim of this work was to describe the manufacturing process for rFIXFc, to assess product quality and to evaluate the capacity of the process to remove impurities and viruses. This manufacturing process utilized a transferable and scalable platform approach established for therapeutic antibody manufacturing and adapted for production of the rFIXFc molecule. rFIXFc was produced using a process free of human- and animal-derived raw materials and a host cell line derived from human embryonic kidney (HEK) 293H cells. The process employed multi-step purification and viral clearance processing, including use of a protein A affinity capture chromatography step, which binds to the Fc portion of the rFIXFc molecule with high affinity and specificity, and a 15 nm pore size virus removal nanofilter. Process validation studies were performed to evaluate identity, purity, activity and safety. The manufacturing process produced rFIXFc with consistent product quality and high purity. Impurity clearance validation studies demonstrated robust and reproducible removal of process-related impurities and adventitious viruses. The rFIXFc manufacturing process produces a highly pure product, free of non-human glycan structures. Validation studies demonstrate that this product is produced with consistent quality and purity. In addition, the scalability and transferability of this process are key attributes to ensure consistent and continuous supply of rFIXFc. © 2014 The Authors. Haemophilia Published by John Wiley & Sons Ltd.

  15. Validation of the manufacturing process used to produce long-acting recombinant factor IX Fc fusion protein

    PubMed Central

    McCue, J; Osborne, D; Dumont, J; Peters, R; Mei, B; Pierce, G F; Kobayashi, K; Euwart, D

    2014-01-01

    Recombinant factor IX Fc (rFIXFc) fusion protein is the first of a new class of bioengineered long-acting factors approved for the treatment and prevention of bleeding episodes in haemophilia B. The aim of this work was to describe the manufacturing process for rFIXFc, to assess product quality and to evaluate the capacity of the process to remove impurities and viruses. This manufacturing process utilized a transferable and scalable platform approach established for therapeutic antibody manufacturing and adapted for production of the rFIXFc molecule. rFIXFc was produced using a process free of human- and animal-derived raw materials and a host cell line derived from human embryonic kidney (HEK) 293H cells. The process employed multi-step purification and viral clearance processing, including use of a protein A affinity capture chromatography step, which binds to the Fc portion of the rFIXFc molecule with high affinity and specificity, and a 15 nm pore size virus removal nanofilter. Process validation studies were performed to evaluate identity, purity, activity and safety. The manufacturing process produced rFIXFc with consistent product quality and high purity. Impurity clearance validation studies demonstrated robust and reproducible removal of process-related impurities and adventitious viruses. The rFIXFc manufacturing process produces a highly pure product, free of non-human glycan structures. Validation studies demonstrate that this product is produced with consistent quality and purity. In addition, the scalability and transferability of this process are key attributes to ensure consistent and continuous supply of rFIXFc. PMID:24811361

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

    PubMed

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

    2017-08-01

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

  17. [Producing recombinant adenovirus encoding green fluorescent protein (Ad-GFP) by suspension cultured HEK-293 N3S cells].

    PubMed

    Tian, Bo; Wu, Bin; Zhang, Qun-Wei; Bi, Jian-Jin; Wang, Lan; Zhu, Bao-Zhen; Geng, Yue; Wu, Zu-Ze

    2007-09-01

    Adenovirus vectors are one of the most promising gene transfer systems. They are of great value for gene therapy because these vectors achieve temporal high-level transgene expression and high gene transfer efficiency. To meet increasing needs of adenovirus vectors for gene therapy programs, parallel development of efficient, scalable and reproducible production processes is required. Perfusion cultivation of 293 cells is one of the most commonly used methods to produce adenovirus vectors and it is suitable for industrialized production specially. Experimental studies had been carried out to produce recombinant adenovirus containing the green fluorescent protein gene (Ad-GFP) by perfusion cultivation of HEK-293 N3S cells in a 5L stirring bioreactors. Perfusion rate was 1-2 volume/day. To infect the 293 N3S cells with Ad-GFP at the density of (2-4) x 10(6) cells/ ml. The time of collecting cells was 48 hours post infection. After three rounds of freeze/thaw and centrifugation, the crude viral lysates were stored at--80 degrees C until use. Then to get the Ad-GFP products by 2 x CsCl-gradient purification. The purity of the products was determined by the A260/A280 ratio and a high performance liquid chromatography (HPLC) assay. The infective titer was determined by a TCID50 assay. The culture term was 10-12 days. The infectious titer, the number of virus particle and the ratio of infectious titer to virus particle for the product were 1.0 x 10(11) IU/mL, 1.68 x 10(12) VP/mL and 6.0% IU/VP respectively. The A260/A280 ratio was 1.33, and the purity determined by HPLC was 99.2%. The cell specific productivity was around 1000 IU/cell. By perfusion cultivation of 293 N3S cells in a 5L stirring bioreactors, we established the production process for Ad-GFP, which paves a way to produce other recombinant adenovirus for gene therapy.

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

    PubMed

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

    2017-07-01

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

  19. [Characterization of hepatitis C virus structural proteins and HCV-like particles produced in recombinant baculovirus infected insect cells].

    PubMed

    Belzhelarskaia, S N; Koroleva, N N; Popenko, V V; Drutsa, V L; Orlova, O V; Rubtsov, P M; Kochetkov, S N

    2010-01-01

    Three proteins, namely: "core" protein C and glycoproteins E1 and E2, are main structural proteins forming a hepatitis C vius (HCV) virion. The virus structure and assembly, a role of the structural proteins in virion morphogenesis remain unknown because of the lack of an efficient culture system for HCV to be grown in vitro. Using recombinant baculoviruses expressing HCV structural protein genes in insect cells the specific structural proteins at the level of 25-35% relative to a common cell protein content, heterodimers of the glcoproteins, and HCV-like particles have been obtained. It has been demonstrated that recombinant proteins C, E1, and E2 go through the posttranslation modification, the glycoproteins form the non-covalent heterodimer, and HCV-like particles are located in endoplasmatic reticulum membrains of infected cells. An ability of the expressed proteins for forming E1E2 dimers and HCV-like particles was used for studying the role of E1 protein glcosylation upon expression and processing of the glycoproteins.

  20. Anti-loxoscelic horse serum produced against a recombinant dermonecrotic protein of Brazilian Loxosceles intermedia spider neutralize lethal effects of Loxosceles laeta venom from Peru.

    PubMed

    Duarte, C G; Bonilla, C; Guimarães, G; Machado de Avila, R A; Mendes, T M; Silva, W; Tintaya, B; Yarleque, A; Chávez-Olórtegui, C

    2015-01-01

    In this work, an anti-loxoscelic serum was produced by immunizing horses with a recombinant dermonecrotic protein from Loxosceles intermedia (rLiD1). Anti-rLiD1 antibodies were able to recognize different species of Loxosceles venoms by Western Blot and ELISA. The efficacy of anti-rLiD1 serum against the toxic effects of Loxosceles laeta (Peru) venom was tested, showing that anti-rLiD1 serum can neutralize those effects. This study confirms that recombinant proteins can be good candidates to replace crude venoms for antivenom production. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Recombinant human milk proteins.

    PubMed

    Lönnerdal, Bo

    2006-01-01

    Human milk provides proteins that benefit newborn infants. They not only provide amino acids, but also facilitate the absorption of nutrients, stimulate growth and development of the intestine, modulate immune function, and aid in the digestion of other nutrients. Breastfed infants have a lower prevalence of infections than formula-fed infants. Since many women in industrialized countries choose not to breastfeed, and an increasing proportion of women in developing countries are advised not to breastfeed because of the risk of HIV transmission, incorporation of recombinant human milk proteins into infant foods is likely to be beneficial. We are expressing human milk proteins known to have anti-infective activity in rice. Since rice is a normal constituent of the diet of infants and children, limited purification of the proteins is required. Lactoferrin has antimicrobial and iron-binding activities. Lysozyme is an enzyme that is bactericidal and also acts synergistically with lactoferrin. These recombinant proteins have biological activities identical to their native counterparts. They are equally resistant to heat processing, which is necessary for food applications, and to acid and proteolytic enzymes which are needed to maintain their biological activity in the gastrointestinal tract of infants. These recombinant human milk proteins may be incorporated into infant formulas, baby foods and complementary foods, and used with the goal to reduce infectious diseases.

  2. 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.

  3. 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

  4. Recombinant DNA production of spider silk proteins.

    PubMed

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

    2013-11-01

    Spider dragline silk is considered to be the toughest biopolymer on Earth due to an extraordinary combination of strength and elasticity. Moreover, silks are biocompatible and biodegradable protein-based materials. Recent advances in genetic engineering make it possible to produce recombinant silks in heterologous hosts, opening up opportunities for large-scale production of recombinant silks for various biomedical and material science applications. We review the current strategies to produce recombinant spider silks.

  5. Fusion of the Dhfr/Mtx and IR/MAR gene amplification methods produces a rapid and efficient method for stable recombinant protein production.

    PubMed

    Noguchi, Chiemi; Araki, Yoshio; 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.

  6. 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

  7. Multiple microfermentor battery: a versatile tool for use with automated parallel cultures of microorganisms producing recombinant proteins and for optimization of cultivation protocols.

    PubMed

    Frachon, Emmanuel; Bondet, Vincent; Munier-Lehmann, Hélène; Bellalou, Jacques

    2006-08-01

    A multiple microfermentor battery was designed for high-throughput recombinant protein production in Escherichia coli. This novel system comprises eight aerated glass reactors with a working volume of 80 ml and a moving external optical sensor for measuring optical densities at 600 nm (OD600) ranging from 0.05 to 100 online. Each reactor can be fitted with miniature probes to monitor temperature, dissolved oxygen (DO), and pH. Independent temperature regulation for each vessel is obtained with heating/cooling Peltier devices. Data from pH, DO, and turbidity sensors are collected on a FieldPoint (National Instruments) I/O interface and are processed and recorded by a LabVIEW program on a personal computer, which enables feedback control of the culture parameters. A high-density medium formulation was designed, which enabled us to grow E. coli to OD600 up to 100 in batch cultures with oxygen-enriched aeration. Accordingly, the biomass and the amount of recombinant protein produced in a 70-ml culture were at least equivalent to the biomass and the amount of recombinant protein obtained in a Fernbach flask with 1 liter of conventional medium. Thus, the microfermentor battery appears to be well suited for automated parallel cultures and process optimization, such as that needed for structural genomics projects.

  8. Multiple Microfermentor Battery: a Versatile Tool for Use with Automated Parallel Cultures of Microorganisms Producing Recombinant Proteins and for Optimization of Cultivation Protocols

    PubMed Central

    Frachon, Emmanuel; Bondet, Vincent; Munier-Lehmann, Hélène; Bellalou, Jacques

    2006-01-01

    A multiple microfermentor battery was designed for high-throughput recombinant protein production in Escherichia coli. This novel system comprises eight aerated glass reactors with a working volume of 80 ml and a moving external optical sensor for measuring optical densities at 600 nm (OD600) ranging from 0.05 to 100 online. Each reactor can be fitted with miniature probes to monitor temperature, dissolved oxygen (DO), and pH. Independent temperature regulation for each vessel is obtained with heating/cooling Peltier devices. Data from pH, DO, and turbidity sensors are collected on a FieldPoint (National Instruments) I/O interface and are processed and recorded by a LabVIEW program on a personal computer, which enables feedback control of the culture parameters. A high-density medium formulation was designed, which enabled us to grow E. coli to OD600 up to 100 in batch cultures with oxygen-enriched aeration. Accordingly, the biomass and the amount of recombinant protein produced in a 70-ml culture were at least equivalent to the biomass and the amount of recombinant protein obtained in a Fernbach flask with 1 liter of conventional medium. Thus, the microfermentor battery appears to be well suited for automated parallel cultures and process optimization, such as that needed for structural genomics projects. PMID:16885269

  9. 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

  10. Production of recombinant proteins by filamentous fungi.

    PubMed

    Ward, Owen P

    2012-01-01

    The initial focus of recombinant protein production by filamentous fungi related to exploiting the extraordinary extracellular enzyme synthesis and secretion machinery of industrial strains, including Aspergillus, Trichoderma, Penicillium and Rhizopus species, was to produce single recombinant protein products. An early recognized disadvantage of filamentous fungi as hosts of recombinant proteins was their common ability to produce homologous proteases which could degrade the heterologous protein product and strategies to prevent proteolysis have met with some limited success. It was also recognized that the protein glycosylation patterns in filamentous fungi and in mammals were quite different, such that filamentous fungi are likely not to be the most suitable microbial hosts for production of recombinant human glycoproteins for therapeutic use. By combining the experience gained from production of single recombinant proteins with new scientific information being generated through genomics and proteomics research, biotechnologists are now poised to extend the biomanufacturing capabilities of recombinant filamentous fungi by enabling them to express genes encoding multiple proteins, including, for example, new biosynthetic pathways for production of new primary or secondary metabolites. It is recognized that filamentous fungi, most species of which have not yet been isolated, represent an enormously diverse source of novel biosynthetic pathways, and that the natural fungal host harboring a valuable biosynthesis pathway may often not be the most suitable organism for biomanufacture purposes. Hence it is expected that substantial effort will be directed to transforming other fungal hosts, non-fungal microbial hosts and indeed non microbial hosts to express some of these novel biosynthetic pathways. But future applications of recombinant expression of proteins will not be confined to biomanufacturing. Opportunities to exploit recombinant technology to unravel the

  11. 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

    2012-04-24

    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.

  12. Recombinant protein expression in Nicotiana.

    PubMed

    Matoba, Nobuyuki; Davis, Keith R; Palmer, Kenneth E

    2011-01-01

    Recombinant protein pharmaceuticals are now widely used in treatment of chronic diseases, and several recombinant protein subunit vaccines are approved for human and veterinary use. With growing demand for complex protein pharmaceuticals, such as monoclonal antibodies, manufacturing capacity is becoming limited. There is increasing need for safe, scalable, and economical alternatives to mammalian cell culture-based manufacturing systems, which require substantial capital investment for new manufacturing facilities. Since a seminal paper reporting immunoglobulin expression in transgenic plants was published in 1989, there have been many technological advances in plant expression systems to the present time where production of proteins in leaf tissues of nonfood crops such as Nicotiana species is considered a viable alternative. In particular, transient expression systems derived from recombinant plant viral vectors offer opportunities for rapid expression screening, construct optimization, and expression scale-up. Extraction of recombinant proteins from Nicotiana leaf tissues can be achieved by collection of secreted protein fractions, or from a total protein extract after grinding the leaves with buffer. After separation from solids, the major purification challenge is contamination with elements of the photosynthetic complex, which can be solved by application of a variety of facile and proven strategies. In conclusion, the technologies required for safe, efficient, scalable manufacture of recombinant proteins in Nicotiana leaf tissues have matured to the point where several products have already been tested in phase I clinical trials and will soon be followed by a rich pipeline of recombinant vaccines, microbicides, and therapeutic proteins.

  13. On-line casein micelle disruption for downstream purification of recombinant human myelin basic protein produced in the milk of transgenic cows.

    PubMed

    Al-Ghobashy, Medhat A; Williams, Martin A K; Brophy, Brigid; Laible, Götz; Harding, David R K

    2009-06-01

    Downstream purification of a model recombinant protein (human myelin basic protein) from milk of transgenic cows is described. The recombinant protein was expressed as a His tagged fusion protein in the milk of transgenic cows and was found associated with the casein micellar phase. While difficulties in obtaining good recoveries were found when employing conventional micelle disruption procedures, direct capture using the cation exchanger SP Sepharose Big Beads was found successful in the extraction of the recombinant protein. Early breakthrough suggested a slow release of the recombinant protein from the micelles and dictated micelle disruption in order to obtain good yields. A new approach for deconstruction of the calcium core of the casein micelles, employing the interaction between the micellar calcium and the active sites of the cation exchanger resin was developed. Milk samples were loaded to the column in aliquots with a column washing step after each aliquot. This sequential loading approach successfully liberated the recombinant protein from the micelles and was found superior to the conventional sample loading approach. It increased the recovery by more than 25%, reduced fouling due to milk components and improved the column hydrodynamic properties as compared to the conventional sample loading approach. Hardware and software modifications to the chromatography system were necessary in order to keep the whole process automated. A second purification step using a Ni2+ affinity column was used to isolate the recombinant protein at purity more than 90% and a recovery percentage of 78%.

  14. Development, evaluation, and laboratory validation of immunoassays for the diagnosis of equine infectious anemia (EIA) using recombinant protein produced from a synthetic p26 gene of EIA virus.

    PubMed

    Singha, Harisankar; Goyal, Sachin K; Malik, Praveen; Khurana, Sandip K; Singh, Raj K

    2013-12-01

    Equine infectious anemia (EIA)-a retroviral disease caused by equine infectious anemia virus (EIAV)-is a chronic, debilitating disease of horses, mules, and donkeys. EIAV infection has been reported worldwide and is recognized as pathogen of significant economic importance to the horse industry. This disease falls under regulatory control program in many countries including India. Control of EIA is based on identification of inapparent carriers by detection of antibodies to EIAV in serologic tests and "Stamping Out" policy. The current internationally accepted test for diagnosis of EIA is the agar gel immune-diffusion test (AGID), which detects antibodies to the major gag gene (p26) product. The objective of this study was to develop recombinant p26 based in-house immunoassays [enzyme linked immunosorbent assays (ELISA), and AGID] for EIA diagnosis. The synthetic p26 gene of EIAV was expressed in Escherichia coli and diagnostic potential of recombinant p26 protein were evaluated in ELISA and AGID on 7,150 and 1,200 equine serum samples, respectively, and compared with commercial standard AGID kit. The relative sensitivity and specificity of the newly developed ELISA were 100 and 98.6 %, respectively. Whereas, relative sensitivity and specificity of the newly developed AGID were in complete agreement in respect to commercial AGID kit. Here, we have reported the validation of an ELISA and AGID on large number of equine serum samples using recombinant p26 protein produced from synthetic gene which does not require handling of pathogenic EIAV. Since the indigenously developed reagents would be economical than commercial diagnostic kit, the rp26 based-immunoassays could be adopted for the sero-diagnosis and control of EIA in India.

  15. Scintillation proximity assay for human DNA topoisomerase I using recombinant biotinyl-fusion protein produced in baculovirus-infected insect cells.

    PubMed

    Lerner, C G; Saiki, A Y

    1996-09-05

    DNA topoisomerases are well-established targets of important therapeutic agents which include the antibacterial quinolones and anticancer camptothecins. Screens for new classes of topoisomerase inhibitors generally employ methods, such as gel electrophoresis, which are not readily amenable to a rapid high-throughput format. We describe here a high-throughput assay to screen for inhibitors of human DNA topoisomerase I based on the scintillation proximity assay. The assay employs recombinant biotinyl-topoisomerase I fusion protein, a hybrid protein which contains a domain that is biotinylated during in vivo expression. The hybrid topoisomerase I fusion protein is found to be biotinylated, active, and nuclear-localized when produced in insect cells using a baculovirus expression system. The biotinyl-topoisomerase I fusion protein can be captured from crude nuclear extracts by immobilization on streptavidin-coated scintillation proximity assay beads. The assay detects binding of 3H-labeled DNA to the bead-immobilized enzyme by scintillation counting. The method is also able to detect stabilization of covalent protein-DNA complexes by camptothecin, an inhibitor previously shown to stabilize covalent intermediates that form during catalysis.

  16. Productivity and quality of recombinant proteins produced by stable CHO cell clones can be predicted by transient expression in HEK cells.

    PubMed

    Diepenbruck, Carolin; Klinger, Matthias; Urbig, Thomas; Baeuerle, Patrick; Neef, Rüdiger

    2013-06-01

    Selection of lead candidates in drug discovery is a complex and time-consuming process. Here, we describe an approach that allows prediction of the productivity and quality of recombinant proteins by stable producer cell clones with the help of transient transfection studies. This is exemplified for three distinct bispecific T cell engager (BiTE(®))-a new class of single-chain antibody-based therapeutics showing very promising results in the treatment of cancer. BiTE(®) titers of transiently transfected HEK cells showed a striking correlation with titers of selected stable CHO cell clones. Likewise, the percentage of the monomeric BiTE(®) fraction in cell culture supernatants correlated well between transiently expressing HEK and stably expressing CHO cell clones. This validates the use of transient transfection studies for the selection of biopharmaceutical lead candidates with desired pharmaceutical properties.

  17. Expression, purification and characterization of yeast protein disulfide isomerase produced by a recombinant baculovirus-mediated silkworm, Bombyx mori, pupae expression system.

    PubMed

    Wang, Liyun; Shimizu, Yuri; Mizunaga, Takemitsu; Matsumoto, Shogo; Otsuka, Yuzuru

    2008-04-01

    Protein disulfide isomerase (PDI) is a multifunctional polypeptide presents in the endoplasmic reticulum of the cell. Silkworm (Bombyx mori) pupae were used as hosts to produce recombinant PDI (rPDI). The concentration-dependent chaperone activity of rPDI was evidenced by the inhibition of the aggregation of rhodanese. Approximately 297 microg rPDI was purified from a single silkworm pupa. Results of rPDI treated with endoglycosidase H and N-glycanase, PNGase F, indicate that non-N-glycosylated rPDI (occupying 90%) and N-glycosylated rPDI are expressed in the silkworm expression system. The difference in glycosylation between silkworm pupae and yeast is discussed.

  18. Recombinant protein polymers in biomaterials.

    PubMed

    Kim, Wookhyun

    2013-01-01

    Naturally occurring protein-based materials have been found that function as critical components in biomechanical response, fibers and adhesives. A relatively small but growing number of recombinant protein-based materials that mimic the desired features of their natural sources, such as collagens, elastins and silks, are considered as an alternative to conventional synthetic polymers. Advances in genetic engineering have facilitated the synthesis of repetitive protein polymers with precise control of molecular weights which are designed by using synthetic genes encoding tandem repeats of oligopeptide originating from a modular domain of natural proteins. Many repeat sequences as protein polymer building blocks adopt a well-defined secondary structure and undergo self-assembly to result in physically cross-linked networks or with chemical cross-linking so that further form three-dimensional architectures similar to natural counterparts. In this review, recombinant protein polymers currently developed will be presented that have emerged as promising class of next generation biomaterials.

  19. Overexpression of Protein Disulfide Isomerase DsbC Stabilizes Multiple-Disulfide-Bonded Recombinant Protein Produced and Transported to the Periplasm in Escherichia coli

    PubMed Central

    Kurokawa, Yoichi; Yanagi, Hideki; Yura, Takashi

    2000-01-01

    Dsb proteins (DsbA, DsbB, DsbC, and DsbD) catalyze formation and isomerization of protein disulfide bonds in the periplasm of Escherichia coli. By using a set of Dsb coexpression plasmids constructed recently, we analyzed the effects of Dsb overexpression on production of horseradish peroxidase (HRP) isozyme C that contains complex disulfide bonds and tends to aggregate when produced in E. coli. When transported to the periplasm, HRP was unstable but was markedly stabilized upon simultaneous overexpression of the set of Dsb proteins (DsbABCD). Whereas total HRP production increased severalfold upon overexpression of at least disulfide-bonded isomerase DsbC, maximum transport of HRP to the periplasm seemed to require overexpression of all DsbABCD proteins, suggesting that excess Dsb proteins exert synergistic effects in assisting folding and transport of HRP. Periplasmic production of HRP also increased when calcium, thought to play an essential role in folding of nascent HRP polypeptide, was added to the medium with or without Dsb overexpression. These results suggest that Dsb proteins and calcium play distinct roles in periplasmic production of HRP, presumably through facilitating correct folding. The present Dsb expression plasmids should be useful in assessing and dissecting periplasmic production of proteins that contain multiple disulfide bonds in E. coli. PMID:10966415

  20. 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

  1. Bactericidal properties of the antimicrobial peptide Ib-AMP4 from Impatiens balsamina produced as a recombinant fusion-protein in Escherichia coli.

    PubMed

    Fan, Xiaobo; Schäfer, Holger; Reichling, Jürgen; Wink, Michael

    2013-10-01

    Antimicrobial peptides (AMPs) represent a novel class of powerful natural antimicrobial agents. As AMPs are bactericidal, production of AMPs in recombinant bacteria is far from trivial. We report the production of Impatiens balsamina antimicrobial peptide 4 (Ib-AMP4, originally isolated from Impatiens balsamina) in Escherichia coli as a fusion protein and investigate Ib-AMP4's antimicrobial effects on human pathogens. A plasmid vector pET32a-Trx-Ib-AMP4 was constructed and transferred into E. coli. After induction, a soluble fusion protein was expressed successfully. The Ib-AMP4 peptide was obtained with a purity of over 90% after nickel affinity chromatography, ultrafiltration, enterokinase cleavage and sephadex size exclusion chromatography. For maximum activity, Ib-AMP4, which possesses two disulfide bonds, required activation with 5 μg/mL H2 O2 . Antimicrobial assays showed that Ib-AMP4 could efficiently target clinical multiresistant isolates including methicillin-resistant Staphylococcus aureus and extended-spectrum β-lactamase-producing E. coli. Time kill experiments revealed that Ib-AMP4 is bactericidal within 10 min after application. Haemolysis and cytotoxicity assays implied selectivity towards bacteria, an important prerequisite for clinical applications. Ib-AMP4 might be an interesting candidate for clinical studies involving patients with septicemia or for coating clinical devices, such as catheters. The method described here may be applicable for expression and purification of other AMPs with multiple disulfide bridges.

  2. 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. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  3. Improving recombinant protein purification yield

    USDA-ARS?s Scientific Manuscript database

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

  4. Recombinant Protein Production by In Vivo Polymer Inclusion Display ▿

    PubMed Central

    Grage, Katrin; Peters, Verena; Rehm, Bernd H. A.

    2011-01-01

    A novel approach to produce purified recombinant proteins was established. The target protein is produced as polyhydroxyalkanoate (PHA) synthase fusion protein, which mediates intracellular formation of PHA inclusions displaying the target protein. After isolation of the PHA inclusions, the pure target protein was released by simple enterokinase digestion. PMID:21803888

  5. Insecticidal activity of recombinant avidin produced in yeast.

    PubMed

    Hinchliffe, Gareth; Bown, David P; Gatehouse, John A; Fitches, Elaine

    2010-06-01

    An expression construct encoding chicken (Gallus gallus) avidin was assembled from amplified fragments of genomic DNA. Recombinant, functional avidin was produced in Pichia pastoris, with yields of up to 80 mg/l of culture supernatant. The recombinant avidin had similar insecticidal activity to egg white avidin when assayed against larvae of a lepidopteran crop pest, cabbage moth (Mamestra brassicae), causing >90% reduction in growth and 100% mortality when fed in optimised diets at levels of 1.5 microM and 15 microM (100 ppm and 1000 ppm wet weight of recombinant protein). The recombinant protein was also highly toxic to a hemipteran pest, the pea aphid (Acyrthosiphon pisum), when fed in liquid artificial diet, causing 100% mortality after 4 days when present at concentrations > or = 3.8 microM (0.25 mg/ml, 250 ppm). Mortality was dose-dependent, with an estimated LC(50) of 2.1 microM. Toxicity to A. pisum was prevented by biotin supplementation of diet. In contrast, avidin had no significant effects on the survival of cereal aphid (Sitobion avenae) at concentrations up to 30 microM in liquid diet. Analysis of genomic DNA showed that symbionts from both aphid species lack the ability to synthesise biotin de novo. Cereal aphids appear to be less sensitive to recombinant avidin in the diet through proteolysis of the ingested protein, which would allow recovery of bound biotin. Crown Copyright 2010. Published by Elsevier Ltd. All rights reserved.

  6. 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. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Antigenic and immunogenic properties of recombinant hemagglutinin proteins from H1N1 A/Brisbane/59/07 and B/Florida/04/06 when produced in various protein expression systems

    PubMed Central

    Santiago, Felix W.; Emo, Kris Lambert; Fitzgerald, Theresa; Treanor, John J.

    2012-01-01

    Antibodies directed against the influenza hemagglutinin (HA) protein largely mediate virus neutralization and confer protection against infection. Consequently, many studies and assays of influenza vaccines are focused on HA-specific immune responses. Recombinant HA (rHA) proteins can be produced in a number of protein expression and cell culture systems. These range from baculovirus infection of insect cell cultures, to transient transfection of plants, to stably transfected human cell lines. Furthermore, the rHA proteins may contain genetic modifications, such as histidine tags or trimerization domains, intended to ease purification or enhance protein stability. However, no systematic study of these different forms of the HA protein have been conducted. It is not clear which, if any, of these different protein expression systems or structural modifications improve or diminish the biological behavior of the proteins as immunogens or antigens in immune assays. Therefore we set out to perform systematic evaluation of rHA produced in different proteins expression systems and with varied modifications. Five rHA proteins based on recent strains of seasonal influenza A and five based on influenza B HA were kindly provided by the Biodefense and Emerging Infections Reagent repository (BEIR). These proteins were evaluated in a combination of biochemical and structural assays, in vitro humoral and cellular immune assays, and in an animal vaccination model. Marked differences in the behavior of the individual proteins was evident suggesting that they are not equal when being used to detect an immune response. They were, nevertheless, similar at eliciting neutralizing antibody responses. PMID:22609035

  8. Streptomyces as host for recombinant production of Mycobacterium tuberculosis proteins.

    PubMed

    Vallin, Carlos; Ramos, Astrid; Pimienta, Elsa; Rodríguez, Caridad; Hernández, Tairí; Hernández, Ivones; Del Sol, Ricardo; Rosabal, Grisel; Van Mellaert, Lieve; Anné, Jozef

    2006-01-01

    The 45/47 kDa APA protein (Rv1860) of Mycobacterium tuberculosis was produced by Streptomyces lividans. The recombinant protein could be recovered from the culture medium of an S. lividans clone containing the apa gene under control of the promoter and signal sequence of the Streptomyces coelicolor agarase gene. The recombinant protein production was further scaled-up using fermentation conditions. The APA protein was subsequently purified from the culture supernatant by means of immunochromatography. About 80 mg of recombinant protein were obtained per liter of culture media. In vivo tests with the APA protein purified from S. lividans TK24/pRGAPA1 revealed that the recombinant protein was antigenic and could induce high titers of specific antibodies in the mouse biological model. Results obtained concerning heterologous production of APA, its immunogenic and antigenic capacity, demonstrated the potential of S. lividans as a valuable host for the production of recombinant proteins from M. tuberculosis.

  9. [Tobacco--a producer of recombinant interleukins].

    PubMed

    Budzianowski, Jaromir

    2012-01-01

    Interleukins are cytokines of highly pleiotropic activity and they have high potential for application in the treatment of cancer and autoimmune diseases. Trials of recombinant interleukin production in plants relate almost exclusively to tobacco, where through the transformation of the nuclear genome (agroinfection) monomeric (IL-2, IL-4, IL-13, IL-18), homodimeric (IL-10) and single-chain heterodimeric (IL-12) interleukins have been obtained. The expression of IL-10 as a homodimer in the chloroplast genome could not be reached. Expression of the given interleukin was obtained in the leaves, cell culture and culture of hairy roots of tobacco. Interleukins obtained in tobacco showed similar in vitro biological activity as commercial ILs produced mostly in E. coli. Glycosylated IL-13 obtained in tobacco was much more resistant to proteolytic digestion than commercial non-glycosylated IL-13; therefore in the case of sufficiently large production it could be suitable for oral administration in the treatment of type I diabetes.

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

    USDA-ARS?s Scientific Manuscript database

    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 ...

  11. 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.

  12. Overview of the purification of recombinant proteins.

    PubMed

    Wingfield, Paul T

    2015-04-01

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

  13. 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

  14. 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

  15. Recombinant human factor IX produced from transgenic porcine milk.

    PubMed

    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.

  16. Expanding the recombinant protein quality in Lactococcus lactis.

    PubMed

    Cano-Garrido, Olivia; Rueda, Fabian L; Sànchez-García, Laura; Ruiz-Ávila, Luis; Bosser, Ramon; Villaverde, Antonio; García-Fruitós, Elena

    2014-12-04

    Escherichia coli has been a main host for the production of recombinant proteins of biomedical interest, but conformational stress responses impose severe bottlenecks that impair the production of soluble, proteolytically stable versions of many protein species. In this context, emerging Generally Recognized As Safe (GRAS) bacterial hosts provide alternatives as cell factories for recombinant protein production, in which limitations associated to the use of Gram-negative microorganisms might result minimized. Among them, Lactic Acid Bacteria and specially Lactococcus lactis are Gram-positive GRAS organisms in which recombinant protein solubility is generically higher and downstream facilitated, when compared to E. coli. However, deep analyses of recombinant protein quality in this system are still required to completely evaluate its performance and potential for improvement. We have explored here the conformational quality (through specific fluorescence emission) and solubility of an aggregation-prone GFP variant (VP1GFP) produced in L. lactis. In this context, our results show that parameters such as production time, culture conditions and growth temperature have a dramatic impact not only on protein yield, but also on protein solubility and conformational quality, that are particularly favored under fermentative metabolism. Metabolic regime and cultivation temperature greatly influence solubility and conformational quality of an aggregation-prone protein in L. lactis. Specifically, the present study proves that anaerobic growth is the optimal condition for recombinant protein production purposes. Besides, growth temperature plays an important role regulating both protein solubility and conformational quality. Additionally, our results also prove the great versatility for the manipulation of this bacterial system regarding the improvement of functionality, yield and quality of recombinant proteins in this species. These findings not only confirm L. lactis as an

  17. 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

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

    PubMed

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

    2016-02-23

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

  19. 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

  20. Production of recombinant proteins by microbes and higher organisms.

    PubMed

    Demain, Arnold L; Vaishnav, Preeti

    2009-01-01

    Large proteins are usually expressed in a eukaryotic system while smaller ones are expressed in prokaryotic systems. For proteins that require glycosylation, mammalian cells, fungi or the baculovirus system is chosen. The least expensive, easiest and quickest expression of proteins can be carried out in Escherichia coli. However, this bacterium cannot express very large proteins. Also, for S-S rich proteins, and proteins that require post-translational modifications, E. coli is not the system of choice. The two most utilized yeasts are Saccharomyces cerevisiae and Pichia pastoris. Yeasts can produce high yields of proteins at low cost, proteins larger than 50 kD can be produced, signal sequences can be removed, and glycosylation can be carried out. The baculoviral system can carry out more complex post-translational modifications of proteins. The most popular system for producing recombinant mammalian glycosylated proteins is that of mammalian cells. Genetically modified animals secrete recombinant proteins in their milk, blood or urine. Similarly, transgenic plants such as Arabidopsis thaliana and others can generate many recombinant proteins.

  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.

  2. Immunological characterization of Mycoplasma hyopneumoniae recombinant proteins.

    PubMed

    Simionatto, Simone; Marchioro, Silvana B; Galli, Vanessa; Brum, Clarice B; Klein, Catia S; Rebelatto, Raquel; Silva, Everton F; Borsuk, Sibele; Conceição, Fabricio R; Dellagostin, Odir A

    2012-03-01

    Mycoplasma hyopneumoniae, the primary pathogen of enzootic pneumonia, is highly prevalent worldwide and causes major economic losses to the pig industry. Commercial vaccines are widely used in the control of this disease, however, they provide only partial protection. The aim of this study was to evaluate 34 recombinant proteins of M. hyopneumoniae expressed in Escherichia coli. Antigenic and immunogenic properties of these proteins were analyzed. For this, the proteins were tested against hyperimmune and convalescent pig sera through ELISA and Western blot. Immunogenicity of the recombinant proteins was evaluated in BALB/c mice following intramuscular inoculation. Most antigens were able to induce a strong immune response and sera from inoculated mice were able to recognize native proteins by cell ELISA and Western blot. Several recombinant proteins were specifically recognized by convalescent pig sera, indicating they are expressed during infection. These data may help to develop more efficacious vaccines against M. hyopneumoniae.

  3. Elastomeric Recombinant Protein-based Biomaterials

    PubMed Central

    Annabi, Nasim; Mithieux, Suzanne M.; Camci-Unal, Gulden; Dokmeci, Mehmet R.; Weiss, Anthony S.; Khademhosseini, Ali

    2013-01-01

    Elastomeric protein-based biomaterials, produced from elastin derivatives, are widely investigated as promising tissue engineering scaffolds due to their remarkable properties including substantial extensibility, long-term stability, self-assembly, high resilience upon stretching, low energy loss, and excellent biological activity. These elastomers are processed from different sources of soluble elastin such as animal-derived soluble elastin, recombinant human tropoelastin, and elastin-like polypeptides into various forms including three dimensional (3D) porous hydrogels, elastomeric films, and fibrous electrospun scaffolds. Elastin-based biomaterials have shown great potential for the engineering of elastic tissues such as skin, lung and vasculature. In this review, the synthesis and properties of various elastin-based elastomers with their applications in tissue engineering are described. PMID:23935392

  4. The Functional Quality of Soluble Recombinant Polypeptides Produced in Escherichia coli Is Defined by a Wide Conformational Spectrum▿

    PubMed Central

    Martínez-Alonso, Mónica; González-Montalbán, Nuria; García-Fruitós, Elena; Villaverde, Antonio

    2008-01-01

    We have observed that a soluble recombinant green fluorescent protein produced in Escherichia coli occurs in a wide conformational spectrum. This results in differently fluorescent protein fractions in which morphologically diverse soluble aggregates abound. Therefore, the functional quality of soluble versions of aggregation-prone recombinant proteins is defined statistically rather than by the prevalence of a canonical native structure. PMID:18836021

  5. Making recombinant proteins in animals--different systems, different applications.

    PubMed

    Dyck, Michael K; Lacroix, Dan; Pothier, François; Sirard, Marc-André

    2003-09-01

    Transgenic animal bioreactors represent a powerful tool to address the growing need for therapeutic recombinant proteins. The ability of transgenic animals to produce complex, biologically active recombinant proteins in an efficient and economic manner has stimulated a great deal of interest in this area. As a result, genetically modified animals of several species, expressing foreign proteins in various tissues, are currently being developed. However, the generation of transgenic animals is a cumbersome process and remains problematic in the application of this technology. The advantages and disadvantages of different transgenic systems in relation to other bioreactor systems are discussed.

  6. 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

  7. Production of a recombinant full-length prion protein in a soluble form without refolding or detergents.

    PubMed

    Arii, Yasuhiro; Oshiro, Satoshi; Wada, Keita; Fukuoka, Shin-ichi

    2011-01-01

    Recombinant prion protein has been produced in insoluble form and refolded following solubilization with denaturants. It is, however, preferable to use a soluble recombinant protein prepared without artificial solubilization. In this study, a soluble recombinant prion protein was produced in Escherichia coli cells by coexpression of neuregulin I-β1 and purified to high purity.

  8. 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

  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. 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.

  11. Trends in recombinant protein use in animal production.

    PubMed

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

    2017-03-04

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

  12. Designing Smart Materials with Recombinant Proteins.

    PubMed

    Hollingshead, Sydney; Lin, Charng-Yu; Liu, Julie C

    2017-07-01

    Recombinant protein design allows modular protein domains with different functionalities and responsive behaviors to be easily combined. Inclusion of these protein domains can enable recombinant proteins to have complex responses to their environment (e.g., temperature-triggered aggregation followed by enzyme-mediated cleavage for drug delivery or pH-triggered conformational change and self-assembly leading to structural stabilization by adjacent complementary residues). These "smart" behaviors can be tuned by amino acid identity and sequence, chemical modifications, and addition of other components. A wide variety of domains and peptides have smart behavior. This review focuses on protein designs for self-assembly or conformational changes due to stimuli such as shifts in temperature or pH. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Lactogenic immunity in transgenic mice producing recombinant antibodies neutralizing coronavirus.

    PubMed

    Castilla, J; Sola, I; Pintado, B; Sánchez-Morgado, J M; Enjuanes, L

    1998-01-01

    Protection against coronavirus infections can be provided by the oral administration of virus neutralizing antibodies. To provide lactogenic immunity, eighteen lines of transgenic mice secreting a recombinant IgG1 monoclonal antibody (rIgG1) and ten lines of transgenic mice secreting recombinant IgA monoclonal antibodies (rIgA) neutralizing transmissible gastroenteritis coronavirus (TGEV) into the milk were generated. Genes encoding the light and heavy chains of monoclonal antibody (MAb) 6A.C3 were expressed under the control of regulatory sequences derived from the mouse genomic DNA encoding the whey acidic protein (WAP) and beta-lactoglobulin (BLG), which are highly abundant milk proteins. The MAb 6A.C3 binds to a highly conserved epitope present in coronaviruses of several species. This MAb does not allow the selection of neutralization escaping virus mutants. The antibody was expressed in the milk of transgenic mice with titers of one million as determined by RIA, and neutralized TGEV infectivity by one million fold corresponding to immunoglobulin concentrations of 5 to 6 mg per ml. Matrix attachment regions (MAR) sequences were not essential for rIgG1 transgene expression, but co-microinjection of MAR and antibody genes led to a twenty to ten thousand-fold increase in the antibody titer in 50% of the rIgG1 transgenic animals generated. Co-microinjection of the genomic BLG gene with rIgA light and heavy chain genes led to the generation of transgenic mice carrying the three transgenes. The highest antibody titers were produced by transgenic mice that had integrated the antibody and BLG genes, although the number of transgenic animals generated does not allow a definitive conclusion on the enhancing effect of BLG co-integration. Antibody expression levels were transgene copy number independent and integration site dependent. The generation of transgenic animals producing virus neutralizing antibodies in the milk could be a general approach to provide protection

  14. Extracellular recombinant protein production from Escherichia coli.

    PubMed

    Ni, Ye; Chen, Rachel

    2009-11-01

    Escherichia coli is the most commonly used host for recombinant protein production and metabolic engineering. Extracellular production of enzymes and proteins is advantageous as it could greatly reduce the complexity of a bioprocess and improve product quality. Extracellular production of proteins is necessary for metabolic engineering applications in which substrates are polymers such as lignocelluloses or xenobiotics since adequate uptake of these substrates is often an issue. The dogma that E. coli secretes no protein has been challenged by the recognition of both its natural ability to secrete protein in common laboratory strains and increased ability to secrete proteins in engineered cells. The very existence of this review dedicated to extracellular production is a testimony for outstanding achievements made collectively by the community in this regard. Four strategies have emerged to engineer E. coli cells to secrete recombinant proteins. In some cases, impressive secretion levels, several grams per liter, were reached. This secretion level is on par with other eukaryotic expression systems. Amid the optimism, it is important to recognize that significant challenges remain, especially when considering the success cannot be predicted a priori and involves much trials and errors. This review provides an overview of recent developments in engineering E. coli for extracellular production of recombinant proteins and an analysis of pros and cons of each strategy.

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

    PubMed Central

    2011-01-01

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

  16. 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.

  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. 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. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Determination and control of low-level amino acid misincorporation in human thioredoxin protein produced in a recombinant Escherichia coli production system.

    PubMed

    Harris, Robert P; Mattocks, Jo; Green, Philip S; Moffatt, Frank; Kilby, Peter M

    2012-08-01

    Escherichia coli is used extensively in the production of proteins within biotechnology for a number of therapeutic applications. Here, we discuss the production and overexpression of the potential biopharmaceutical human thioredoxin protein (rhTRX) within E. coli. Overexpression of foreign molecules within the cell can put an enormous amount of stress on the translation machinery. This can lead to a misfiring in the construction of a protein resulting in populations differing slightly in amino acid composition. Whilst this may still result in a population of active molecules being expressed, it does present significant problems with molecules that are destined for clinical applications. Amino acid misincorporation of this subset could potentially result in antibodies being raised to these unnatural proteins. Cross-reaction with a patient's endogenous thioredoxin could then lead to an autoimmune phenomena and serious health implications. Generally, the issue of misincorporation appears not to be a routine regulatory concern (see ICH Q6B guidelines). Therefore, amino acid misincorporation may not have been detected, much less explored in the clinic as the occurrence or absence of these random errors is not routinely reported. Using current technologies based on proteomics, the ability to find misincorporation critically depends upon the criteria for matching theoretical and experimental mass spectrometry data. Additionally, isolation and extraction of these mistranslated proteins from the production process is both difficult and expensive. Therefore, it is advantageous to find routes for removing their production during the upstream phase. In this study, we show how modern proteomic technology can be used to identify and quantify amino acid misincorporation. Using these techniques we have shown how manipulation of gene sequence and scoping of fermentation media composition can lead to the reduction and elimination of these misincorporations in rhTRX. Copyright

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

    PubMed

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

    2009-01-01

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

  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. [Polyclonal antibodies against recombinant dengue virus NS3 protein].

    PubMed

    Morales, Liliana; Velandia, Myriam L; Calderon, María Angélica; Castellanos, Jaime E; Chaparro-Olaya, Jacqueline

    2017-01-24

    Dengue is a disease caused by one of four serotypes of the dengue virus (DENV) and is endemic in approximately 130 countries. The incidence of dengue has increased dramatically in recent decades, as well as the frequency and magnitude of outbreaks. Despite all efforts, there are no prophylactic or therapeutic treatments for the disease. Accordingly, research on the processes governing the DENV infection cycle is essential to develop vaccines or antiviral therapies. One of themost attractive DENV molecules to investigate is nonstructural protein 3 (NS3), which is essential for viral replication and a major immune target for infection. To produce antibodies to support future studies on NS3 and its cellular interactions with other proteins. Two recombinant proteins of the helicase domain of DENV NS3 serotype 2 were expressed, and used to immunize mice and produce polyclonal antibodies. The antibodies produced were useful in Western blot and immunofluorescence tests. We report an NS3 antibody that immunoprecipitates the viral protein and detects it in Western blot with no need to over-express it or use cell extracts with metabolic radiolabeling. The recombinant proteins expressed and the antibodies produced constitute valuable tools for studying DENV infectious processes involving NS3 and for evaluating tests designed to interfere with its functions.

  4. 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.

  5. A Multi-Omics Analysis of Recombinant Protein Production in Hek293 Cells

    PubMed Central

    Dietmair, Stefanie; Hodson, Mark P.; Quek, Lake-Ee; Timmins, Nicholas E.; Gray, Peter; Nielsen, Lars K.

    2012-01-01

    Hek293 cells are the predominant hosts for transient expression of recombinant proteins and are used for stable expression of proteins where post-translational modifications performed by CHO cells are inadequate. Nevertheless, there is little information available on the key cellular features underpinning recombinant protein production in Hek293 cells. To improve our understanding of recombinant protein production in Hek293 cells and identify targets for the engineering of an improved host cell line, we have compared a stable, recombinant protein producing Hek293 cell line and its parental cell line using a combination of transcriptomics, metabolomics and fluxomics. Producer cultures consumed less glucose than non-producer cultures while achieving the same growth rate, despite the additional burden of recombinant protein production. Surprisingly, there was no indication that producer cultures compensated for the reduction in glycolytic energy by increasing the efficiency of glucose utilization or increasing glutamine consumption. In contrast, glutamine consumption was lower and the majority of genes involved in oxidative phosphorylation were downregulated in producer cultures. We observed an overall downregulation of a large number of genes associated with broad cellular functions (e.g., cell growth and proliferation) in producer cultures, and therefore speculate that a broad adaptation of the cellular network freed up resources for recombinant protein production while maintaining the same growth rate. Increased abundance of genes associated with endoplasmic reticulum stress indicated a possible bottleneck at the point of protein folding and assembly. PMID:22937046

  6. The production of recombinant proteins in transgenic barley grains.

    PubMed

    Horvath, H; Huang, J; Wong, O; Kohl, E; Okita, T; Kannangara, C G; von Wettstein, D

    2000-02-15

    The grain of the self-pollinating diploid barley species offers two modes of producing recombinant enzymes or other proteins. One uses the promoters of genes with aleurone-specific expression during germination and the signal peptide code for export of the protein into the endosperm. The other uses promoters of the structural genes for storage proteins deposited in the developing endosperm. Production of a protein-engineered thermotolerant (1, 3-1, 4)-beta-glucanase with the D hordein gene (Hor3-1) promoter during endosperm development was analyzed in transgenic plants with four different constructs. High expression of the enzyme and its activity in the endosperm of the mature grain required codon optimization to a C+G content of 63% and synthesis as a precursor with a signal peptide for transport through the endoplasmic reticulum and targeting into the storage vacuoles. Synthesis of the recombinant enzyme in the aleurone of germinating transgenic grain with an alpha-amylase promoter and the code for the export signal peptide yielded approximately 1 microgram small middle dotmg(-1) soluble protein, whereas 54 microgram small middle dotmg(-1) soluble protein was produced on average in the maturing grain of 10 transgenic lines with the vector containing the gene for the (1, 3-1, 4)-beta-glucanase under the control of the Hor3-1 promoter.

  7. 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

  8. 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

    PubMed Central

    2010-01-01

    Background The insect cell line is a critical component in the production of recombinant proteins in the baculovirus expression system and new cell lines hold the promise of increasing both quantity and quality of protein production. Results Seventy cell lines were established by single-cell cloning from a primary culture of cells derived from eggs of the black witch moth (Ascalapha odorata; Lepidoptera, Noctuidae). Among 8 rapidly growing lines, cell line 38 (Ao38) was selected for further analysis, based on susceptibility to AcMNPV infection and production of secreted alkaline phosphatase (SEAP) from a baculovirus expression vector. In comparisons with low-passage High Five (BTI-Tn-5B1-4) cells, infected Ao38 cells produced β-galactosidase and SEAP at levels higher (153% and 150%, respectively) than those measured from High Five cells. Analysis of N-glycans of SEAP produced in Ao38 cells revealed two N-glycosylation sites and glycosylation patterns similar to those reported for High Five and Sf9 cells. Glycopeptide isoforms consisted of pauci- or oligomannose, with and without fucose on N-acetylglucosamine(s) linked to asparagine residues. Estimates of Ao38 cell volume suggest that Ao38 cells are approximately 2.5× larger than Sf9 cells but only approximately 74% of the size of High Five cells. Ao38 cells were highly susceptible to AcMNPV infection, similar to infectivity of Sf9 cells. Production of infectious AcMNPV budded virions from Ao38 cells peaked at approximately 4.5 × 107 IU/ml, exceeding that from High Five cells while lower than that from Sf9 cells. Ao38 cells grew rapidly in stationary culture with a population doubling time of 20.2 hr, and Ao38 cells were readily adapted to serum-free medium (Sf-900III) and to a suspension culture system. Analysis of Ao38 and a parental Ascalapha odorata cell line indicated that these lines were free of the alphanodavirus that was recently identified as an adventitious agent in High Five cell lines. Conclusions Ao

  9. Optimization of Salmonella enteritidis recombinant heat shock protein 60 production.

    PubMed

    Rainczak, K; Bajzert, J; Galli, J; Selera, A; Wieliczko, A; Borkowski, J; Stefaniak, T

    2011-01-01

    The aim of the study was to optimize conditions for producing Salmonella Enteritidis recombinant heat shock protein 60 (rHsp60). Seven Escherichia coli host strains (Rosetta, Turner, C41, C43, Origami, BL21pLys, Rosetta pLys) were transformed by a recombinant plasmid containing Hsp60 gene from Salmonella Enteritidis, and then cultured and induced by isopropyl-beta-D-thiogalactopyranoside (IPTG). The highest S. Enteritidis rHsp60 yield was obtained using E. coli strain C41. Induction of this strain using IPTG allowed the yield 400 microg of S. Enteritidis Hsp60 protein/2L of culture, but by autoinduction the yield exceeded 800 microg/2L.

  10. [An effective scheme to produce recombinant uracil-DNA glycosylase of Escherichia coli for PCR diagnostics].

    PubMed

    Dmitrochenko, A E; Turiianskaia, O M; Gilep, A A; Usanov, S A; Iantsevich, A V

    2014-01-01

    An effective scheme has been developed to produce recombinant uracil-DNA glycosylase of Escherichia coli K12 intended to be used for PCR diagnostics, making it possible to achieve a high yield of the end product using a two-stage purification. The gene encoding this enzyme was cloned into the pCWori vector within the same reading frame with six residues of histidine in the C-erminal sequence. Using this vector and the E. coli DH5alpha, a host-vector expression system has been developed and conditions for protein synthesis have been optimized. To purify the protein, metal affinity chromatography with further dialysis was used to remove imidazole. The enzyme yield was no less than 60 mg of the end protein per 1 L of the culture medium. The concordance between amino acid sequences of the recombinant and native enzymes was proved by peptide mass fingerprinting and mass spectrometry. A rapid test to determine the activity of the enzyme preparation was suggested. It was found that the activity of 1.0 mg of the recombinant protein is no less than 3 x 10(3) units. The recombinant enzyme was most stable at pH 8.0 and an ionic strength of the solution equal to 200 mM; it lost its activity completely for 10 min at 60 degrees C. Storage during 1 h at 20 degrees C resulted in the loss of no more than 30% of activity. In the enzyme preparation, the activity of DNase was absent. The free energy of the unfolding of the protein globule of the recombinant uracil-DNA glycosylase is 23.1 +/- 0.2 kJ/mol. The data obtained indicate that the recombinant enzyme may be recommended for use in PCR diagnostics to prevent the appearance of false positive results caused by pollution of the reaction mixture by products of the preceding reactions.

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

    PubMed

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

    2015-01-01

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

  12. Characterization of the Recombinant BADH2 Protein

    USDA-ARS?s Scientific Manuscript database

    The compound which is responsible for the flavor of aromatic rice is formed when a particular protein (BADH2) is not produced correctly in the plant. In this research, we characterize a synthetic copy of the protein using a specialized technique that will provide information on identifying specific...

  13. 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.

  14. Recombinant avidin and avidin-fusion proteins.

    PubMed

    Airenne, K J; Marjomäki, V S; Kulomaa, M S

    1999-12-31

    Both chicken egg-white avidin and its bacterial relative streptavidin are well known for their extraordinary high affinity with biotin (Kd approximately 10(-15) M). They are widely used as tools in a number of affinity-based separations, in diagnostic assays and in a variety of other applications. These methods have collectively become known as (strept)avidin-biotin technology. Biotin can easily and effectively be attached to different molecules, termed binders and probes, without destroying their biological activity. The exceptional stability of the avidin-biotin complex and the wide range of commercially available reagents explain the popularity of this system. In order by genetic engineering to modify the unwanted properties of avidin and to further expand the existing avidin-biotin technology, production systems for recombinant avidin and avidin-fusion proteins have been established. This review article presents an overview of the current status of these systems. Future trends in the production and applications of recombinant avidin and avidin-fusion proteins are also discussed.

  15. Recent advances in recombinant protein production

    PubMed Central

    Kunert, Renate; Casanova, Emilio

    2013-01-01

    Designing appropriate expression vectors is one of the critical steps in the generation of stable cell lines for recombinant protein production. Conventional expression vectors are severely affected by the chromatin environment surrounding their integration site into the host genome, resulting in low expression levels and transgene silencing. In the past, a new generation of expression vectors and different strategies was developed to overcome the chromatin effects. Bacterial artificial chromosomes (BACs) are cloning vectors capable of accommodating up to 350 Kb. Thus, BACs can carry a whole eukaryotic locus with all the elements controlling the expression of a gene; therefore, BACs harbor their own chromatin environment. Expression vectors based on BACs containing open/permissive chromatin loci are not affected by the chromatin surrounding their integration site in the host cell genome. Consequently, BAC-based expression vectors containing the appropriate loci confer predictable and high levels of expression over time. These properties make BAC-based expression vectors a very attractive tool applied to the recombinant protein production field. PMID:23680894

  16. Recombinant Brucella abortus gene expressing immunogenic protein

    SciTech Connect

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

    1991-06-11

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

  17. Recent advances in production of recombinant spider silk proteins.

    PubMed

    Chung, Hannah; Kim, Tae Yong; Lee, Sang Yup

    2012-12-01

    Spider silk has been drawing much attention as a great biomaterial having many applications in biotechnology and biomedicine owing to its several desired material characteristics such as outstanding strength, toughness, and elasticity as well as biodegradability and biocompatibility. With various applications foreseeable in industry, there has been much effort to produce recombinant spider silk protein in large amounts. However, owing to the difficulties in its production using spiders, alternative host systems and engineering methods have been investigated to develop suitable production systems that can efficiently produce spider silk protein. Here, we review recent advances in production of spider silk proteins in various heterologous host systems with focus given on the development of metabolic and cellular engineering strategies. Copyright © 2012 Elsevier Ltd. All rights reserved.

  18. Statistical approaches to maximize recombinant protein expression in Escherichia coli: a general review.

    PubMed

    Papaneophytou, Christos P; Kontopidis, George

    2014-02-01

    The supply of many valuable proteins that have potential clinical or industrial use is often limited by their low natural availability. With the modern advances in genomics, proteomics and bioinformatics, the number of proteins being produced using recombinant techniques is exponentially increasing and seems to guarantee an unlimited supply of recombinant proteins. The demand of recombinant proteins has increased as more applications in several fields become a commercial reality. Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, producing soluble proteins in E. coli is still a major bottleneck for structural biology projects. One of the most challenging steps in any structural biology project is predicting which protein or protein fragment will express solubly and purify for crystallographic studies. The production of soluble and active proteins is influenced by several factors including expression host, fusion tag, induction temperature and time. Statistical designed experiments are gaining success in the production of recombinant protein because they provide information on variable interactions that escape the "one-factor-at-a-time" method. Here, we review the most important factors affecting the production of recombinant proteins in a soluble form. Moreover, we provide information about how the statistical design experiments can increase protein yield and purity as well as find conditions for crystal growth.

  19. Mismatch repair proteins: key regulators of genetic recombination.

    PubMed

    Surtees, J A; Argueso, J L; Alani, E

    2004-01-01

    Mismatch repair (MMR) systems are central to maintaining genome stability in prokaryotes and eukaryotes. MMR proteins play a fundamental role in avoiding mutations, primarily by removing misincorporation errors that occur during DNA replication. MMR proteins also act during genetic recombination in steps that include repairing mismatches in heteroduplex DNA, modulating meiotic crossover control, removing 3' non-homologous tails during double-strand break repair, and preventing recombination between divergent sequences. In this review we will, first, discuss roles for MMR proteins in repairing mismatches that occur during recombination, particularly during meiosis. We will also explore how studying this process has helped to refine models of double-strand break repair, and particularly to our understanding of gene conversion gradients. Second, we will examine the role of MMR proteins in repressing homeologous recombination, i.e. recombination between divergent sequences. We will also compare the requirements for MMR proteins in preventing homeologous recombination to the requirements for these proteins in mismatch repair.

  20. Proteins involved in meiotic recombination: a role in male infertility?

    PubMed

    Sanderson, Matthew L; Hassold, Terry J; Carrell, Douglas T

    2008-01-01

    Meiotic recombination results in the formation of crossovers, by which genetic information is exchanged between homologous chromosomes during prophase I of meiosis. Recombination is a complex process involving many proteins. Alterations in the genes involved in recombination may result in infertility. Molecular studies have improved our understanding of the roles and mechanisms of the proteins and protein complexes involved in recombination, some of which have function in mitotic cells as well as meiotic cells. Human gene sequencing studies have been performed for some of these genes and have provided further information on the phenotypes observed in some infertile individuals. However, further studies are needed to help elucidate the particular role(s) of a given protein and to increase our understanding of these protein systems. This review will focus on our current understanding of proteins involved in meiotic recombination from a genomic perspective, summarizing our current understanding of known mutations and single nucleotide polymorphisms that may affect male fertility by altering meiotic recombination.

  1. Protein expression in yeast as an approach to production of recombinant malaria antigens.

    PubMed

    Bathurst, I C

    1994-01-01

    The selection of a system suitable for expression of recombinant malaria antigens for vaccine development is, in the final analysis, empirical. However, experience gained with both malaria antigens and other recombinant proteins has provided helpful guidelines. Recombinant DNA technology has been successfully applied to the development of vaccines against a number of human diseases. For example, recombinant DNA-derived hepatitis B virus surface antigen has been produced from both prokaryotic and eukaryotic systems. Yeast has been demonstrated to be an excellent host for the expression of recombinant proteins with uses in diagnostics, therapeutics, and vaccine production. Both intracellular and secretory systems have been developed and optimized for the production of high levels of recombinant proteins. Recombinant DNA technology, and in particular yeast expression systems, have been successfully used to produce malaria antigens, several of which have been protective in various animal models. In contrast, attempts to produce sufficient quantities of antigens for a malaria vaccine from in vitro cultures of the malaria parasite have been unsuccessful. Recombinant proteins can be produced and purified from yeast in large quantities and at low cost, each being requirements for a vaccine to be used in a global vaccination program against malaria.

  2. Producing proteins in transgenic plants and animals.

    PubMed

    Larrick, J W; Thomas, D W

    2001-08-01

    The requirement for large quantities of therapeutic proteins has fueled interest in the production of recombinant proteins in plants and animals. The first commercial products to be made in this way have experienced much success, and it is predicted that in the future a plethora of protein products will be made using these 'natural' bioreactors.

  3. A gene responsible for prolyl-hydroxylation of moss-produced recombinant human erythropoietin

    PubMed Central

    Parsons, Juliana; Altmann, Friedrich; Graf, Manuela; Stadlmann, Johannes; Reski, Ralf; Decker, Eva L.

    2013-01-01

    Recombinant production of pharmaceutical proteins is crucial, not only for personalized medicine. While most biopharmaceuticals are currently produced in mammalian cell culture, plant-made pharmaceuticals gain momentum. Post-translational modifications in plants are similar to those in humans, however, existing differences may affect quality, safety and efficacy of the products. A frequent modification in higher eukaryotes is prolyl-4-hydroxylase (P4H)-catalysed prolyl-hydroxylation. P4H sequence recognition sites on target proteins differ between humans and plants leading to non-human posttranslational modifications of recombinant human proteins produced in plants. The resulting hydroxyprolines display the anchor for plant-specific O-glycosylation, which bears immunogenic potential for patients. Here we describe the identification of a plant gene responsible for non-human prolyl-hydroxylation of human erythropoietin (hEPO) recombinantly produced in plant (moss) bioreactors. Targeted ablation of this gene abolished undesired prolyl-hydroxylation of hEPO and thus paves the way for plant-made pharmaceuticals humanized via glyco-engineering in moss bioreactors. PMID:24145658

  4. A gene responsible for prolyl-hydroxylation of moss-produced recombinant human erythropoietin.

    PubMed

    Parsons, Juliana; Altmann, Friedrich; Graf, Manuela; Stadlmann, Johannes; Reski, Ralf; Decker, Eva L

    2013-10-22

    Recombinant production of pharmaceutical proteins is crucial, not only for personalized medicine. While most biopharmaceuticals are currently produced in mammalian cell culture, plant-made pharmaceuticals gain momentum. Post-translational modifications in plants are similar to those in humans, however, existing differences may affect quality, safety and efficacy of the products. A frequent modification in higher eukaryotes is prolyl-4-hydroxylase (P4H)-catalysed prolyl-hydroxylation. P4H sequence recognition sites on target proteins differ between humans and plants leading to non-human posttranslational modifications of recombinant human proteins produced in plants. The resulting hydroxyprolines display the anchor for plant-specific O-glycosylation, which bears immunogenic potential for patients. Here we describe the identification of a plant gene responsible for non-human prolyl-hydroxylation of human erythropoietin (hEPO) recombinantly produced in plant (moss) bioreactors. Targeted ablation of this gene abolished undesired prolyl-hydroxylation of hEPO and thus paves the way for plant-made pharmaceuticals humanized via glyco-engineering in moss bioreactors.

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

    PubMed

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

    2010-10-13

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

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

    SciTech Connect

    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.

  7. Purification of secreted recombinant proteins from Escherichia coli.

    PubMed

    Le, H V; Trotta, P P

    1991-01-01

    Secretion systems engineered for the expression of heterologous protein in E. coli provide several advantages for subsequent isolation of purified product. Proteins released from the periplasmic space, which represent a small fraction (i.e., 4-10%) of total cell protein, can readily be separated from other cellular proteins by centrifugation of the remaining cellular debris or cross-flow ultrafiltration. The starting material derived from secretion systems is generally of higher purity than comparable material produced from strains expressing cytoplasmically for systems exhibiting similar expression levels. The available evidence suggests that recombinant proteins derived from the periplasm are generally, but not always (44-46), soluble in a nonaggregated form. Consequently, simple purification protocols can be effectively employed for producing homogeneous product with a high yield. The majority of the secreted recombinant proteins reviewed in this chapter were purified by simple one- or two-step chromatography procedures. High-resolution techniques such as reversed phase HPLC were found necessary only in cases where the secreted polypeptides were contaminated with proteolytic degradation variants, e.g., hirudin (51) and beta-endorphin (22). The fact that a high level of biological activity has been shown to be characteristic of purified recombinant proteins secreted into the periplasmic space suggests the presence of a native conformation stabilized by the expected disulfide linkages. Intramolecular disulfide bonds most probably form either as the polypeptide is translocated through the cytoplasmic membrane into the periplasm or within the periplasmic compartment, which has a higher oxidation potential than that found in the cytoplasm (57). Studies performed with hGH (31) and muIL-2 (35) provide excellent examples of differences observed in protein folding and disulfide bond formation between heterologous proteins expressed in the cytoplasmic and periplasmic

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

    PubMed

    Fabrick, Jeffrey A; Hull, J Joe

    2017-04-20

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

  9. 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.

  10. 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.

  11. Producing recombinant therapeutic glycoproteins with enhanced sialylation using CHO-gmt4 glycosylation mutant cells

    PubMed Central

    Goh, John SY; Liu, Yingwei; Chan, Kah Fai; Wan, Corrine; Teo, Gavin; Zhang, Peiqing; Zhang, Yuanxing; Song, Zhiwei

    2014-01-01

    Recombinant glycoprotein drugs require proper glycosylation for optimal therapeutic efficacy. Glycoprotein therapeutics are rapidly removed from circulation and have reduced efficacy if they are poorly sialylated. Ricinus communis agglutinin-I (RCA-I) was found highly toxic to wild-type CHO-K1 cells and all the mutants that survived RCA-I treatment contained a dysfunctional N-acetylglucosaminyltransferase I (GnT I) gene. These mutants are named CHO-gmt4 cells. Interestingly, upon restoration of GnT I, the sialylation of a model glycoprotein, erythropoietin, produced in CHO-gmt4 cells was shown to be superior to that produced in wild-type CHO-K1 cells. This addendum summarizes the applicability of this cell line, from transient to stable expression of the recombinant protein, and from a lab scale to an industrial scale perfusion bioreactor. In addition, CHO-gmt4 cells can be used to produce glycoproteins with mannose-terminated N-glycans. Recombinant glucocerebrosidase produced by CHO-gmt4 cells will not require glycan remodeling and may be directly used to treat patients with Gaucher disease. CHO-gmt4 cells can also be used to produce other glycoprotein therapeutics which target cells expressing mannose receptors. PMID:24911584

  12. Producing recombinant therapeutic glycoproteins with enhanced sialylation using CHO-gmt4 glycosylation mutant cells.

    PubMed

    Goh, John S Y; Liu, Yingwei; Chan, Kah Fai; Wan, Corrine; Teo, Gavin; Zhang, Peiqing; Zhang, Yuanxing; Song, Zhiwei

    2014-01-01

    Recombinant glycoprotein drugs require proper glycosylation for optimal therapeutic efficacy. Glycoprotein therapeutics are rapidly removed from circulation and have reduced efficacy if they are poorly sialylated. Ricinus communis agglutinin-I (RCA-I) was found highly toxic to wild-type CHO-K1 cells and all the mutants that survived RCA-I treatment contained a dysfunctional N-acetylglucosaminyltransferase I (GnT I) gene. These mutants are named CHO-gmt4 cells. Interestingly, upon restoration of GnT I, the sialylation of a model glycoprotein, erythropoietin, produced in CHO-gmt4 cells was shown to be superior to that produced in wild-type CHO-K1 cells. This addendum summarizes the applicability of this cell line, from transient to stable expression of the recombinant protein, and from a lab scale to an industrial scale perfusion bioreactor. In addition, CHO-gmt4 cells can be used to produce glycoproteins with mannose-terminated N-glycans. Recombinant glucocerebrosidase produced by CHO-gmt4 cells will not require glycan remodeling and may be directly used to treat patients with Gaucher disease. CHO-gmt4 cells can also be used to produce other glycoprotein therapeutics which target cells expressing mannose receptors.

  13. [Human bone morphogenetic protein-2: recombinant expression in E. coli].

    PubMed

    Ma, Q; Dang, G; Ma, D

    1998-04-01

    To explore the way of producing human bone morphogenetic protein-2(hBMP-2) for bone healing by using the gene engeneering techniques. hBMP-2 cDNA fragment, which consists of 3' end partial propeptide and mature peptide sequence, was inserted into the multiple cloning site of expression vector pkpL-3a via ligation. The recombinant plasmid pkpL-3a/hBMP-2 was transformed into E. coli pOp2136. By the method of restriction map, the positive expression clone was selected. SDS-PAGE analysis showed a new foreign protein band near 27,000 after induction. The yield of induced hBMP-2 accouned for 10% of the total bacterial proteins. The partial purified recombinant hBMP-2 was implanted into Wistar rat thigh. After 4 weeks, histological analysis showed that it induced the proliferation of mesenchymal type cells and formation of new cartilage and bone in the implantation area. The hBMP-2 produced by gene engeneering techniques has the biologic capacity of ectopic bone formation.

  14. Towards the molecular characterization of the stable producer phenotype of recombinant antibody-producing NS0 myeloma cells.

    PubMed

    Prieto, Y; Rojas, L; Hinojosa, L; González, I; Aguiar, D; de la Luz, K; Castillo, A; Pérez, R

    2011-08-01

    The loss of heterologous protein expression is one of the major problems faced by industrial cell line developers and has been reported by several authors. Therefore, the understanding of the mechanisms involved in the generation of stable and high producer cell lines is a critical issue, especially for those processes based on long term continuous cultures. We characterized two recombinant NS0 myeloma cell lines expressing Nimotuzumab, a humanized anti-human epidermal growth factor receptor (EGFR) antibody. The hR3/H7 clone is a stable producer obtained from the unstable hR3/t16 clone. The unstable clone was characterized by a bimodal distribution of intracellular immunoglobulin staining using flow cytometry. Loss of antibody production was due to the emergence of a non-producer cell subpopulation that increased with cell generation number. Immunoglobulin heavy chain (HC) and light chain (LC) ratio (HC/LC) was lower for the unstable phenotype. Proteomic maps using two dimensional gel electrophoresis (2DE) were obtained for both clones, at initial cell culture time and after 40 generations. Fifteen proteins potentially associated with the phenomenon of production stability were identified. The hR3/H7 stable clone showed an up-regulated expression pattern for most of these proteins. The regulation of recombinant antibody production by the host NS0 myeloma cell line most likely involves simultaneously cellular processes such as DNA transcription, mRNA processing, protein synthesis and folding, vesicular transport, glycolysis and energy production, according to the proteins identified in the present proteomic study.

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

    SciTech Connect

    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.

  16. 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.

    2017-02-21

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

  17. 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

  18. Expression and purification of recombinant human EGFL7 protein.

    PubMed

    Picuric, Srdjan; Friedrich, Matthias; Oess, Stefanie

    2009-11-01

    The secreted epidermal growth factor-like protein 7 (EGFL7) plays an important role in angiogenesis, especially in the recruitment of endothelial and smooth muscle cells to the site of the nascent vessel and their ordered assembly into functional vasculature. However, progress in the understanding of the underlying mechanisms is to date greatly hindered by the lack of recombinant EGFL7 protein in a stable, soluble, native state, thus preventing e.g. the characterization of the proposed functional receptor as well as investigation of additional biological effects of EGFL7. So far all attempts to produce sufficient amounts of recombinant EGFL7 protein by various groups have failed. In this study we describe a procedure for the expression and purification of human EGFL7 from Sf9 cells and for the first time provide means to isolate biologically functional EGFL7 protein in sufficient quantities for its further biological characterization. We believe that the availability of EGFL7 will greatly accelerate our understanding of the precise role of EGFL7 and the underlying molecular mechanisms of EGFL7 action in the fundamentally important process of angiogenesis.

  19. 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.

  20. 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. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. 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.

  2. 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.

  3. 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.

  4. Amino acid consumption in naïve and recombinant CHO cell cultures: producers of a monoclonal antibody.

    PubMed

    Carrillo-Cocom, L M; Genel-Rey, T; Araíz-Hernández, D; López-Pacheco, F; López-Meza, J; Rocha-Pizaña, M R; Ramírez-Medrano, A; Alvarez, M M

    2015-10-01

    Most commercial media for mammalian cell culture are designed to satisfy the amino acid requirements for cell growth, but not necessarily those for recombinant protein production. In this study, we analyze the amino acid consumption pattern in naïve and recombinant Chinese hamster ovary (CHO) cell cultures. The recombinant model we chose was a CHO-S cell line engineered to produce a monoclonal antibody. We report the cell concentration, product concentration, and amino acid concentration profiles in naïve and recombinant cell cultures growing in CD OptiCHO™ medium with or without amino acid supplementation with a commercial supplement (CHO CD EfficientFeed™ B). We quantify and discuss the amino acid demands due to cell growth and recombinant protein production during long term fed batch cultivation protocols. We confirmed that a group of five amino acids, constituting the highest mass fraction of the product, shows the highest depletion rates and could become limiting for product expression. In our experiments, alanine, a non-important mass constituent of the product, is in high demand during recombinant protein production. Evaluation of specific amino acid demands could be of great help in the design of feeding/supplementation strategies for recombinant mammalian cell cultures.

  5. High-throughput purification of affinity-tagged recombinant proteins.

    PubMed

    Wiesler, Simone C; Weinzierl, Robert O J

    2012-08-26

    generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase. Altogether, we generated, purified and analysed 381 mutants--a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results. This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

  6. Thermal adaptability of Kluyveromyces marxianus in recombinant protein production

    PubMed Central

    2013-01-01

    Background Kluyveromyces marxianus combines the ease of genetic manipulation and fermentation with the ability to efficiently secrete high molecular weight proteins, performing eukaryotic post-translational modifications. It is able to grow efficiently in a wide range of temperatures. The secretion performances were analyzed in the host K. marxianus L3 in the range between 5°C and 40°C by means of 3 different reporter proteins, since temperature appears a key parameter for production and secretion of recombinant proteins. Results The recombinant strains were able to grow up to 40°C and, along the tested temperature interval (5-40°C), the specific growth rates (μ) were generally lower as compared to those of the untransformed strain. Biomass yields were slightly affected by temperature, with the highest values reached at 15°C and 30°C. The secretion of the endogenous β-fructofuranosidase, used as an internal control, was efficient in the range of the tested temperature, as evaluated by assaying the enzyme activity in the culture supernatants. The endogenous β-fructofuranosidase production was temperature dependent, with the highest yield at 30°C. The heterologous proteins HSA, GAA and Sod1p were all successfully produced and secreted between 5°C and 40°C, albeit each one presented a different optimal production temperature (15, 40, 5-30°C for HSA, GAA and Sod1p, respectively). Conclusions K. marxianus L3 has been identified as a promising and flexible cell factory. In a sole host, the optimization of growth temperatures for the efficient secretion of each individual protein can be carried out over a wide range of temperatures. PMID:23587421

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

    USDA-ARS?s Scientific Manuscript database

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

  8. The adhesive properties of coacervated recombinant hybrid mussel adhesive proteins.

    PubMed

    Lim, Seonghye; Choi, Yoo Seong; Kang, Dong Gyun; Song, Young Hoon; Cha, Hyung Joon

    2010-05-01

    Marine mussels attach to substrates using adhesive proteins. It has been suggested that complex coacervation (liquid-liquid phase separation via concentration) might be involved in the highly condensed and non-water dispersed adhesion process of mussel adhesive proteins (MAPs). However, as purified natural MAPs are difficult to obtain, it has not been possible to experimentally validate the coacervation model. In the present work, we demonstrate complex coacervation in a system including recombinant MAPs and hyaluronic acid (HA). Our recombinant hybrid MAPs, fp-151 and fp-131, can be produced in large quantities, and are readily purified. We observed successful complex coacervation using cationic fp-151 or fp-131, and an anionic HA partner. Importantly, we found that highly condensed complex coacervates significantly increased the bulk adhesive strength of MAPs in both dry and wet environments. In addition, oil droplets were successfully engulfed using a MAP-based interfacial coacervation process, to form microencapsulated particles. Collectively, our results indicate that a complex coacervation system based on MAPs shows superior adhesive properties, combined with additional valuable features including liquid/liquid phase separation and appropriate viscoelasticity. Our microencapsulation system could be useful in the development of new adhesive biomaterials, including self-adhesive microencapsulated drug carriers, for use in biotechnological and biomedical applications.

  9. Optimization of elastin-like polypeptide fusions for expression and purification of recombinant proteins in plants.

    PubMed

    Conley, Andrew J; Joensuu, Jussi J; Jevnikar, Anthony M; Menassa, Rima; Brandle, Jim E

    2009-06-15

    The demand for recombinant proteins for medical and industrial use is expanding rapidly and plants are now recognized as an efficient, inexpensive means of production. Although the accumulation of recombinant proteins in transgenic plants can be low, we have previously demonstrated that fusions with an elastin-like polypeptide (ELP) tag can significantly enhance the production yield of a range of different recombinant proteins in plant leaves. ELPs are biopolymers with a repeating pentapeptide sequence (VGVPG)(n) that are valuable for bioseparation, acting as thermally responsive tags for the non-chromatographic purification of recombinant proteins. To determine the optimal ELP size for the accumulation of recombinant proteins and their subsequent purification, various ELP tags were fused to green fluorescent protein, interleukin-10, erythropoietin and a single chain antibody fragment and then transiently expressed in tobacco leaves. Our results indicated that ELP tags with 30 pentapeptide repeats provided the best compromise between the positive effects of small ELP tags (n = 5-40) on recombinant protein accumulation and the beneficial effects of larger ELP tags (n = 80-160) on recombinant protein recovery during inverse transition cycling (ITC) purification. In addition, the C-terminal orientation of ELP fusion tags produced higher levels of target proteins, relative to N-terminal ELP fusions. Importantly, the ELP tags had no adverse effect on the receptor binding affinity of erythropoietin, demonstrating the inert nature of these tags. The use of ELP fusion tags provides an approach for enhancing the production of recombinant proteins in plants, while simultaneously assisting in their purification.

  10. 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. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. High Yield of Recombinant Protein in Shaken E. coli Cultures with Enzymatic Glucose Release Medium EnPresso B.

    PubMed

    Ukkonen, Kaisa; Neubauer, Antje; Pereira, Vinit J; Vasala, Antti

    2017-01-01

    Expression of recombinant proteins in sufficient quantities is essential for protein structure-function studies. The most commonly used method for recombinant protein production is overexpression in E. coli cultures. However, producing high yields of functional proteins in E. coli can be a challenge in conventional shaken cultures. This is often due to nonoptimal growth conditions, which result in low cell yields and insoluble or incorrectly folded target protein. To overcome the shortcomings of shake flask cultivation, we present a cultivation method based on enzymatic glucose delivery. This system mimics the fed-batch principle used in bioreactor cultivations and provides high yields of biomass and recombinant proteins in shaken cultivations.

  12. [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.

  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. 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.

  15. Key determinants affecting sheep wool biodegradation directed by a keratinase-producing Bacillus subtilis recombinant strain.

    PubMed

    Zaghloul, Taha I; Embaby, Amira M; Elmahdy, Ahmed R

    2011-02-01

    OVAT (one variable at a time) approach was applied in this study to screen the most important physicochemical key determinants involved in the process of sheep wool biodegradation. The process was directed by a keratinase-producing Bacillus subtilis DB 100 (p5.2) recombinant strain. Data indicate that, sheep wool could be degraded efficiently in cultures incubated at 30°C, with initial pH of 7 with agitation at 150 rpm. Two times autoclaved alkali treated and undefatted chopped sheep wool is more accessible to biodegradation. B. subtilis recombinant cells could utilize sheep wool as a sole source of carbon and nitrogen. Sheep wool-based modified basal medium II, lacking NH₄Cl and yeast extract, could greatly support the growth of these bacterial cells. Sheep wool biodegradation was conducted efficiently in the absence of kanamycin consequently; high stability of the recombinant plasmid (p5.2) represents a great challenge upon scaling up this process. Three key determinants (sheep wool concentration, incubation time and inoculum size) imposing considerable constraints on the process are highlighted. Sheep wool-based tap water medium and sheep wool-based distilled water medium were formulated in this study. High levels of released end products, produced from sheep wool biodegradation are achieved upon using these two sheep wool-based water media. Data indicate that, sheep wool hydrolysate is rich in some amino acids, such as tyrosine, phenylalanine, lysine, proline, isoleucine, leucine, valine, aspartic acid and glutamic acid. Moreover, the resulting sheep wool hydrolysate contains soluble proteins of high and intermediate molecular weights. The present study demonstrates a feasible, cheap, reproducible, efficient and rapid biotechnological approach towards utilization of raw sheep wool waste through a recombinant bacterium.

  16. 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.

  17. 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.

  18. 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

    2013-02-06

    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. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

    PubMed

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

    2015-09-01

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

  20. Assessment of stable isotope incorporation into recombinant proteins.

    PubMed

    Zhang, Xin; Luo, Quanzhou; Apostol, Izydor; Luo, Shun; Jerums, Matthew; Huang, Gang; Jiang, Xinzhao Grace; Gastwirt, Jessica; Savjani, Nimesh; Lewis, Jeffrey; Keener, Ronald; Wypych, Jette

    2013-02-15

    Stable isotope labeling combined with mass spectrometry has been widely used in a diverse set of applications in the biochemistry and biomedical fields. When stable isotope-labeled proteins are produced via metabolic labeling of cell culture, a comprehensive assessment of the labeling pattern is imperative. In this study, we present a set of mass spectrometry-based bioanalytical tools developed for quantitatively tracing the levels of the stable isotopes incorporated into the recombinant proteins (monoclonal antibodies and Fc fusion proteins expressed in different host systems) that include total mass analysis, peptide mapping analysis, and amino acid analysis. We show that these three mass spectrometry-based analytical methods have distinctive advantages and limitations and that they are mutually complementary in evaluating the quality of stable isotope-labeled proteins. In addition, we show that the analytical techniques developed here are powerful tools to provide valuable insights into studying cell metabolism and performing flux analysis during cell culture. Copyright © 2012 Elsevier Inc. All rights reserved.

  1. Application of recombinant fusion proteins for tissue engineering.

    PubMed

    Nagaoka, Masato; Jiang, Hu-Lin; Hoshiba, Takashi; Akaike, Toshihiro; Cho, Chong-Su

    2010-03-01

    Extracellular matrix (ECM) plays important roles in tissue engineering because cellular growth and differentiation, in the two-dimensional cell culture as well as in the three-dimensional space of the developing organism, require ECM with which the cells can interact. Also, the development of new synthetic ECMs is very important because ECMs facilitate the localization and delivery of cells to the specific sites in the body. Therefore, the development of synthetic ECMs to replace the natural ECMs is increasingly essential and promising in tissue engineering. Recombinant genetic engineering method has enabled the synthesis of protein-based polymers with precisely controlled functionalities for the development of new synthetic ECMs. In this review, the design and construction of structure-based recombinant fusion proteins such as elastin-like polymers (ELPs) and silk-like polymers (SLPs), cell-bound growth factor-based recombinant fusion proteins such as basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), hybrid system composed of recombinant protein and synthetic polymer, and E-cadherin-based fusion protein by recombinant genetic engineering were explained for application of the synthetic ECMs. Modulation of mechanical properties, stimuli-sensitivity, biodegradation and cell recognition can be achieved through precise control of sequence, length, hydrophobicity and cell binding domain by recombinant genetic engineering.

  2. Heat shock protein produced by Helicobacter pylori.

    PubMed

    Yokota, K; Hirai, Y; Haque, M; Hayashi, S; Isogai, H; Sugiyama, T; Nagamachi, E; Tsukada, Y; Fujii, N; Oguma, K

    1994-01-01

    The cells of Helicobacter pylori were suspended in the medium containing 35S-methionine. After a heat shock of the cells at 42 C for 5, 10, and 30 min, the production of proteins was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Out of many proteins produced by the cells, only 66 kDa protein production was dramatically increased by heat treatment. The N-terminal amino acid sequence of 66 kDa protein was quite similar to that of 62 kDa and 54 kDa proteins previously suggested as heat shock protein (HSP) of H. pylori based on the reaction with polyclonal and monoclonal antibodies against HSP 60 family proteins produced by other bacteria. Therefore, it was concluded that H. pylori produces the 66 kDa protein as its major heat shock protein which belongs to HSP 60 family.

  3. Production of a recombinant industrial protein using barley cell cultures.

    PubMed

    Ritala, A; Wahlström, E H; Holkeri, H; Hafren, A; Mäkeläinen, K; Baez, J; Mäkinen, K; Nuutila, A M

    2008-06-01

    The use of recombinant DNA-based protein production using genetically modified plants could provide a reproducible, consistent quality, safe, animal-component free, origin-traceable, and cost-effective source for industrial proteins required in large amounts (1000s of metric tons) and at low cost (below US$100/Kg). The aim of this work was to demonstrate the feasibility of using barley suspension cell culture to support timely testing of the genetic constructs and early product characterization to detect for example post-translational modifications within the industrial protein caused by the selected recombinant system. For this study the human Collagen I alpha 1 (CIa1) chain gene encoding the complete helical region of CIa1 optimized for monocot expression was fused to its N- and C-terminal telopeptide and to a bacteriophage T4 fibritin foldon peptide encoding sequences. The CIa1 accumulation was targeted to the endoplasmic reticulum (ER) by fusing the CIa1 gene to an ER-directing signal peptide sequence and an ER retention signal HDEL. The construct containing the CIa1 gene was then introduced into immature barley half embryos or barley cells by particle bombardment. Transgenic barley cells resulting from these transformations were grown as suspension cultures in flasks and in a Wave bioreactor producing CIa1 similar to CIa1 purified from the yeast Pichia pastoris based on Western blotting, pepsin resistance, and mass spectroscopy analysis. The barley cell culture derived-CIa1 intracellular accumulation levels ranged from 2 to 9 microg/l illustrating the need for further process improvement in order to use this technology to supply material for product development activities.

  4. A new hybrid protein for production of recombinant bacteriorhodopsin in Escherichia coli.

    PubMed

    Nekrasova, Oksana V; Wulfson, Andrey N; Tikhonov, Roman V; Yakimov, Sergey A; Simonova, Tatyana N; Tagvey, Anna I; Dolgikh, Dmitry A; Ostrovsky, Mikhail A; Kirpichnikov, Mikhail P

    2010-06-01

    Unique properties of bacteriorhodopsin, namely, photochromism and high thermal stability, make this protein an attractive target for physico-chemical studies, as well as for various biotechnological applications. Using Mistic as a suitable carrier for insertion of recombinant membrane proteins into cytoplasmic membrane of Escherichia coli, we developed a system for overexpression of bacteriorhodopsin and worked out an efficient procedure for its purification and renaturation with the final yield of 120 mg/l of refolded protein, which is the highest value reported to date for bacteriorhodopsin produced in E. coli. Functional activity of recombinant bacteriorhodopsin was confirmed by spectroscopic and electrochemical assays. 2010 Elsevier B.V. All rights reserved.

  5. Vaccinia virus vectors: new strategies for producing recombinant vaccines.

    PubMed Central

    Hruby, D E

    1990-01-01

    The development and continued refinement of techniques for the efficient insertion and expression of heterologous DNA sequences from within the genomic context of infectious vaccinia virus recombinants are among the most promising current approaches towards effective immunoprophylaxis against a variety of protozoan, viral, and bacterial human pathogens. Because of its medical relevance, this area is the subject of intense research interest and has evolved rapidly during the past several years. This review (i) provides an updated overview of the technology that exists for assembling recombinant vaccinia virus strains, (ii) discusses the advantages and disadvantages of these approaches, (iii) outlines the areas of outgoing research directed towards overcoming the limitations of current techniques, and (iv) provides some insight (i.e., speculation) about probable future refinements in the use of vaccinia virus as a vector. PMID:2187593

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

    PubMed Central

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

    2016-01-01

    Background: As a drug target and an antigenic agent, HIV-1 protease (HIV-1 PR) is at the center of attention for designing anti-AIDS inhibitors and diagnostic tests. In previous studies, the production of the recombinant protease has been faced with several difficulties; therefore, the aims of this study were the easy production, purification of the soluble form of protease in E. coli and investigation of its immunoreactivity. Methods: Protease coding region was isolated from the serum of an infected individual, amplified by RT-PCR and cloned into PTZ57R using TA-cloning. Protease coding frame was isolated by PCR and cloned in pET102/D. TOPO expression vector and cloned protease was expressed in Escherichia coli (E. coli) BL21. Produced recombinant protein was purified by affinity Ni-NTA column and protein concentration was checked by BCA protein assay kit. Subsequently, immunoreactivity of recombinant protease (rPR) was assayed by Western blotting and ELISA. Results: Cloning of the HIV protease by TOPO cloning system in pET102/D.TOPO was confirmed with PCR and sequencing. The concentration range of purified recombinant protein was 85 to 100 μg/ml. Immunogenicity of rPR was confirmed by Western blotting and ELISA. Conclusion: Soluble production of recombinant HIV-1 protease (HIV-1 rPR) was performed successfully. This recombinant protein disclosed 86% specificity and 90% sensitivity in immunoassay tests. PMID:27920885

  7. Chaperokine function of recombinant Hsp72 produced in insect cells using a baculovirus expression system is retained.

    PubMed

    Zheng, Hongying; Nagaraja, Ganachari M; Kaur, Punit; Asea, Edwina E; Asea, Alexzander

    2010-01-01

    Extracellular heat shock protein 72 (Hsp72; inducible form of the 70-kDa heat shock protein) plays a critical role in innate and adaptive immune responses and has shown promise as an ideal adjuvant for the optimization of antigen-specific anti-tumor vaccines. Recent studies suggest that to correctly elucidate the mechanisms by which Hsp72 exerts its beneficial effects in vitro, great care must be taken to ensure that endotoxin by-products do not invalidate the findings. In this study, we have taken advantage of the baculovirus expression vector system for production of endotoxin-free recombinant Hsp72. The coding sequence of human hsp72 was recombined into the baculovirus immediately downstream of the strong polyhedron gene promoter. Ninety-six h post-infection of Sf9 insect cells with recombinant baculovirus, maximal levels of Hsp72 protein were detected. The recombinant human Hsp72 was purified by affinity chromatography from insect cells, and purity was confirmed by SDS-PAGE and mass spectrometry. The purified human recombinant Hsp72(bv) (Hsp72 produced using the BEVS) was demonstrated to have no endotoxin contamination and was shown to have stimulated potent calcium flux in the human monocytic cell line. Furthermore, recombinant Hsp72(bv) enhanced the tolerance of neuroblastoma cells to heat stress-induced cell death and displayed classical chaperokine functions including augmentation of inflammatory cytokine productions in mouse splenocytes. The production of functional, endotoxin-free recombinant human Hsp72(bv) in insect cells is inexpensive and convenient and eliminates the need of special procedures for endotoxin depletion. Endotoxin-free recombinant human Hsp72(bv) can now be used to unlock the important role Hsp72 plays in modulating immune function.

  8. Meiotic recombination proteins localize to linear elements in Schizosaccharomyces pombe.

    PubMed

    Lorenz, Alexander; Estreicher, Anna; Kohli, Jürg; Loidl, Josef

    2006-08-01

    In fission yeast, meiotic prophase nuclei develop structures known as linear elements (LinEs), instead of a canonical synaptonemal complex. LinEs contain Rec10 protein. While Rec10 is essential for meiotic recombination, the precise role of LinEs in this process is unknown. Using in situ immunostaining, we show that Rec7 (which is required for meiosis-specific DNA double-strand break (DSB) formation) aggregates in foci on LinEs. The strand exchange protein Rad51, which is known to mark the sites of DSBs, also localizes to LinEs, although to a lesser degree. The number of Rec7 foci corresponds well with the average number of genetic recombination events per meiosis suggesting that Rec7 marks the sites of recombination. Rec7 and Rad51 foci do not co-localize, presumably because they act sequentially on recombination sites. The localization of Rec7 is dependent on Rec10 but independent of the DSB-inducing protein Rec12/Spo11. Neither Rec7 nor Rad51 localization depends on the LinE-associated proteins Hop1 and Mek1, but the formation of Rad51 foci depends on Rec10, Rec7, and, as expected, Rec12/Spo11. We propose that LinEs form around designated recombination sites before the induction of DSBs and that most, if not all, meiotic recombination initiates within the setting provided by LinEs.

  9. One single method to produce native and Tat-fused recombinant human α-synuclein in Escherichia coli

    PubMed Central

    2013-01-01

    Background Human α-synuclein is a small-sized, natively unfolded protein that in fibrillar form is the primary component of Lewy bodies, the pathological hallmark of Parkinson’s disease. Experimental evidence suggests that α-synuclein aggregation is the key event that triggers neurotoxicity although additional findings have proposed a protective role of α-synuclein against oxidative stress. One way to address the mechanism of this protective action is to evaluate α-synuclein-mediated protection by delivering this protein inside cells using a chimeric protein fused with the Tat-transduction domain of HIV Tat, named TAT-α-synuclein. Results A reliable protocol was designed to efficiently express and purify two different forms of human α-synuclein. The synthetic cDNAs encoding for the native α-synuclein and the fusion protein with the transduction domain of Tat protein from HIV were overexpressed in a BL21(DE3) E. coli strain as His-tagged proteins. The recombinant proteins largely localized (≥ 85%) to the periplasmic space. By using a quick purification protocol, based on recovery of periplasmic space content and metal-chelating chromatography, the recombinant α-synuclein protein forms could be purified in a single step to ≥ 95% purity. Both α-synuclein recombinant proteins form fibrils and the TAT-α-synuclein is also cytotoxic in the micromolar concentration range. Conclusions To further characterize the molecular mechanisms of α-synuclein neurotoxicity both in vitro and in vivo and to evaluate the relevance of extracellular α-synuclein for the pathogenesis and progression of Parkinson’s disease, a suitable method to produce different high-quality forms of this pathological protein is required. Our optimized expression and purification procedure offers an easier and faster means of producing different forms (i.e., both the native and the TAT-fusion form) of soluble recombinant α-synuclein than previously described procedures. PMID:23557146

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

    PubMed

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

    2017-01-01

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

  11. Preparation of the Mgm101 recombination protein by MBP-based tagging strategy.

    PubMed

    Wang, Xiaowen; Mbantenkhu, MacMillan; Wierzbicki, Sara; Chen, Xin Jie

    2013-06-25

    The MGM101 gene was identified 20 years ago for its role in the maintenance of mitochondrial DNA. Studies from several groups have suggested that the Mgm101 protein is involved in the recombinational repair of mitochondrial DNA. Recent investigations have indicated that Mgm101 is related to the Rad52-type recombination protein family. These proteins form large oligomeric rings and promote the annealing of homologous single stranded DNA molecules. However, the characterization of Mgm101 has been hindered by the difficulty in producing the recombinant protein. Here, a reliable procedure for the preparation of recombinant Mgm101 is described. Maltose Binding Protein (MBP)-tagged Mgm101 is first expressed in Escherichia coli. The fusion protein is initially purified by amylose affinity chromatography. After being released by proteolytic cleavage, Mgm101 is separated from MBP by cationic exchange chromatography. Monodispersed Mgm101 is then obtained by size exclusion chromatography. A yield of ~0.87 mg of Mgm101 per liter of bacterial culture can be routinely obtained. The recombinant Mgm101 has minimal contamination of DNA. The prepared samples are successfully used for biochemical, structural and single particle image analyses of Mgm101. This protocol may also be used for the preparation of other large oligomeric DNA-binding proteins that may be misfolded and toxic to bacterial cells.

  12. 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.

  13. 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.

  14. 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

  15. 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

    2014-09-08

    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.

  16. Agroinfiltration contributes to VP1 recombinant protein degradation.

    PubMed

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

    2016-11-01

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

  17. 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. © 2014 American Institute of Chemical Engineers.

  18. 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 and...

  19. 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 and...

  20. 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 and...

  1. 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 and...

  2. 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 and...

  3. Self-Assembled Materials Made from Functional Recombinant Proteins.

    PubMed

    Jang, Yeongseon; Champion, Julie A

    2016-10-18

    Proteins are potent molecules that can be used as therapeutics, sensors, and biocatalysts with many advantages over small-molecule counterparts due to the specificity of their activity based on their amino acid sequence and folded three-dimensional structure. However, they also have significant limitations in their stability, localization, and recovery when used in soluble form. These opportunities and challenges have motivated the creation of materials from such functional proteins in order to protect and present them in a way that enhances their function. We have designed functional recombinant fusion proteins capable of self-assembling into materials with unique structures that maintain or improve the functionality of the protein. Fusion of either a functional protein or an assembly domain to a leucine zipper domain makes the materials design strategy modular, based on the high affinity between leucine zippers. The self-assembly domains, including elastin-like polypeptides (ELPs) and defined-sequence random coil polypeptides, can be fused with a leucine zipper motif in order to promote assembly of the fusion proteins into larger structures upon specific stimuli such as temperature and ionic strength. Fusion of other functional domains with the counterpart leucine zipper motif endows the self-assembled materials with protein-specific functions such as fluorescence or catalytic activity. In this Account, we describe several examples of materials assembled from functional fusion proteins as well as the structural characterization, functionality, and understanding of the assembly mechanism. The first example is zipper fusion proteins containing ELPs that assemble into particles when introduced to a model extracellular matrix and subsequently disassemble over time to release the functional protein for drug delivery applications. Under different conditions, the same fusion proteins can self-assemble into hollow vesicles. The vesicles display a functional protein on

  4. Straightforward approach to produce recombinant scorpion toxins-Pore blockers of potassium channels.

    PubMed

    Nekrasova, Oksana; Kudryashova, Ksenia; Fradkov, Arkadiy; Yakimov, Sergey; Savelieva, Maria; Kirpichnikov, Mikhail; Feofanov, Alexey

    2017-01-10

    Scorpion venom peptide blockers (KTx) of potassium channels are a valuable tool for structure-functional studies and prospective candidates for medical applications. Low yields of recombinant KTx hamper their wide application. We developed convenient and efficient bioengineering approach to a large-scale KTx production that meets increasing demands for such peptides. Maltose-binding protein was used as a carrier for cytoplasmic expression of folded disulfide-rich KTx in E. coli. TEV protease was applied for in vitro cleavage of the target peptide from the carrier. To produce KTx with retained native N-terminal sequence, the last residue of TEV protease cleavage site (CSTEV) was occupied by the native N-terminal residue of a target peptide. It was shown that decreased efficiency of hydrolysis of fusion proteins with non-canonical CSTEV can be overcome without by-product formation. Disulfide formation and folding of a target peptide occurred in cytoplasm eliminating the need for renaturation procedure in vitro. Advantages of this approach were demonstrated by producing six peptides with three disulfide bonds related to four KTx sub-families and achieving peptide yields of 12-22mg per liter of culture. The developed approach can be of general use for low-cost production of various KTx, as well as other disulfide-rich peptides and proteins.

  5. The transcriptional response of Escherichia coli to recombinant protein insolubility.

    PubMed

    Smith, Harold E

    2007-03-01

    Bacterial production of recombinant proteins offers several advantages over alternative expression methods and remains the system of choice for many structural genomics projects. However, a large percentage of targets accumulate as insoluble inclusion bodies rather than soluble protein, creating a significant bottleneck in the protein production pipeline. Numerous strategies have been reported that can improve in vivo protein solubility, but most do not scale easily for high-throughput expression screening. To understand better the host cell response to the accumulation of insoluble protein, we determined genome-wide changes in bacterial gene expression upon induction of either soluble or insoluble target proteins. By comparing transcriptional profiles for multiple examples from the soluble or insoluble class, we identified a pattern of gene expression that correlates strongly with protein solubility. Direct targets of the sigma32 heat shock sigma factor, which includes genes involved in protein folding and degradation, were highly expressed in response to induction of insoluble protein. This same group of genes was also upregulated by insoluble protein accumulation under a different growth regime, indicating that sigma32-mediated gene expression is a general response to protein insolubility. This knowledge provides a starting point for the rational design of growth parameters and host strains with improved protein solubility characteristics. Summary Problems with protein solubility are frequently encountered when recombinant proteins are expressed in E. coli. The bacterial host responds to this problem by increasing expression of the protein folding machinery via the heat shock sigma factor sigma32. Manipulation of the sigma32 regulon might provide a general mechanism for improving recombinant protein solubility.

  6. [Immunogenic and protective characteristics of recombinant Lassa virus NP protein].

    PubMed

    Ignat'ev, G M

    2002-01-01

    Recombinant fragment of Lassa virus (strain Josiah) nucleocapsid protein (corresponding to amino acid residues 141 + 569) constructed by Dr. Jan ter Meulen (Tropenmedizine, Hamburg) was used for immunizing CBA/calac mice. The preparation was injected intraperitoneally twice with 2-week interval in a dose of 10 micrograms. The parameters of the resultant specific humoral and cell-mediated immunity were comparable to those in reference animals immunized with inactivated Lassa virus. Challenge with Lassa virus (10,000 PFU) resulted in 100% death of the reference animals, while of 15 animals immunized with the recombinant NP protein 8 survived.

  7. Conformational features of tau fibrils from Alzheimer's disease brain are faithfully propagated by unmodified recombinant protein.

    PubMed

    Morozova, Olga A; March, Zachary M; Robinson, Anne S; Colby, David W

    2013-10-08

    Fibrils composed of tau protein are a pathological hallmark of several neurodegenerative disorders including Alzheimer's disease (AD). Here we show that when recombinant tau protein is seeded with paired helical filaments (PHFs) isolated from AD brain, the amyloid formed shares many of the structural features of AD PHFs. In contrast, tau amyloids formed with heparin as an inducing agent-a common biochemical model of tau misfolding-are structurally distinct from brain-derived PHFs. Using ultrastructural analysis by electron microscopy, circular dichroism, and chemical denaturation, we found that AD seeded recombinant tau fibrils were not significantly different than tau fibrils isolated from AD brain tissue. Tau fibrils produced by incubating recombinant tau with heparin had significantly narrower fibrils with a longer periodicity, higher chemical stability, and distinct secondary structure compared to AD PHFs. The addition of heparin to the reaction of recombinant tau and AD PHFs also corrupted the templating process, resulting in a mixture of fibril conformations. Our results suggest that AD-isolated PHFs act as a conformational template for the formation of recombinant tau fibrils. Therefore, the use of AD PHFs as seeds to stimulate recombinant tau amyloid formation produces synthetic tau fibers that closely resemble those associated with AD pathology and provides a biochemical model of tau misfolding that may be of improved utility for structural studies and drug screening. These results also demonstrate that post-translational modifications such as phosphorylation are not a prerequisite for the propagation of the tau fibril conformation found in AD.

  8. Production of two vaccinating recombinant rotavirus proteins in the milk of transgenic rabbits.

    PubMed

    Soler, Eric; Le Saux, Agnès; Guinut, Frédéric; Passet, Bruno; Cohen, Ruxandra; Merle, Christine; Charpilienne, Annie; Fourgeux, Cynthia; Sorel, Véronique; Piriou, Antoine; Schwartz-Cornil, Isabelle; Cohen, Jean; Houdebine, Louis-Marie

    2005-12-01

    Rotaviruses are the main cause of infantile viral gastroenteritis worldwide leading to approximately 500,000 deaths each year mostly in the developing world. For unknown reasons, live attenuated viruses used in classical vaccine strategies were shown to be responsible for intussusception (a bowel obstruction). New strategies allowing production of safe recombinant non-replicating rotavirus candidate vaccine are thus clearly needed. In this study we utilized transgenic rabbit milk as a source of rotavirus antigens. Individual transgenic rabbit lines were able to produce several hundreds of micrograms per ml of secreted recombinant VP2 and VP6 proteins in their milk. Viral proteins expressed in our model were immunogenic and were shown to induce a significant reduction in viral antigen shedding after challenge with virulent rotavirus in the adult mouse model. To our knowledge, this is the first report of transgenic mammal bioreactors allowing the rapid co-production of two recombinant viral proteins in milk to be used as a vaccine.

  9. Recombinant Jembrana disease virus proteins as antigens for the detection of antibody to bovine lentiviruses.

    PubMed

    Burkala, E J; Narayani, I; Hartaningsih, N; Kertayadnya, G; Berryman, D I; Wilcox, G E

    1998-09-01

    Jembrana disease virus (JDV) is a recently identified bovine lentivirus causing an acute severe disease syndrome in banteng cattle (Bos javanicus) and a milder disease syndrome in Bos taurus cattle in Indonesia. The virus is closely related genetically to the previously identified bovine lentivirus, bovine immunodeficiency virus (BIV). Recombinant clones were produced which contained the capsid (CA) and transmembrane (TM) subunits of the respective gag and env open reading frames of JDV. The proteins were expressed as fusions to the glutathione-s-transferase (GST) enzyme in Escherichia coli and purification was achieved using affinity chromatography via immobilized reduced glutathione. The soluble recombinant CA and TM antigens of JDV were reacted in western immunoblots with both serum antibodies from JDV-infected Bos javanicus cattle and Bos taurus cattle immunized with BIV. The recombinant CA protein of JDV reacted equally well with both the JDV and BIV antisera. The recombinant TM protein of JDV also reacted with antibody from the JDV infected cattle and with the BIV antisera. The results indicated conservation of immunogenic epitopes of the CA and TM proteins of the two viruses. The production of the recombinant proteins should enable the development of rapid and sensitive serological tests for JDV and BIV, and tools for further study of the immune response to JDV and the differential epidemiology of JDV infections in cattle.

  10. Thermolabile antifreeze protein produced in Escherichia coli for structural analysis.

    PubMed

    Lin, Feng-Hsu; Sun, Tianjun; Fletcher, Garth L; Davies, Peter L

    2012-03-01

    The only hyperactive antifreeze protein (AFP) found to date in fishes is an extreme variant of the 3-kDa, alpha-helical, alanine-rich type I AFP, which is referred to here as type Ih. Purification of the 33-kDa homodimeric AFP Ih from a natural source was hampered by its low levels in fish plasma; by the need to remove the more abundant smaller isoforms; and by its extreme thermolability. Moreover, ice affinity as a purification tool was spoiled by the tendency of fish IgM antibodies to bind to ice in the presence of AFPs. In order to produce enough protein for crystallography we expressed AFP Ih as a recombinant protein in the Arctic Express® strain of Escherichia coli at 12 °C, just below the thermal denaturation temperature of 16-18 °C. His-tags were not useful because they compromised the activity and yield of AFP Ih. But in the absence of fish antibodies we were able to recover 10-mg quantities of the antifreeze protein using two cycles of ice affinity purification followed by anion-exchange chromatography to remove contaminating chaperones. The purified recombinant AFP Ih yielded diffraction-quality crystals with an extremely asymmetrical unit cell. By transferring the genes of the chaperones into a methionine auxotroph we were able to grow this host at low temperatures and produce sufficient selenomethionine-labeled AFP Ih for crystallography.

  11. Recombinant Structural Proteins and Their Use in Future Materials.

    PubMed

    Sutherland, Tara D; Rapson, Trevor D; Huson, Mickey G; Church, Jeffrey S

    2017-01-01

    Recombinant proteins are polymers that offer the materials engineer absolute control over chain length and composition: key attributes required for design of advanced polymeric materials. Through this control, these polymers can be encoded to contain information that enables them to respond as the environment changes. However, despite their promise, protein-based materials are under-represented in materials science. In this chapter we investigate why this is and describe recent efforts to address this. We discuss constraints limiting rational design of structural proteins for advanced materials; advantages and disadvantages of different recombinant expression platforms; and, methods to fabricate proteins into solid-state materials. Finally, we describe the silk proteins used in our laboratory as templates for information-containing polymers.

  12. Spider Silk Fibers Spun from Soluble Recombinant Silk Produced in Mammalian Cells

    NASA Astrophysics Data System (ADS)

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

    2002-01-01

    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.

  13. Factor VIII--Baxter: rAHF-PFM, recombinant anti-haemophilic factor--protein-free method, recombinant factor VIII--protein-free.

    PubMed

    2003-01-01

    Baxter Healthcare is developing a next-generation recombinant factor VIII for the treatment of haemophilia A that is produced using a totally protein-free manufacturing process. By excluding proteins or raw materials derived from human or animal sources in the final product, the risk of transmission of potentially infectious agents is removed. All of the recombinant factor VIII products currently on the market incorporate proteins or raw materials derived from either human or animal sources, as part of the nutrients required for the cells to produce the protein. Baxter has exclusive rights to Quadrant Healthcare's factor VIII stabilisation technology. Quadrant received 1 million US dollars under the agreement, and was to receive milestone payments in excess of 2 million US dollars, together with royalties on sales should a new factor VIII product using Quadrant's technology reach the market. In December 2000, Quadrant Healthcare was acquired by Elan Drug Delivery (Elan Corporation), which continued to develop the proprietary formulation and stabilisation technologies of Quadrant. In July 2003, the newly formed company Quadrant Technologies acquired Elan Drug Delivery from Elan Corporation and renamed it Quadrant Drug Delivery. In July 2003, the US FDA approved the use of ADVATE for the prevention and control of bleeding episodes in people with haemophilia A. At the Bear Stearns 16th Annual Healthcare Conference held in September 2003, Baxter reported that ADVATE was launched in August 2003 in the US. A pivotal phase III clinical trial of this recombinant factor VIII therapy, initiated in 89 previously untreated patients with haemophilia A, has been completed at sites in the US, UK, Sweden, France, Belgium, Germany, Denmark and Italy. This trial compared the pharmacokinetics, tolerability and immunogenicity of this therapy with Baxter's currently marketed recombinant factor VIII preparation, Recombinate trade mark. The first clinical data that were presented on this

  14. Chimeragenesis of distantly-related proteins by noncontiguous recombination

    PubMed Central

    Smith, Matthew A; Romero, Philip A; Wu, Timothy; Brustad, Eric M; Arnold, Frances H

    2013-01-01

    We introduce a method for identifying elements of a protein structure that can be shuffled to make chimeric proteins from two or more homologous parents. Formulating recombination as a graph-partitioning problem allows us to identify noncontiguous segments of the sequence that should be inherited together in the progeny proteins. We demonstrate this noncontiguous recombination approach by constructing a chimera of β-glucosidases from two different kingdoms of life. Although the protein's alpha–beta barrel fold has no obvious subdomains for recombination, noncontiguous SCHEMA recombination generated a functional chimera that takes approximately half its structure from each parent. The X-ray crystal structure shows that the structural blocks that make up the chimera maintain the backbone conformations found in their respective parental structures. Although the chimera has lower β-glucosidase activity than the parent enzymes, the activity was easily recovered by directed evolution. This simple method, which does not rely on detailed atomic models, can be used to design chimeras that take structural, and functional, elements from distantly-related proteins. PMID:23225662

  15. 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.

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

    PubMed

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

    2012-03-07

    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.

  17. Using Vaccinia's innate ability to introduce DNA into mammalian cells for production of recombinant proteins.

    PubMed

    Jester, Brian C; Drillien, Robert; Ruff, Marc; Florentz, Catherine

    2011-12-10

    Production of recombinant protein in mammalian cells is time-consuming, labor-intensive and costly. While seeking to overcome these limitations, we discovered that Vaccinia virus has the innate ability to transfer exogenous plasmid DNA into mammalian cells during the infection process. Parameters influencing the efficiency of this event were characterized and a quick, simple and inexpensive way to produce eukaryotic proteins was established.

  18. Receptor specificity and functional comparison of recombinant sea bass (Dicentrarchus labrax) gonadotropins (FSH and LH) produced in different host systems.

    PubMed

    Molés, Gregorio; Zanuy, Silvia; Muñoz, Iciar; Crespo, Berta; Martínez, Iago; Mañanós, Evaristo; Gómez, Ana

    2011-06-01

    Different yields, biopotency, and in vivo pharmacokinetics are obtained for recombinant sea bass gonadoltropins depending on the production system and DNA construct, but they show specific activation of their corresponding receptors. Gonadotropins (GTHs) are glycoprotein hormones that play a major role in the regulation of gonadal functions. Recently, we succeeded in isolating the native sea bass Fsh from sea bass pituitaries, but to ensure the availability of bioactive GTHs and no cross-contamination with other related glycoproteins, recombinant sea bass GTHs were produced using two expression systems-insect and mammalian cells-and different constructs that yielded tethered or noncovalently bound dimers. Their production levels, binding specificity to their homologous cognate receptors, and bioactivity were investigated and compared. Both expression systems were successful in the generation of bioactive recombinant GTHs, but insect Sf9 cells yielded higher amounts of recombinant proteins than mammalian Chinese Hamster Ovary (CHO) stable clones. All recombinant GTHs activated their cognate receptors without cross-ligand binding and were able to stimulate sea bass gonadal steroidogenesis in vitro, although with different biopotencies. To assess their use for in vivo applications, their half-life in sea bass plasma was evaluated. Sf9-GTHs had a lower in vivo stability compared with CHO-GTHs due to their rapid clearance from the blood circulation. Cell-dependent glycosylation could be contributing to the final in vivo stability and biopotency of these recombinant glycoproteins. In conclusion, both insect and mammalian expression systems produced bioactive sea bass recombinant gonadotropins, although with particular features useful for different proposes (e.g., antibody production or in vivo studies, respectively).

  19. Mapping the yeast host cell response to recombinant membrane protein production: relieving the biological bottlenecks.

    PubMed

    Ashe, Mark P; Bill, Roslyn M

    2011-06-01

    Understanding the structures and functions of membrane proteins is an active area of research within bioscience. Membrane proteins are key players in essential cellular processes such as the uptake of nutrients, the export of waste products, and the way in which cells communicate with their environment. It is therefore not surprising that membrane proteins are targeted by over half of all prescription drugs. Since most membrane proteins are not abundant in their native membranes, it is necessary to produce them in recombinant host cells to enable further structural and functional studies. Unfortunately, achieving the required yields of functional recombinant membrane proteins is still a bottleneck in contemporary bioscience. This has highlighted the need for defined and rational optimization strategies based upon experimental observation rather than relying on trial and error. We have published a transcriptome and subsequent genetic analysis that has identified genes implicated in high-yielding yeast cells. These results have highlighted a role for alterations to a cell's protein synthetic capacity in the production of high yields of recombinant membrane protein: paradoxically, reduced protein synthesis favors higher yields. These results highlight a potential bottleneck at the protein folding or translocation stage of protein production.

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

    PubMed

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

    2014-01-25

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

  1. Micro-algae come of age as a platform for recombinant protein production

    PubMed Central

    Specht, Elizabeth; Miyake-Stoner, Shigeki

    2010-01-01

    A complete set of genetic tools is still being developed for the micro-alga Chlamydomonas reinhardtii. Yet even with this incomplete set, this photosynthetic single-celled plant has demonstrated significant promise as a platform for recombinant protein expression. In recent years, techniques have been developed that allow for robust expression of genes from both the nuclear and plastid genome. With these advances, many research groups have examined the pliability of this and other micro-algae as biological machines capable of producing recombinant peptides and proteins. This review describes recent successes in recombinant protein production in Chlamydomonas, including production of complex mammalian therapeutic proteins and monoclonal antibodies at levels sufficient for production at economic parity with existing production platforms. These advances have also shed light on the details of algal protein production at the molecular level, and provide insight into the next steps for optimizing micro-algae as a useful platform for the production of therapeutic and industrially relevant recombinant proteins. PMID:20556634

  2. Immunoreactivity evaluation of a new recombinant chimeric protein against Brucella in the murine model

    PubMed Central

    Abdollahi, Abbas; Mansouri, Shahla; Amani, Jafar; Fasihi-Ramandi, Mahdi; Moradi, Mohammad

    2016-01-01

    Background and Objectives: Brucellosis is an important health problem in developing countries and no vaccine is available for the prevention of infection in humans. Because of clinically infectious diseases and their economic consequences in human and animals, designing a proper vaccine against Brucella is desirable. In this study, we evaluated the immune responses induced by a designed recombinant chimera protein in murine model. Materials and Methods: Three immunodominant antigens of Brucella have been characterized as potential immunogenic and protective antigens including: trigger factor (TF), Omp31 and Bp26 were fused together by EAAAK linkers to produce a chimera (structure were designed in silico), which was synthesized, cloned, and expressed in E. coli BL21 (DE3). The purification of recombinant protein was performed using Ni-NTA agarose. SDS-PAGE and anti-His antibody was used for confirmation purified protein (Western blot). BALB/c immunization was performed by purified protein and adjuvant, and sera antibody levels were measured by ELISA. otted. Results: SDS-PAGE and Western blotting results indicated the similarity of in silico designing and in vitro experiments. ELISA result proved that the immunized sera of mice contain high levels of antibodies (IgG) against recombinant chimeric protein. Conclusion: The recombinant chimeric protein could be a potential antigen candidate for the development of a subunit vaccine against Brucella. PMID:27928487

  3. Micro-algae come of age as a platform for recombinant protein production.

    PubMed

    Specht, Elizabeth; Miyake-Stoner, Shigeki; Mayfield, Stephen

    2010-10-01

    A complete set of genetic tools is still being developed for the micro-alga Chlamydomonas reinhardtii. Yet even with this incomplete set, this photosynthetic single-celled plant has demonstrated significant promise as a platform for recombinant protein expression. In recent years, techniques have been developed that allow for robust expression of genes from both the nuclear and plastid genome. With these advances, many research groups have examined the pliability of this and other micro-algae as biological machines capable of producing recombinant peptides and proteins. This review describes recent successes in recombinant protein production in Chlamydomonas, including production of complex mammalian therapeutic proteins and monoclonal antibodies at levels sufficient for production at economic parity with existing production platforms. These advances have also shed light on the details of algal protein production at the molecular level, and provide insight into the next steps for optimizing micro-algae as a useful platform for the production of therapeutic and industrially relevant recombinant proteins.

  4. Identification, expression and antigenic analysis of recombinant hemagglutinin proteins of canine distemper virus.

    PubMed

    Chan, Kun-Wei; Hsieh, Hsien-Hua; Wang, Hsien-Chi; Lee, Ya-Jane; Sung, Ming-Hua; Wong, Min-Liang; Hsu, Wei-Li

    2009-01-01

    Canine distemper (CD) is a widely distributed disease of dogs, caused by the canine distemper virus (CDV). In the present study, the gene encoding the hemagglutinin (H) protein of a CDV isolate from central Taiwan was sequenced and compared with other strains. Sequence variations were noticed in the H gene from the field CDV strain that had previously been implicated in the increasing incidence of CD. To establish a serology-based diagnostic test, the full-length H protein, as well as five deletion mutants of a recombinant H protein of the local isolate, were produced using an E. coli expression system. Three truncated recombinant proteins with relatively high expression levels, designated HM3, HM4 and HM5, were used as antigens to examine their reactivity with canine sera. By using three negative sera and 17 CD-positive sera, the high specificity of recombinant H proteins was observed by ELISA. In addition, immunoblotting demonstrated that all three purified recombinant proteins exhibit an antigenic property recognized by the serum of a CD-suspected dog.

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

    PubMed

    Huber, Victor C

    2014-01-01

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

  6. Recombination of protein fragments: a promising approach toward engineering proteins with novel nanomechanical properties.

    PubMed

    Balamurali, M M; Sharma, Deepak; Chang, Anderson; Khor, Dingyue; Chu, Ricky; Li, Hongbin

    2008-10-01

    Combining single molecule atomic force microscopy (AFM) and protein engineering techniques, here we demonstrate that we can use recombination-based techniques to engineer novel elastomeric proteins by recombining protein fragments from structurally homologous parent proteins. Using I27 and I32 domains from the muscle protein titin as parent template proteins, we systematically shuffled the secondary structural elements of the two parent proteins and engineered 13 hybrid daughter proteins. Although I27 and I32 are highly homologous, and homology modeling predicted that the hybrid daughter proteins fold into structures that are similar to that of parent protein, we found that only eight of the 13 daughter proteins showed beta-sheet dominated structures that are similar to parent proteins, and the other five recombined proteins showed signatures of the formation of significant alpha-helical or random coil-like structure. Single molecule AFM revealed that six recombined daughter proteins are mechanically stable and exhibit mechanical properties that are different from the parent proteins. In contrast, another four of the hybrid proteins were found to be mechanically labile and unfold at forces that are lower than the approximately 20 pN, as we could not detect any unfolding force peaks. The last three hybrid proteins showed interesting duality in their mechanical unfolding behaviors. These results demonstrate the great potential of using recombination-based approaches to engineer novel elastomeric protein domains of diverse mechanical properties. Moreover, our results also revealed the challenges and complexity of developing a recombination-based approach into a laboratory-based directed evolution approach to engineer novel elastomeric proteins.

  7. A protocol for the production of recombinant spider silk-like proteins for artificial fiber spinning

    PubMed Central

    Teulé, Florence; Cooper, Alyssa R; Furin, William A; Bittencourt, Daniela; Rech, Elibio L; Brooks, Amanda; Lewis, Randolph V

    2009-01-01

    The extreme strength and elasticity of spider silks originate from the modular nature of their repetitive proteins. To exploit such materials and mimic spider silks, comprehensive strategies to produce and spin recombinant fibrous proteins are necessary. This protocol describes silk gene design and cloning, protein expression in bacteria, recombinant protein purification and fiber formation. With an improved gene construction and cloning scheme, this technique is adaptable for the production of any repetitive fibrous proteins, and ensures the exact reproduction of native repeat sequences, analogs or chimeric versions. The proteins are solubilized in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) at 25–30% (wt/vol) for extrusion into fibers. This protocol, routinely used to spin single micrometer-size fibers from several recombinant silk-like proteins from different spider species, is a powerful tool to generate protein libraries with corresponding fibers for structure–function relationship investigations in protein-based biomaterials. This protocol may be completed in 40 d. PMID:19229199

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

    PubMed Central

    Martínez-Alonso, Mónica; Villaverde, Antonio

    2010-01-01

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

  9. Remotely activated protein-producing nanoparticles.

    PubMed

    Schroeder, Avi; Goldberg, Michael S; Kastrup, Christian; Wang, Yingxia; Jiang, Shan; Joseph, Brian J; Levins, Christopher G; Kannan, Sneha T; Langer, Robert; Anderson, Daniel G

    2012-06-13

    The development of responsive nanomaterials, nanoscale systems that actively respond to stimuli, is one general goal of nanotechnology. Here we develop nanoparticles that can be controllably triggered to synthesize proteins. The nanoparticles consist of lipid vesicles filled with the cellular machinery responsible for transcription and translation, including amino acids, ribosomes, and DNA caged with a photolabile protecting group. These particles served as nanofactories capable of producing proteins including green fluorescent protein (GFP) and enzymatically active luciferase. In vitro and in vivo, protein synthesis was spatially and temporally controllable, and could be initiated by irradiating micrometer-scale regions on the time scale of milliseconds. The ability to control protein synthesis inside nanomaterials may enable new strategies to facilitate the study of orthogonal proteins in a confined environment and for remotely activated drug delivery.

  10. 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

  11. 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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Therapeutic advances in rheumatology with the use of recombinant proteins.

    PubMed

    Rothe, Achim; Power, Barbara E; Hudson, Peter J

    2008-11-01

    Antibody engineering and protein design have led to the creation of a new era of targeted anti-inflammatory therapies in rheumatology. Recombinant DNA technologies have enabled the selection and humanization of specific antibody fragments in order to develop therapeutic reagents of any specificity that can be 'armed' to deliver effective anti-inflammatory 'payloads'. Antibodies and antibody-like proteins provide the opportunity to block key soluble mediators of inflammation in their milieu, or alternatively to block intracellular inflammation-triggering pathways by binding to an upstream cell-surface receptor. These designer proteins can be tuned for desired pharmacokinetic and pharmacodynamic effects, and represent tools for specific therapeutic intervention by delivering precisely the required immunosuppressive effect. The extent of desired and undesired effects of a particular biologic therapy, however, can be broader than initially predicted and require careful evaluation during clinical trials. This Review highlights advances in recombinant technologies for the development of novel biologic therapies in rheumatology.

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

    PubMed

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

    2015-12-01

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

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

    PubMed

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

    2017-04-01

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

  15. Recombinant protein-based viral disease diagnostics in veterinary medicine.

    PubMed

    Balamurugan, Vinayagamurthy; Venkatesan, Gnanavel; Sen, Arnab; Annamalai, Lakshmanan; Bhanuprakash, Veerakyathappa; Singh, Raj Kumar

    2010-09-01

    Identification of pathogens or antibody response to pathogens in human and animals modulates the treatment strategies for naive population and subsequent infections. Diseases can be controlled and even eradicated based on the epidemiology and effective prophylaxis, which often depends on development of efficient diagnostics. In addition, combating newly emerging diseases in human as well as animal healthcare is challenging and is dependent on developing safe and efficient diagnostics. Detection of antibodies directed against specific antigens has been the method of choice for documenting prior infection. Other than zoonosis, development of inexpensive vaccines and diagnostics is a unique problem in animal healthcare. The advent of recombinant DNA technology and its application in the biotechnology industry has revolutionized animal healthcare. The use of recombinant DNA technology in animal disease diagnosis has improved the rapidity, specificity and sensitivity of various diagnostic assays. This is because of the absence of host cellular proteins in the recombinant derived antigen preparations that dramatically decrease the rate of false-positive reactions. Various recombinant products are used for disease diagnosis in veterinary medicine and this article discusses recombinant-based viral disease diagnostics currently used for detection of pathogens in livestock and poultry.

  16. Improvement of thermostability of recombinant collagen-like protein by incorporating a foldon sequence.

    PubMed

    Du, Chunling; Wang, Mingqi; Liu, Jinying; Pan, Mingli; Cai, Yurong; Yao, Juming

    2008-05-01

    Collagen is a popular biomaterial in many specific biological interactions as well as a structural element. In this work, the recombinant collagen-like proteins were synthesized using Escherichia coli expression system. A foldon sequence, GYIPEAPRDGQAYVRKDG EWVLLSTFL, derived from the native T4 phage fibritin was incorporated at the C-terminal of collagen-like protein molecules to stabilize the triple helix formed in the proteins. The differential scanning calorimetry and thermogravimetric analysis measurements showed that the thermostability of the recombinant collagen-like proteins was significantly improved when compared with those without the foldon sequence at the C-terminal. Fourier transform infrared and scanning electron microscopy observations indicated that the collagen-like proteins forms the triple helix structure and prefer to aggregate as fibrils, same as the native collagen. Moreover, the mice fibroblasts L929 cells could attach and grew very well on the recombinant collage-like proteins. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that the cell biocompatibility of collagen-like proteins produced in this work was even better than that of native collagen, suggesting that the collagen-like proteins may be a satisfactory candidate for the future applications as a biomaterial.

  17. Expression and properties of the recombinant lumazine (riboflavin) protein from Photobacterium leiognathi.

    PubMed

    Illarionov, B; Illarionova, V; Lee, J; van Dongen, W; Vervoort, J

    1994-11-11

    Photobacterium leiognathi lumazine protein has been expressed in Escherichia coli in high yield, 30 mg/l. The cloned gene was one previously reported by Illarionov (EMBL X56534), that had a similar sequence and was located in the same position as the lumazine protein gene in P. phosphoreum. This gene was placed downstream of the T7 gene 10 promoter of the plasmid pT7-7. When the E. coli are grown at 37 degrees C the protein accumulates in inclusion bodies but solubilization can be achieved in 6 M urea. By a simple procedure of dialysis in the presence of riboflavin and centrifugation, without any chromatography, the recombinant holoprotein is purified to 95% homogeneity. The spectral properties of this recombinant riboflavin protein are the same as those of a fluorescent riboflavin-bound protein produced by many strains of P. leiognathi. The absorption spectrum has the same maxima, 276, 386, 464 nm, the circular dichroism is also the same, and both absorption spectrum and CD are the same as that of apo-lumazine protein having riboflavin bound. The riboflavin on the recombinant can be easily replaced by 6,7-dimethyl-8-ribityllumazine. The absorption and fluorescence spectra, fluorescence yield, and bioluminescence properties of this rebound protein identify it as authentic lumazine protein.

  18. Spider silks: recombinant synthesis, assembly, spinning, and engineering of synthetic proteins

    PubMed Central

    Scheibel, Thomas

    2004-01-01

    Since thousands of years humans have utilized insect silks for their own benefit and comfort. The most famous example is the use of reeled silkworm silk from Bombyx mori to produce textiles. In contrast, despite the more promising properties of their silk, spiders have not been domesticated for large-scale or even industrial applications, since farming the spiders is not commercially viable due to their highly territorial and cannibalistic nature. Before spider silks can be copied or mimicked, not only the sequence of the underlying proteins but also their functions have to be resolved. Several attempts to recombinantly produce spider silks or spider silk mimics in various expression hosts have been reported previously. A new protein engineering approach, which combines synthetic repetitive silk sequences with authentic silk domains, reveals proteins that closely resemble silk proteins and that can be produced at high yields, which provides a basis for cost-efficient large scale production of spider silk-like proteins. PMID:15546497

  19. 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

  20. 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

  1. 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.

  2. 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.

  3. Production of recombinant TRAIL and TRAIL receptor: Fc chimeric proteins.

    PubMed

    Schneider, P

    2000-01-01

    The tumor necrosis factor (TNF)/TNF receptor (TNFR) families of ligands and receptors are implicated in a variety of physiological and pathological processes and regulate cellular functions as diverse as proliferation, differentiation, and death. Recombinant forms of these ligands and receptors can act to agonize or antagonize these functions and are therefore useful for laboratory studies and may have clinical applications. A protocol is presented for the expression and purification of dimeric soluble receptors fused to the Fc portion of human IgG1 and of soluble, N-terminally Flag-tagged ligands. Soluble recombinant proteins are easier to handle than membrane-bound proteins and the use of tags greatly facilitates their detection and purification. In addition, some tags may provide enhanced biological activity to the recombinant proteins (mainly by oligomerization and stabilization effects) and facilitate their functional characterization. Expression in bacterial (for selected ligands) and eukaryotic expression systems (for ligands and receptors) was performed using M15 pREP4 bacteria and human embryonic kidney 293 cells, respectively. The yield of purified protein is about 1 mg/liter for the mammalian expression system and several milligrams per liter for the bacterial expression system. Protocols are given for a specific ligand-receptor pair, namely TRAIL (Apo-2L) and TRAIL receptor 2 (DR5), but can be applied to other ligands and receptors of the TNF family.

  4. 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.

  5. 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.

  6. A general method of protein purification for recombinant unstructured non-acidic proteins.

    PubMed

    Campos, Francisco; Guillén, Gabriel; Reyes, José L; Covarrubias, Alejandra A

    2011-11-01

    Typical late embryogenesis abundant (LEA) proteins accumulate in response to water deficit imposed by the environment or by plant developmental programs. Because of their physicochemical properties, they can be considered as hydrophilins and as a paradigm of intrinsically unstructured proteins (IUPs) in plants. To study their biophysical and biochemical characteristics large quantities of highly purified protein are required. In this work, we report a fast and simple purification method for non-acidic recombinant LEA proteins that does not need the addition of tags and that preserves their in vitro protective activity. The method is based on the enrichment of the protein of interest by boiling the bacterial protein extract, followed by a differential precipitation with trichloroacetic acid (TCA). Using this procedure we have obtained highly pure recombinant LEA proteins of groups 1, 3, and 4 and one recombinant bacterial hydrophilin. This protocol will facilitate the purification of this type of IUPs, and could be particularly useful in proteomic projects/analyses.

  7. Detection of recombinant human lactoferrin and lysozyme produced in a bitransgenic cow.

    PubMed

    Kaiser, Germán G; Mucci, Nicolás C; González, Vega; Sánchez, Lourdes; Parrón, José A; Pérez, María D; Calvo, Miguel; Aller, Juan F; Hozbor, Federico A; Mutto, Adrián A

    2017-03-01

    Lactoferrin and lysozyme are 2 glycoproteins with great antimicrobial activity, being part of the nonspecific defensive system of human milk, though their use in commercial products is difficult because human milk is a limited source. Therefore, many investigations have been carried out to produce those proteins in biological systems, such as bacteria, yeasts, or plants. Mammals seem to be more suitable as expression systems for human proteins, however, especially for those that are glycosylated. In the present study, we developed a bicistronic commercial vector containing a goat β-casein promoter and an internal ribosome entry site fragment between the human lactoferrin and human lysozyme genes to allow the introduction of both genes into bovine adult fibroblasts in a single transfection. Embryos were obtained by somatic cell nuclear transfer, and, after 6 transferences to recipients, 3 pregnancies and 1 viable bitransgenic calf were obtained. The presence of the vector was confirmed by fluorescent in situ hybridization of skin cells. At 13 mo of life and after artificial induction of lactation, both recombinant proteins were found in the colostrum and milk of the bitransgenic calf. Human lactoferrin concentration in the colostrum was 0.0098 mg/mL and that in milk was 0.011 mg/mL; human lysozyme concentration in the colostrum was 0.0022 mg/mL and that in milk was 0.0024 mg/mL. The molar concentration of both human proteins revealed no differences in protein production of the internal ribosome entry site upstream and downstream protein. The enzymatic activity of lysozyme in the transgenic milk was comparable to that of human milk, being 6 and 10 times higher than that of bovine lysozyme present in milk. This work represents an important step to obtain multiple proteins or enhance single protein production by using animal pharming and fewer regulatory and antibiotic-resistant foreign sequences, allowing the design of humanized milk with added biological value for

  8. Integrated approach to produce a recombinant, His-tagged human α-galactosidase A in mammalian cells.

    PubMed

    Corchero, José Luis; Mendoza, Rosa; Lorenzo, Julia; Rodríguez-Sureda, Victor; Domínguez, Carmen; Vázquez, Esther; Ferrer-Miralles, Neus; Villaverde, Antonio

    2011-01-01

    Successful production of recombinant proteins (r-proteins) by transient gene expression (TGE) depends on several parameters (including producer cells, culture conditions, transfection procedure, or expression vector) that should be optimized when producing any recombinant product. In this work, TGE-based production of human α-galactosidase A (GLA) is described. Producer cells, expression vectors, and parameters influencing cell metabolism after transfection have been tested. The enzyme is secreted, has the right molecular weight, and is enzymatically active. Productivities of up to 30-40 mg/L have been achieved, with a simple, fast procedure. A 6 × His tag allows enzyme purification in a single step, rendering a highly pure product. We propose a TGE-based protocol able to produce up to several milligrams per liter of highly pure, active GLA in a time as short as a few days. By this, enough amounts of engineered versions of the enzyme can be easily produced to be tested in vitro or in preclinical trials.

  9. Endotoxin depletion of recombinant protein preparations through their preferential binding to histidine tags.

    PubMed

    Mack, Laura; Brill, Boris; Delis, Natalia; Groner, Bernd

    2014-12-01

    The presence of endotoxins in preparations of recombinantly produced therapeutic proteins poses serious problems for patients. Endotoxins can cause fever, respiratory distress syndromes, intravascular coagulation, or endotoxic shock. A number of methods have been devised to remove endotoxins from protein preparations using separation procedures based on molecular mass or charge properties. Most of the methods are limited in their endotoxin removal capacities and lack general applicability. We are describing a biotechnological approach for endotoxin removal. This strategy exploits the observation that endotoxins form micelles that expose negative charges on their surface, leading to preferential binding of endotoxins to cationic surfaces, allowing the separation from their resident protein. Endotoxins exhibit high affinity to stretches of histidines, which are widely used tools to facilitate the purification of recombinant proteins. They bind to nickel ions and are the basis for protein purification from cellular extracts by immobilized metal affinity chromatography. We show that the thrombin-mediated cleavage of two histidine tags from the purified recombinant protein and the adsorption of these histidine tags and their associated endotoxins to a nickel affinity column result in an appreciable depletion of the endotoxins in the purified protein fraction.

  10. Automated recombinant protein expression screening in Escherichia coli.

    PubMed

    Busso, Didier; Stierlé, Matthieu; Thierry, Jean-Claude; Moras, Dino

    2008-01-01

    To fit the requirements of structural genomics programs, new as well as classical methods have been adapted to automation. This chapter describes the automated procedure developed within the Structural Biology and Genomics Platform, Strasbourg for performing recombinant protein expression screening in Escherichia coli. The procedure consists of parallel competent cells transformation, cell plating, and liquid culture inoculation, implemented for up to 96 samples at a time.

  11. Purification of recombinant nacre-associated mineralization protein AP7 fused with maltose-binding protein.

    PubMed

    Huang, Yu-Chieh; Chang, Hsun-Hui; Mou, Yun; Chi, Peter; Chan, Jerry Chun Chung; Luo, Shih-Chi

    2014-08-01

    Formation of biominerals often involves specific proteins that modulate the process of matrix assembly, nucleation, and crystal growth. AP7 is an aragonite-associated protein of 7 kDa and is intrinsically disordered. The structural disorder of AP7 makes it very difficult to express in Escherchiacoli. In this work, we report the first successful expression and purification of recombinant AP7 using the maltose-binding protein (MBP) fusion approach. We obtain a high-yield production of recombinant MBP-AP7 protein inE. coli (∼60 mg/L). We also establish an efficient protocol to remove the MBP fusion protein by Factor Xa, followed by purification using size-exclusion chromatography. Characterization of the recombinant AP7 protein has been carried out using MALDI-TOF, peptide mass fingerprinting, and circular dichroism (CD). The mass data confirm that the purified recombinant protein is AP7. The CD data suggest that the recombinant AP7 protein exists as partially disordered structure at neutral pH. The calcium carbonate precipitation assay shows that both MBP-AP7 and AP7 exhibit morphological modification on calcite crystallites. The co-precipitation of MBP-tagged AP7 derivatives and calcium carbonate generate different types of AP7 composite calcite and vaterite crystals. This system should be helpful to establish a model for understanding the structure/function relationship between the protein and inorganic mineral interaction.

  12. Arabinosylation of recombinant human immunoglobulin-based protein therapeutics.

    PubMed

    Hossler, Patrick; Chumsae, Christopher; Racicot, Christopher; Ouellette, David; Ibraghimov, Alexander; Serna, Daniel; Mora, Alessandro; McDermott, Sean; Labkovsky, Boris; Scesney, Susanne; Grinnell, Christine; Preston, Gregory; Bose, Sahana; Carrillo, Ralf

    Protein glycosylation is arguably the paramount post-translational modification on recombinant glycoproteins, and highly cited in the literature for affecting the physiochemical properties and the efficacy of recombinant glycoprotein therapeutics. Glycosylation of human immunoglobulins follows a reasonably well-understood metabolic pathway, which gives rise to a diverse range of asparagine-linked (N-linked), or serine/threonine-linked (O-linked) glycans. In N-linked glycans, fucose levels have been shown to have an inverse relationship with the degree of antibody-dependent cell-mediated cytotoxicity, and high mannose levels have been implicated in potentially increasing immunogenicity and contributing to less favorable pharmacokinetic profiles. Here, we demonstrate a novel approach to potentially reduce the presence of high-mannose species in recombinant human immunoglobulin preparations, as well as facilitate an approximate 100% replacement of fucosylation with arabinosylation in Chinese hamster ovary cell culture through media supplementation with D-arabinose, an uncommonly used mammalian cell culture sugar substrate. The replacement of fucose with arabinose was very effective and practical to implement, since no cell line engineering or cellular adaptation strategies were required. Arabinosylated recombinant IgGs and the accompanying reduction in high mannose glycans, facilitated a reduction in dendritic cell uptake, increased FcγRIIIa signaling, and significantly increased the levels of ADCC. These aforementioned effects were without any adverse changes to various structural or functional attributes of multiple recombinant human antibodies and a bispecific DVD-Ig. Protein arabinosylation represents an expansion of the N-glycan code in mammalian expressed glycoproteins.

  13. 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.

  14. Generation of polyclonal antibodies against recombinant human glucocerebrosidase produced in Escherichia coli.

    PubMed

    Novo, Juliana Branco; Oliveira, Maria Leonor Sarno; Magalhães, Geraldo Santana; Morganti, Ligia; Raw, Isaías; Ho, Paulo Lee

    2010-11-01

    Deficiency of the lysosomal glucocerebrosidase (GCR) enzyme results in Gaucher's disease, the most common inherited storage disorder. Treatment consists of enzyme replacement therapy by the administration of recombinant GCR produced in Chinese hamster ovary cells. The production of anti-GCR antibodies has already been described with placenta-derived human GCR that requires successive chromatographic procedures. Here, we report a practical and efficient method to obtain anti-GCR polyclonal antibodies against recombinant GCR produced in Escherichia coli and further purified by a single step through nickel affinity chromatography. The purified GCR was used to immunize BALB/c mice and the induction of anti-GCR antibodies was evaluated by enzyme-linked immunosorbent assay. The specificity of the antiserum was also evaluated by western blot analysis against recombinant GCR produced by COS-7 cells or against endogenous GCR of human cell lines. GCR was strongly recognized by the produced antibodies, either as cell-associated or as secreted forms. The detected molecular masses of 59-66 kDa are in accordance to the expected size for glycosylated GCR. The GCR produced in E. coli would facilitate the production of polyclonal (shown here) and monoclonal antibodies and their use in the characterization of new biosimilar recombinant GCRs coming in the near future.

  15. A novel method for increasing the expression level of recombinant proteins.

    PubMed

    Wang, Aijun; Clapper, Jonathan; Guderian, Jeffery A; Foy, Teresa M; Fanger, Gary R; Retter, Marc W; Skeiky, Yasir A W

    2003-07-01

    Expression of recombinant proteins is an important step towards elucidating the functions of many genes discovered through genomic sequencing projects. It is also critical for validating gene targets and for developing effective therapies for many diseases. Here we describe a novel method to express recombinant proteins that are extremely difficult to produce otherwise. The increased protein expression level is achieved by using a fusion partner, MTB32-C, which is the carboxyl terminal fragment of the Mycobacterium tuberculosis antigen, MTB32 (Rv0125). By fusing MTB32-C to the N-termini of target genes, we have demonstrated significant enhancement of recombinant protein expression level in Escherichia coli. The inclusion of a 6xHis tag and the 128-amino acid of MTB32-C will add 13.5 kDa to the fusion molecule. Comparison of the mRNA levels of the fusion and non-fusion proteins indicated that the increased fusion protein expression may be regulated at translational or post-translational steps. There are many potential applications for the generated fusion proteins. For example, MTB32-C fusion proteins have been used successfully as immunogens to generate both polyclonal and monoclonal antibodies. These antibodies have been used to characterize cellular localization of the proteins and to validate gene targets at protein level. In addition, these antibodies may be useful in diagnostic and therapeutic applications for many diseases. If desired, the MTB32-C portion in the fusion protein can be removed after protein expression, making it possible to study protein structure and function as well as to screen for potential drugs. Thus, this novel fusion expression system has become a powerful tool for many applications.

  16. Recombination Proteins Mediate Meiotic Spatial Chromosome Organization and Pairing

    PubMed Central

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

    2010-01-01

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

  17. Characterization and biological activities of recombinant human plasminogen kringle 1-3 produced in Escherichia coli.

    PubMed

    You, Weon-Kyoo; So, Seung-Ho; Sohn, Young-Doug; Lee, Hyosil; Park, Doo-Hong; Chung, Soo-Il; Chung, Kwang-Hoe

    2004-07-01

    Angiogenesis, the formation of new capillaries from preexisting blood vessels, is involved in many pathological conditions, for example, tumorigenesis, diabetic retinopathy, and rheumatoid arthritis. Angiostatin, which contains the kringle 1-4 domains of plasminogen, is known to be a potent inhibitor of angiogenesis and a strong suppressor of various solid tumors. In this study, we expressed recombinant protein containing the kringle 1-3 domains of human plasminogen in Escherichia coli and investigated its biological activities. The protein was successfully refolded from inclusion bodies and purified at a 30% overall yield, as a single peak by HPLC. The purified recombinant protein had biochemical properties that were similar to those of the native form, which included molecular size, lysine-binding capacity, and immunoreactivity with a specific antibody. The recombinant protein was also found to strongly inhibit the proliferation of bovine capillary endothelial cells in vitro, and the formation of new capillaries on chick embryos. In addition, it suppressed the growth of primary Lewis lung carcinoma and B16 melanoma in an in vivo mouse model. Our findings suggest that the recombinant kringle 1-3 domains in a prokaryote expression system have anti-angiogenic activities, which may be useful in clinical and basic research in the field of angiogenesis.

  18. Enhancing recombinant protein quality and yield by protein stability profiling.

    PubMed

    Mezzasalma, Tara M; Kranz, James K; Chan, Winnie; Struble, Geoffrey T; Schalk-Hihi, Céline; Deckman, Ingrid C; Springer, Barry A; Todd, Matthew J

    2007-04-01

    The reliable production of large amounts of stable, high-quality proteins is a major challenge facing pharmaceutical protein biochemists, necessary for fulfilling demands from structural biology, for high-throughput screening, and for assay purposes throughout early discovery. One strategy for bypassing purification challenges in problematic systems is to engineer multiple forms of a particular protein to optimize expression, purification, and stability, often resulting in a nonphysiological sub-domain. An alternative strategy is to alter process conditions to maximize wild-type construct stability, based on a specific protein stability profile (PSP). ThermoFluor, a miniaturized 384-well thermal stability assay, has been implemented as a means of monitoring solution-dependent changes in protein stability, complementing the protein engineering and purification processes. A systematic analysis of pH, buffer or salt identity and concentration, biological metals, surfactants, and common excipients in terms of an effect on protein stability rapidly identifies conditions that might be used (or avoided) during protein production. Two PSPs are presented for the kinase catalytic domains of Akt-3 and cFMS, in which information derived from a ThermoFluor PSP led to an altered purification strategy, improving the yield and quality of the protein using the primary sequences of the catalytic domains.

  19. 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

  20. 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.

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

    PubMed

    Gupta, Sanjeev K; Shukla, Pratyoosh

    2016-12-01

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

  2. Variability in the Immunodetection of His-tagged Recombinant Proteins

    PubMed Central

    Debeljak, Nataša; Feldman, Laurie; Davis, Kerry L.; Komel, Radovan; Sytkowski, Arthur J.

    2006-01-01

    Labeling of recombinant proteins with polypeptide fusion partners, or affinity tagging, is a useful method to facilitate subsequent protein purification and detection. Poly-histidine tags (His-tags) are among the most commonly used affinity tags. We report strikingly variable immunodetection of two His-tagged recombinant human erythropoietins (Epo), wild type Epo (Epowt) and Epo containing an R103A mutation (EpoR103A). Both were engineered to contain a C-terminal six residue His-tag. The cDNA constructs were stably transfected into CHO cells and COS-7 cells. Clones from the CHO cell transfections were selected for further characterization and larger-scale protein expression. Three chromatographic steps were utilized to achieve pharmacologically pure Epo. Conditioned media from the Epo-expressing cell lines and protein-containing samples from each step of purification were analyzed by SDS-PAGE and dot blot, using both monoclonal anti-human Epo antibody (AE7A5) and anti-His antibodies. While the successful incorporation of the His-tag into our constructs was confirmed by Epo binding to Ni2+-NTA resin and by μLC/MS/MS amino acid sequencing, the levels of immunodetection of His-tagged protein varied markedly depending on the particular anti His-tag antibody used. Such variability in His-tag immunorecognition can lead to critical adverse effects on several analytical methods. PMID:17081490

  3. A glycosylated recombinant human granulocyte colony stimulating factor produced in a novel protein production system (AVI-014) in healthy subjects: a first-in human, single dose, controlled study.

    PubMed

    Varki, Roslyn; Pequignot, Ed; Leavitt, Mark C; Ferber, Andres; Kraft, Walter K

    2009-01-28

    AVI-014 is an egg white-derived, recombinant, human granulocyte colony-stimulating factor (G-CSF). This healthy volunteer study is the first human investigation of AVI-014. 24 male and female subjects received a single subcutaneous injection of AVI-014 at 4 or 8 mcg/kg. 16 control subjects received 4 or 8 mcg/kg of filgrastim (Neupogen, Amgen) in a partially blinded, parallel fashion. The Geometric Mean Ratio (GMR) (90% CI) of 4 mcg/kg AVI-014/filgrastim AUC(0-72 hr) was 1.00 (0.76, 1.31) and Cmax was 0.86 (0.66, 1.13). At the 8 mcg/kg dose, the AUC(0-72) GMR was 0.89 (0.69, 1.14) and Cmax was 0.76 (0.58, 0.98). A priori pharmacokinetic bioequivalence was defined as the 90% CI of the GMR bounded by 0.8-1.25. Both the white blood cell and absolute neutrophil count area under the % increase curve AUC(0-9 days) and Cmax (maximal % increase from baseline)GMR at 4 and 8 mcg/kg fell within the 0.5-2.0 a priori bound set for pharmacodynamic bioequivalence. The CD 34+ % increase curve AUC(0-9 days) and Cmax GMR for both doses was approximately 1, but 90% confidence intervals were large due to inherent variance, and this measure did not meet pharmacodynamic bioequivalence. AVI-014 demonstrated a side effect profile similar to that of filgrastim. AVI-014 has safety, pharmacokinetic, and pharmacodynamic properties comparable to filgrastim at an equal dose in healthy volunteers. These findings support further investigation in AVI-014.

  4. A glycosylated recombinant human granulocyte colony stimulating factor produced in a novel protein production system (AVI-014) in healthy subjects: a first-in human, single dose, controlled study

    PubMed Central

    Varki, Roslyn; Pequignot, Ed; Leavitt, Mark C; Ferber, Andres; Kraft, Walter K

    2009-01-01

    Background AVI-014 is an egg white-derived, recombinant, human granulocyte colony-stimulating factor (G-CSF). This healthy volunteer study is the first human investigation of AVI-014. Methods 24 male and female subjects received a single subcutaneous injection of AVI-014 at 4 or 8 mcg/kg. 16 control subjects received 4 or 8 mcg/kg of filgrastim (Neupogen, Amgen) in a partially blinded, parallel fashion. Results The Geometric Mean Ratio (GMR) (90% CI) of 4 mcg/kg AVI-014/filgrastim AUC(0–72 hr) was 1.00 (0.76, 1.31) and Cmax was 0.86 (0.66, 1.13). At the 8 mcg/kg dose, the AUC(0–72) GMR was 0.89 (0.69, 1.14) and Cmax was 0.76 (0.58, 0.98). A priori pharmacokinetic bioequivalence was defined as the 90% CI of the GMR bounded by 0.8–1.25. Both the white blood cell and absolute neutrophil count area under the % increase curve AUC(0–9 days) and Cmax (maximal % increase from baseline)GMR at 4 and 8 mcg/kg fell within the 0.5–2.0 a priori bound set for pharmacodynamic bioequivalence. The CD 34+ % increase curve AUC(0–9 days) and Cmax GMR for both doses was ~1, but 90% confidence intervals were large due to inherent variance, and this measure did not meet pharmacodynamic bioequivalence. AVI-014 demonstrated a side effect profile similar to that of filgrastim. Conclusion AVI-014 has safety, pharmacokinetic, and pharmacodynamic properties comparable to filgrastim at an equal dose in healthy volunteers. These findings support further investigation in AVI-014. PMID:19175929

  5. Fermentation and downstream process for high yield production of Plasmodium falciparum recombinant HRP II protein and its application in diagnosis.

    PubMed

    Singh, Anil K; Athmaram, T N; Shrivastava, Saurabh; Merwyn, S; Agarwal, G S; Gopalan, N

    2013-07-01

    Malaria represents the world's greatest public health problem in terms of number of people affected, levels of morbidity and mortality in tropical and subtropical countries. Malaria parasites are members of the Apicomplexa, family of Plasmodiidae. Histidine-rich protein-II secreted by Plasmodium falciparum is known to be a compelling marker in malaria diagnosis and follow-up. In our present study, we have optimized the batch fermentation and downstream process for large scale production of recombinant P. falciparum HRP-II 62 kDa protein for diagnostic application. The culture broth was effectively induced with IPTG twice at different time intervals to sustain induction for a long period. Batch fermentation resulted in a wet weight of 61.34 g/L and dry cell biomass 12.81 g/L. With the improved downstream process, purified recombinant protein had a yield of 304.60 mg/L. The authenticity of the purified recombinant protein was confirmed via western blotting using indigenously developed HRP-II specific monoclonal antibodies and known positive human clinical sera samples. Further, the reactivity of recombinant HRP-II protein was validated using commercially available immuno chromatographic strips. Indirect ELISA using recombinant purified protein recognized the P. falciparum specific antibodies in suspected human sera samples. Our results clearly suggest that the recombinant HRP-II protein produced via batch fermentation has immense potential for routine diagnostic application.

  6. Recombinant bacterial expression and purification of human fragile X mental retardation protein isoform 1.

    PubMed

    Evans, Timothy L; Mihailescu, Mihaela-Rita

    2010-12-01

    The loss of expression of the fragile X mental retardation protein (FMRP) leads to fragile X syndrome. FMRP has two types of RNA binding domains, two K-homology domains and an arginine-glycine-glycine box domain, and it is proposed to act as a translation regulator of specific messenger RNA. The interest to produce sufficient quantities of pure recombinant FMRP for biochemical and biophysical studies is high. However, the recombinant bacterial expression of FMRP has had limited success, and subsequent recombinant eukaryotic and in vitro expression has also resulted in limited success. In addition, the in vitro and eukaryotic expression systems may produce FMRP which is posttranslationally modified, as phosphorylation and arginine methylation have been shown to occur on FMRP. In this study, we have successfully isolated the conditions for recombinant expression, purification and long-term storage of FMRP using Escherichia coli, with a high yield. The expression of FMRP using E. coli renders the protein devoid of the posttranslational modifications of phosphorylation and arginine methylation, allowing the study of the direct effects of these modifications individually and simultaneously. In order to assure that FMRP retained activity throughout the process, we used fluorescence spectroscopy to assay the binding activity of the FMRP arginine-glycine-glycine box for the semaphorin 3F mRNA and confirmed that FMRP remained active.

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

    PubMed

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

    2005-01-01

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

  8. 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

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

    PubMed

    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.

  10. Serotype specificity of recombinant fusion protein containing domain III and capsid protein of dengue virus 2.

    PubMed

    Izquierdo, Alienys; Valdés, Iris; Gil, Lázaro; Hermida, Lisset; Gutiérrez, Sheila; García, Angélica; Bernardo, Lidice; Pavón, Alekis; Guillén, Gerardo; Guzmán, María G

    2012-07-01

    Recombinant fusion protein containing domain III of the dengue envelope protein fused to capsid protein from dengue 2 virus was immunogenic and conferred protection in mice against lethal challenge in previously report. Here, the antigenic specificity of this recombinant protein using anti-dengue antibodies from mice and humans and the cross-reactive humoral and cellular response induced in immunized mice were evaluated. The homologous anti-dengue antibodies showed a higher reactivity to the recombinant protein compared to the wide cross-reactivity observed for viral antigen as determined by ELISA. The IgG anti-dengue and functional antibodies, induced by the recombinant proteins in mice, were highly serotype specific by ELISA, hemaglutination inhibition and plaque reduction neutralizing tests. Accordingly, the cellular immune response determined by the IFNγ and TNFα secretion, was serotype specific. The specificity of serotype associated to this recombinant protein in addition to its high antigenicity, immunogenicity and protecting capacity suggest its advantage as a possible functional and safe vaccine candidate against dengue in a future tetravalent formulation. Copyright © 2012 Elsevier B.V. All rights reserved.

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

    PubMed Central

    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

  12. 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.

  13. 2-Hydroxyglutarate produced by neomorphic IDH mutations suppresses homologous recombination and induces PARP inhibitor sensitivity

    PubMed Central

    Sulkowski, Parker L.; Corso, Christopher D.; Robinson, Nathaniel D.; Scanlon, Susan E.; Purshouse, Karin R.; Bai, Hanwen; Liu, Yanfeng; Sundaram, Ranjini K.; Hegan, Denise C.; Fons, Nathan R.; Breuer, Gregory A.; Song, Yuanbin; Mishra-Gorur, Ketu; De Feyter, Henk; de Graaf, Robin A.; Surovtseva, Yulia V.; Kachman, Maureen; Halene, Stephanie; Günel, Murat; Glazer, Peter M.; Bindra, Ranjit S.

    2017-01-01

    2-Hydroxyglutarate (2HG) exists as two enantiomers, (R)-2HG and (S)-2HG, and both are implicated in tumor progression via their inhibitory effects on α-ketoglutarate (αKG)-dependent dioxygenases. The former is an oncometabolite that is induced by the neomorphic activity conferred by isocitrate dehydrogenase-1 and -2 (IDH1/2) mutations, whereas the latter is produced under pathologic processes such as hypoxia. Here, we report that IDH1/2 mutations induce a homologous recombination (HR) defect that renders tumor cells exquisitely sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors. This “BRCAness” phenotype of IDH mutant cells can be completely reversed by treatment with small molecule inhibitors of the mutant IDH1 enzyme, and, conversely, it can be entirely recapitulated by treatment with either 2HG enantiomer alone in cells with intact IDH1/2 proteins. We demonstrate IDH1-dependent PARP inhibitor sensitivity in a range of clinically relevant models, including primary patient-derived glioma cells in culture and genetically matched tumor xenografts in vivo. These findings provide the basis for a possible therapeutic strategy exploiting the biological consequences of mutant IDH, rather than attempting to block 2HG production, by targeting the 2HG-dependent HR-deficiency with PARP inhibition. Furthermore, our results uncover an unexpected link between oncometabolites, altered DNA repair, and genetic instability. PMID:28148839

  14. Cloning, expression, and purification of recombinant protein from a single synthetic multivalent construct of Mycobacterium tuberculosis.

    PubMed

    Fang, Chee-Mun; Zainuddin, Zainul F; Musa, Mustaffa; Thong, Kwai-Lin

    2006-06-01

    Tuberculosis remains a major infectious disease with over 8 million new cases and 2 million deaths annually. Therefore, a vaccine more potent than BCG is desperately needed. In this regard, an approximately 800 bp DNA encoding a mycobacterial synthetic gene designated as VacIII (containing ubiquitin gene UbGR and four immunogenic mycobacterial epitopes or genes of ESAT-6, Phos1, Hsp 16.3, and Mtb8.4) was sub-cloned into a bacterial expression vector of pRSET-B resulting in a 6 x His-VacIII fusion gene construction. This recombinant clone was over expressed in Escherichia coli BL-21 (DE-3). The expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in cell lysate. The inclusion bodies were solubilized with 8M urea and the recombinant protein was purified by Ni-NTA column and dialyzed by urea gradient dialysis. This method produced a relatively high yield of recombinant VacIII protein and the cloned VacIII gene offers the potential development of other vaccine formats such as DNA vaccine and recombinant vaccine.

  15. Metabolic engineering of recombinant protein secretion by Saccharomyces cerevisiae.

    PubMed

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

    2012-08-01

    The yeast Saccharomyces cerevisiae is a widely used cell factory for the production of fuels and chemicals, and it is also provides a platform for the production of many heterologous proteins of medical or industrial interest. Therefore, many studies have focused on metabolic engineering S. cerevisiae to improve the recombinant protein production, and with the development of systems biology, it is interesting to see how this approach can be applied both to gain further insight into protein production and secretion and to further engineer the cell for improved production of valuable proteins. In this review, the protein post-translational modification such as folding, trafficking, and secretion, steps that are traditionally studied in isolation will here be described in the context of the whole system of protein secretion. Furthermore, examples of engineering secretion pathways, high-throughput screening and systems biology applications of studying protein production and secretion are also given to show how the protein production can be improved by different approaches. The objective of the review is to describe individual biological processes in the context of the larger, complex protein synthesis network.

  16. Physical studies of conformational plasticity in a recombinant prion protein.

    PubMed

    Zhang, H; Stockel, J; Mehlhorn, I; Groth, D; Baldwin, M A; Prusiner, S B; James, T L; Cohen, F E

    1997-03-25

    PrP(Sc) is known to be the major, if not the only, component of the infectious prion. Limited proteolysis of PrP(Sc) produces an N-terminally truncated polypeptide of about 142 residues, designated PrP 27-30. Recently, a recombinant protein (rPrP) of 142 residues corresponding to the Syrian hamster PrP 27-30 was expressed in Escherichia coli and purified (Mehlhorn et al., 1996). rPrP has been refolded into both alpha-helical and beta-sheet structures as well as various intermediates in aqueous buffers. The beta-sheet state and two pH-dependent alpha-helical states were characterized by CD and NMR. The alpha-helical conformation occurred only after the formation of an intramolecular disulfide bond, whereas the beta-sheet form was accessible either with or without the disulfide. Of the different alpha-helical forms studied, only those refolded in the pH range 5-8 were substantially soluble at physiological pH, exhibiting similar conformations and monomeric analytical sedimentation profiles throughout the above pH range. Furthermore, refolded alpha-rPrP showed NMR chemical shift dispersion typical of proteins with native conformations, although 2D NMR indicated large segments of conformational flexibility. It displayed a cooperative thermal denaturation transition; at elevated temperatures, it converted rapidly and irreversibly to the thermodynamically more stable beta-sheet form. Unfolding of alpha-rPrP by GdnHCl revealed a two-phase transition with a relatively stable folding intermediate at 2 M GdnHCl. The deltaG values were estimated to be 1.9 +/- 0.4 kcal/mol for the first phase and 6.5 +/- 1.2 kcal/mol for the second, consistent with a folding core surrounded by significant segments of flexible conformation. By NMR, alpha-rPrP(acid) isolated at pH 2 without refolding exhibited heterogeneous line widths, consistent with an acid-denatured molten globular state. We conclude that to the extent that rPrP constitutes a relevant folding domain of PrP(C), the various

  17. Tailoring recombinant protein quality by rational media design.

    PubMed

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

    2015-01-01

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

  18. Comparison of recombinant protein expression in a baculovirus system in insect cells (Sf9) and silkworm.

    PubMed

    Usami, Akihiro; Ishiyama, Seiji; Enomoto, Chiaki; Okazaki, Hironobu; Higuchi, Keiko; Ikeda, Mashahiro; Yamamoto, Takeshi; Sugai, Mutsumi; Ishikawa, Yukiko; Hosaka, Yumiko; Koyama, Teruyuki; Tobita, Yoneko; Ebihara, Syoko; Mochizuki, Toshiko; Asano, Yoshimi; Nagaya, Hidekazu

    2011-02-01

    Using a hybrid baculovirus system, we compared the expression of 45 recombinant proteins from six categories using two models: silkworm (larvae and pupae) and an Sf9 cell line. A total of 45 proteins were successfully expressed; preparation of hybrid baculovirus was unsuccessful for one protein, and two proteins were not expressed. A similar pattern of expression was seen in both silkworm and Sf9 cells, with double and multiple bands found in immunoblotting of the precipitate of both hosts. Degraded proteins were seen only in the silkworm system (particularly in the larvae). Production was more efficient in silkworms; a single silkworm produced about 70 times more protein than 10(6) Sf9 cells in 2 ml of culture medium.

  19. Enhancing effect of a protein from Lonomia obliqua hemolymph on recombinant protein production

    PubMed Central

    Greco, Katia N.; Sousa, Alvaro P. B.; Moraes, Roberto H. P.; Astray, Renato M.; Pereira, Carlos A.

    2008-01-01

    Gene expression in animal cells allows large scale production of proteins used for either structure and function studies or therapeutic purposes. Maximizing recombinant protein production is necessary to optimize cell growth and protein expression. Some studies have demonstrated the presence of pharmacologically active substances in insect hemolymph. In this work, we have identified and purified a protein from Lonomia obliqua hemolymph able to increase the production of the rabies virus glycoprotein, expressed in Drosophila melanogaster S2 cells, by about 59%. PMID:19003176

  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. High-throughput process development for recombinant protein purification.

    PubMed

    Rege, Kaushal; Pepsin, Mike; Falcon, Brandy; Steele, Landon; Heng, Meng

    2006-03-05

    Methods development in chromatographic purification processes is a complex operation and has traditionally relied on trial and error approaches. The availability of a large number of commercial media, choice of different modes of chromatography, and diverse operating conditions contribute to the challenging task of accelerating methods development. In this paper, we describe a novel microtiter-plate based screening method to identify the appropriate sequence of chromatographic steps that result in high purities of bioproducts from their respective culture broths. Protein mixtures containing the bioproduct were loaded on aliquots of different chromatographic media in microtiter plates. Serial step elution of the proteins, in concert with bioproduct-specific assays, resulted in the identification of "active fractions" containing the bioproduct. The identification of a successful chromatographic step was based on the purity of the active fractions, which were then pooled and used as starting material for screening the next chromatographic dimension. This procedure was repeated across subsequent dimensions until single band purities of the protein were obtained. The sequence of chromatographic steps and the corresponding operating conditions identified from the screen were validated under scaled-up conditions. Various modes of chromatography including hydrophobic interaction, ion exchange (cation and anion exchange) and hydrophobic charge-induction chromatography (HCIC), and different operating conditions (pH, salt concentration and type, etc.) were employed in the screen. This approach was employed to determine the sequence of chromatographic steps for the purification of recombinant alpha-amylase from its cell-free culture broth. Recommendations from the screen resulted in single-band purity of the protein under scaled-up conditions. Similar results were observed for an scFv-beta-lactamase fusion protein. The use of a miniaturized screen enables the parallel

  2. Real time detection of anthrax spores using highly specific anti-EA1 recombinant antibodies produced by competitive panning.

    PubMed

    Love, Tracey E; Redmond, Caroline; Mayers, Carl N

    2008-05-20

    We describe a targeted approach for the production of biological recognition elements capable of fast, specific detection of anthrax spores on biosensor surfaces. The aim was to produce single chain antibodies (scFvs) to EA1, a Bacillus anthracis S-layer protein that is also present, although not identical, in related to Bacillus species. The aim of the work was to produce antibodies that would detect B. anthracis EA1 protein and intact spores with a high degree of specificity, but would not detect other Bacillus species. Existing monoclonal antibodies were evaluated and found to recognise B. anthracis EA1 and S-layer proteins from other closely related Bacillus species. Recombinant anti-EA1 scFvs were isolated from B. anthracis immune library that contained antibody genes raised against B. anthracis spores and purified exosporium. Two approaches for scFv selection were used; standard (non-competitive) panning, and competitive panning. The non-competitive biopanning strategy isolated scFvs that recognised EA1 from B. anthracis, but also cross-reacted with other Bacillus species. In contrast, the competitive panning approach used S-layer proteins from other Bacillus species to generate scFvs that were highly specific to B. anthracis EA1 and demonstrated apparent nanomolar binding affinities. Specific, real time detection of B. anthracis spores was demonstrated with these scFvs using an evanescent wave biosensor, the Resonant Mirror. The approach described can be used to generate specific antibodies to any desired target where homologous proteins also exist in closely related species, and demonstrates clear advantages to using recombinant technology to produce biological recognition elements for detection of biological threat agents.

  3. A Recombinant Fungal Chitin Deacetylase Produces Fully Defined Chitosan Oligomers with Novel Patterns of Acetylation

    PubMed Central

    Naqvi, Shoa; Cord-Landwehr, Stefan; Singh, Ratna; Bernard, Frank; Kolkenbrock, Stephan; El Gueddari, Nour Eddine

    2016-01-01

    . tritici in the periplasm of E. coli as a fusion protein with the maltose-binding protein; this strategy allows the production of these difficult-to-express enzymes in sufficient quantities for them to be characterized and optimized through protein engineering. Here, the recombinant enzyme was used to produce partially acetylated chitosan oligosaccharides from chitin oligomers, whereby the pronounced regioselectivity of the enzyme led to the production of defined products with novel patterns of acetylation. This approach widens the scope for both the production and functional analysis of chitosan oligomers and thus will eventually allow the detailed molecular structure-function relationships of biologically active chitosans to be studied, which is essential for developing applications for these functional biopolymers for a circular bioeconomy, e.g., in agriculture, medicine, cosmetics, and food sciences. PMID:27590819

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

    PubMed

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

    2017-07-27

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

  5. 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.

  6. Recombinant Human Peptidoglycan Recognition Proteins Reveal Antichlamydial Activity

    PubMed Central

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

    2016-01-01

    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

  7. The recombinant EHV-1 vector producing CDV hemagglutinin as potential vaccine against canine distemper.

    PubMed

    Pan, Zihao; Liu, Jin; Ma, Jiale; Jin, Qiuli; Yao, Huochun; Osterrieder, Nikolaus

    2017-10-01

    Canine distemper virus (CDV), is a pantropic agent of morbillivirus that causes fetal disease in dogs. Base on a broad host rang of CDV, the continued vaccines inoculation is unavoidable to pose gene recombination risk in vaccine virus and wild virus. The current study presents the construction of novel vectors, using equine herpesvirus type 1 (EHV-1) expressing the canine distemper virus (CDV). The recent field strain hemagglutinin protein and nucleoprotein were used for the construction of the viral vector vaccines. Based on the Bacterial artificial chromosome (BAC) genomes of EHV-1 RacH strain, the recombinant EHV-1 vaccine virus encoding CDV hemagglutinin protein (EHV-H) or CDV nucleoprotein (EHV-N) was constructed separately. The constructed BACs were rescued after 72 h post infection, and the expression of H or N in the recombinant viruses was confirmed by western-blotting. Furthermore, high levels of neutralizing antibodies were induced persistently following vaccination in the groups EHV-H&EHV-N and EHV-H, but the EHV-N group. The groups of vaccinated EHV-H and EHV-H&EHV-N pups were monitored for clinical signs, whereas the vaccinated EHV-N group developed moderate symptoms. The present study demonstrated that EHV-1 based recombinant virus carrying CDV H could be a promising vaccine candidate against canine distemper. Copyright © 2017. Published by Elsevier Ltd.

  8. 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

  9. 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.

  10. Quantification of metabolic limitations during recombinant protein production in Escherichia coli.

    PubMed

    Heyland, Jan; Blank, Lars M; Schmid, Andreas

    2011-09-10

    Escherichia coli is one of the major microorganisms for recombinant protein production because it has been best characterized in terms of molecular genetics and physiology, and because of the availability of various expression vectors and strains. The synthesis of proteins is one of the most energy consuming processes in the cell, with the result that cellular energy supply may become critical. Indeed, the so called metabolic burden of recombinant protein synthesis was reported to cause alterations in the operation of the host's central carbon metabolism. To quantify these alterations in E. coli metabolism in dependence of the rate of recombinant protein production, (13)C-tracer-based metabolic flux analysis in differently induced cultures was used. To avoid dilution of the (13)C-tracer signal by the culture history, the recombinant protein produced was used as a flux probe, i.e., as a read out of intracellular flux distributions. In detail, an increase in the generation rate rising from 36 mmol(ATP)g(CDW)(-1)h(-1) for the reference strain to 45 mmol(ATP)g(CDW)(-1)h(-1) for the highest yielding strain was observed during batch cultivation. Notably, the flux through the TCA cycle was rather constant at 2.5±0.1 mmol g(CDW)(-1)h(-1), hence was independent of the induced strength for gene expression. E. coli compensated for the additional energy demand of recombinant protein synthesis by reducing the biomass formation to almost 60%, resulting in excess NADPH. Speculative, this excess NADPH was converted to NADH via the soluble transhydrogenase and subsequently used for ATP generation in the electron transport chain. In this study, the metabolic burden was quantified by the biomass yield on ATP, which constantly decreased from 11.7g(CDW)mmol(ATP)(-1) for the reference strain to 4.9g(CDW)mmol(ATP)(-1) for the highest yielding strain. The insights into the operation of the metabolism of E. coli during recombinant protein production might guide the optimization of

  11. Regulatory Elements in Vectors for Efficient Generation of Cell Lines Producing Target Proteins

    PubMed Central

    Maksimenko, O.; Gasanov, N. B.; Georgiev, P.

    2015-01-01

    To date, there has been an increasing number of drugs produced in mammalian cell cultures. In order to enhance the expression level and stability of target recombinant proteins in cell cultures, various regulatory elements with poorly studied mechanisms of action are used. In this review, we summarize and discuss the potential mechanisms of action of such regulatory elements. PMID:26483956

  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. A recombinant envelope protein from Dengue virus purified by IMAC is bioequivalent with its immune-affinity chromatography purified counterpart.

    PubMed

    Hermida, L; Rodríguez, R; Lazo, L; López, C; Márquez, G; Páez, R; Suárez, C; Espinosa, R; García, J; Guzmán, G; Guillén, G

    2002-03-28

    Semi-purified DEN-4 envelope protein, obtained in Pichia pastoris, was capable of generating neutralising and protecting antibodies after immunisation in mice. Here we compared two purification processes of this recombinant protein using two chromatographic steps: immune-affinity chromatography and immobilised metal ion adsorption chromatography (IMAC). The protein purified by both methods produced functional antibodies reflected by titres of haemagglutination inhibition and neutralisation. IMAC could be used as an alternative for high scale purification.

  14. The recombinant globular head domain of the measles virus hemagglutinin protein as a subunit vaccine against measles.

    PubMed

    Lobanova, Liubov M; Eng, Nelson F; Satkunarajah, Malathy; Mutwiri, George K; Rini, James M; Zakhartchouk, Alexander N

    2012-04-26

    Despite the availability of live attenuated measles virus (MV) vaccines, a large number of measles-associated deaths occur among infants in developing countries. The development of a measles subunit vaccine may circumvent the limitations associated with the current live attenuated vaccines and eventually contribute to global measles eradication. Therefore, the goal of this study was to test the feasibility of producing the recombinant globular head domain of the MV hemagglutinin (H) protein by stably transfected human cells and to examine the ability of this recombinant protein to elicit MV-specific immune responses. The recombinant protein was purified from the culture supernatant of stably transfected HEK293T cells secreting a tagged version of the protein. Two subcutaneous immunizations with the purified recombinant protein alone resulted in the production of MV-specific serum IgG and neutralizing antibodies in mice. Formulation of the protein with adjuvants (polyphosphazene or alum) further enhanced the humoral immune response and in addition resulted in the induction of cell-mediated immunity as measured by the production of MV H-specific interferon gamma (IFN-γ) and interleukin 5 (IL-5) by in vitro re-stimulated splenocytes. Furthermore, the inclusion of polyphosphazene into the vaccine formulation induced a mixed Th1/Th2-type immune response. In addition, the purified recombinant protein retained its immunogenicity even after storage at 37°C for 2 weeks.

  15. [Refolding of the fusion protein of recombinant enterokinase light chain rEKL].

    PubMed

    Yi, Jin-Hua; Zhang, Yuan-Xing

    2006-09-01

    The fusion protein of enterokinase light chain, DsbA-rEKL, was expressed mainly in inclusion body in E. coli. The recombinant bacteria was fermented to high density, with high expression of the fusion protein. After being washed with 0.5% Triton X-100 and 4mol/L urea, the inclusion body was dissolved in 6mol/L guanidine and 100mmol/L DTP, derivatized by cystine and refolded by pulse refolding. The strategy of pulse refolding involved the addition of 0.03mg/mL of fusion protein until its final concentration reached 0.3mg/mL. The refolded protein was autocleaved and the active EKL molecule was released after adding 2mmol/L CaCl2. Using the two-step purification processes of IDA-Sepharose chromatography and Q-Sepharose chromatography, the purity of rEKL was found to be above 95%, with a high activity to cleave the recombinant reteplase fusion protein Trx-rPA. The yield of purified rEKL was more than 60mg/L of cultures. As a result, the therapeutic proteins like rPA could be produced on a large-scale in a way such as expressed in the form of fusion proteins.

  16. Multi-bit biomemory consisting of recombinant protein variants, azurin.

    PubMed

    Yagati, Ajay Kumar; Kim, Sang-Uk; Min, Junhong; Choi, Jeong-Woo

    2009-01-01

    In this study a protein-based multi-bit biomemory device consisting of recombinant azurin with its cysteine residue modified by site-directed mutagenesis method has been developed. The recombinant azurin was directly immobilized on four different gold (Au) electrodes patterned on a single silicon substrate. Using cyclic voltammetry (CV), chronoamperometry (CA) and open circuit potential amperometry (OCPA) methods the memory function of the fabricated biodevice was validated. The charge transfer occurs between protein molecules and Au electrode enables a bi-stable electrical conductivity allowing the system to be used as a digital memory device. Data storage is achieved by applying redox potentials which are within the range of 200mV. Oxidation and open circuit potentials with current sensing were used for writing and reading operations respectively. Applying oxidation potentials in different combinations to each Au electrodes, multi-bit information was stored in to the azurin molecules. Finally, the switching robustness and reliability of the proposed device has been examined. The results suggest that the proposed device has a function of memory and can be used for the construction of nano-scale multi-bit information storage device.

  17. Purification of IFT particle proteins and preparation of recombinant proteins for structural and functional analysis.

    PubMed

    Behal, Robert H; Betleja, Ewelina; Cole, Douglas G

    2009-01-01

    Intraflagellar transport (IFT) is characterized by a robust bidirectional movement of large proteinaceous particles along the length of eukaryotic cilia and flagella. Essential for the assembly and function of the organelle, IFT is believed to transport a large array of ciliary components in and out of the organelle. Biochemical analysis of the proteins involved with this transport has been largely dependent on the ability to isolate suitable quantities of intact cilia or flagella. One model organism, Chlamydomonas reinhardtii, has proven to be especially well-suited for such endeavors. Indeed, many of the IFT particle proteins were initially identified through biochemical analysis of green algae. This chapter describes some of the most effective methods for the purification of IFT particle proteins from Chlamydomonas flagella. This chapter also describes complementary approaches where recombinant IFT proteins are generated with affinity tags that allow rapid and specific purification. The recombinant proteins can be used to analyze protein-protein interactions and can be directly delivered to mutant cells to analyze functional domains. Although the techniques described here are focused entirely on Chlamydomonas IFT proteins, the approaches, especially regarding recombinant proteins, should be applicable to the study of IFT machinery in other model organisms.

  18. 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

  19. Recombinant probes for visualizing endogenous synaptic proteins in living neurons

    PubMed Central

    Gross, Garrett G.; Junge, Jason A.; Mora, Rudy J.; Kwon, Hyung-Bae; Olson, C. Anders; Takahashi, Terry T.; Liman, Emily R.; Ellis-Davies, Graham C.R.; McGee, Aaron W.; Sabatini, Bernardo L.; Roberts, Richard W.; Arnold, Don B.

    2013-01-01

    Summary The ability to visualize endogenous proteins in living neurons provides a powerful means to interrogate neuronal structure and function. Here we generate recombinant antibody-like proteins, termed FingRs (Fibronectin intrabodies generated with mRNA display), that bind endogenous neuronal proteins PSD-95 and Gephyrin with high affinity and which, when fused to GFP, allow excitatory and inhibitory synapses to be visualized in living neurons. Design of the FingR incorporates a novel transcriptional regulation system that ties FingR expression to the level of the target and reduces background fluorescence. In dissociated neurons and brain slices FingRs generated against PSD-95 and Gephyrin did not affect the expression patterns of their endogenous target proteins or the number or strength of synapses. Together, our data indicate that PSD-95 and Gephyrin FingRs can report the localization and amount of endogenous synaptic proteins in living neurons and thus may be used to study changes in synaptic strength in vivo. PMID:23791193

  20. 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

  1. Phosphorylation of chloramphenicol by a recombinant protein Yhr2 from Streptomyces avermitilis MA4680.

    PubMed

    Rajesh, Thangamani; Sung, Changmin; Kim, Hyeonjeong; Song, Eunjung; Park, Hyung-Yeon; Jeon, Jong-Min; Yoo, Dongwon; Kim, Hyun Joong; Kim, Yong Hyun; Choi, Kwon-Young; Song, Kyung-Guen; Yang, Yung-Hun

    2013-06-15

    Although phosphorylation of chloramphenicol has been shown to occur in the chloramphenicol producer, Streptomyces venezuelae, there are no reports on the existence of chloramphenicol phosphorylase in other Streptomyces species. In the present study, we report the modification of chloramphenicol by a recombinant protein, designated as Yhr2 (encoded by SAV_877), from Streptomyces avermitilis MA4680. Recombinant Yhr2 was expressed in Escherichia coli BL21 (DE3) and the cells expressing this recombinant protein were shown to phosphorylate chloramphenicol to a 3'-O-phosphoryl ester derivative, resulting in an inactivated form of the antibiotic. Expression of yhr2 conferred chloramphenicol resistance to E. coli cells up to 25 μg/mL and in an in vitro reaction, adenosine triphosphate (ATP), guanosine triphosphate (GTP), adenosine diphosphate (ADP) and guanosine diphosphate (GDP) were shown to be the phosphate donors for phosphorylation of chloramphenicol. This study highlights that antibiotic resistance conferring genes could be easily expressed and functionalized in other organisms that do not produce the respective antibiotic.

  2. Transient expression and cellular localization of recombinant proteins in cultured insect cells

    USDA-ARS?s Scientific Manuscript database

    Heterologous protein expression systems are used for production of recombinant proteins, interpretation of cellular trafficking/localization, and for the determination of biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for ...

  3. Dissecting limiting factors of the Protein synthesis Using Recombinant Elements (PURE) system.

    PubMed

    Li, Jun; Zhang, Chi; Huang, Poyi; Kuru, Erkin; Forster-Benson, Eliot T C; Li, Taibo; Church, George M

    2017-01-01

    Reconstituted cell-free protein synthesis systems such as the Protein synthesis Using Recombinant Elements (PURE) system give high-throughput and controlled access to in vitro protein synthesis. Here we show that compared with the commercial S30 crude extract based RTS 100 E. coli HY system, the PURE system has less mRNA degradation and produces up to ∼6-fold full-length proteins. However the majority of polypeptides PURE produces are partially translated or inactive since the signal from firefly luciferase (Fluc) translated in PURE is only ∼2/3(rd) of that measured using the RTS 100 E. coli HY S30 system. Both of the 2 batch systems suffer from low ribosome recycling efficiency when translating proteins from 82 kD to 224 kD. A systematic fed-batch analysis of PURE shows replenishment of 6 small molecule substrates individually or in combination before energy depletion increased Fluc protein yield by ∼1.5 to ∼2-fold, while creatine phosphate and magnesium have synergistic effects when added to the PURE system. Additionally, while adding EF-P to PURE reduced full-length protein translated, it increased the fraction of functional protein and reduced partially translated protein probably by slowing down the translation process. Finally, ArfA, rather than YaeJ or PrfH, helped reduce ribosome stalling when translating Fluc and improved system productivity in a template-dependent fashion.

  4. Dissecting limiting factors of the Protein synthesis Using Recombinant Elements (PURE) system

    DOE PAGES

    Li, Jun; Zhang, Chi; Huang, Poyi; ...

    2017-05-09

    Reconstituted cell-free protein synthesis systems such as the Protein synthesis Using Recombinant Elements (PURE) system give high-throughput and controlled access to in vitro protein synthesis. Here we show that compared with the commercial S30 crude extract based RTS 100 E. coli HY system, the PURE system has less mRNA degradation and produces up to ~6-fold full-length proteins. However the majority of polypeptides PURE produces are partially translated or inactive since the signal from firefly luciferase (Fluc) translated in PURE is only ~2/3rd of that measured using the RTS 100 E. coli HY S30 system. Both of the 2 batch systemsmore » suffer from low ribosome recycling efficiency when translating proteins from 82 kD to 224 kD. A systematic fed-batch analysis of PURE shows replenishment of 6 small molecule substrates individually or in combination before energy depletion increased Fluc protein yield by ~1.5 to ~2-fold, while creatine phosphate and magnesium have synergistic effects when added to the PURE system. Additionally, while adding EF-P to PURE reduced full-length protein translated, it increased the fraction of functional protein and reduced partially translated protein probably by slowing down the translation process. Finally, ArfA, rather than YaeJ or PrfH, helped reduce ribosome stalling when translating Fluc and improved system productivity in a template-dependent fashion.« less

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

    PubMed

    Cheng, Chung-Hsien; Lee, Wen-Chien

    2010-08-28

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

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

    PubMed Central

    2010-01-01

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

  7. Transcriptome analysis of Corynebacterium glutamicum in the process of recombinant protein expression in bioreactors

    PubMed Central

    Sun, Yang; Guo, Wenwen; Wang, Fen; Zhan, Chunjun; Yang, Yankun; Liu, Xiuxia; Bai, Zhonghu

    2017-01-01

    Corynebacterium glutamicum (C. glutamicum) is a favorable host cell for the production of recombinant proteins, such as important enzymes and pharmaceutical proteins, due to its excellent potential advantages. Herein, we sought to systematically explore the influence of recombinant protein expression on the transcription and metabolism of C. glutamicum. Two C. glutamicum strains, the wild-type strain and an engineered strain expressing enhanced green fluorescent protein (EGFP), were cultured in parallel in 5-L bioreactors to study the change in metabolism in the process of EGFP expression. The results revealed that EGFP expression had great effects on the growth and metabolism of C. glutamicum and contributed to metabolism-like anaerobic conditions as follows: glycolysis was enhanced, the TCA cycle was shunted, and Glu, Val, Met, lactate and acetate were accumulated to produce sufficient ATP for EGFP production and transfer. Many differentially expressed genes related to ribosomal protein, transcriptional regulators, and energy metabolism were found to be expressed in the presence of EGFP, laying the foundation for identifying genomic loci to change the flow of the host cell metabolism to improve the ability of expressing foreign proteins in C. glutamicum. PMID:28369109

  8. Inflammatory response of endothelial cells to hepatitis C virus recombinant envelope glycoprotein 2 protein exposure

    PubMed Central

    Urbaczek, Ana Carolina; Ribeiro, Lívia Carolina de Abreu; Ximenes, Valdecir Farias; Afonso, Ana; Nogueira, Camila Tita; Generoso, Wesley Cardoso; Alberice, Juliana Vieira; Rudnicki, Martina; Ferrer, Renila; da Fonseca, Luiz Marcos; da Costa, Paulo Inácio

    2014-01-01

    The hepatitis C virus (HCV) encodes approximately 10 different structural and non-structural proteins, including the envelope glycoprotein 2 (E2). HCV proteins, especially the envelope proteins, bind to cell receptors and can damage tissues. Endothelial inflammation is the most important determinant of fibrosis progression and, consequently, cirrhosis. The aim of this study was to evaluate and compare the inflammatory response of endothelial cells to two recombinant forms of the HCV E2 protein produced in different expression systems (Escherichia coli and Pichia pastoris). We observed the induction of cell death and the production of nitric oxide, hydrogen peroxide, interleukin-8 and vascular endothelial growth factor A in human umbilical vein endothelial cells (HUVECs) stimulated by the two recombinant E2 proteins. The E2-induced apoptosis of HUVECs was confirmed using the molecular marker PARP. The apoptosis rescue observed when the antioxidant N-acetylcysteine was used suggests that reactive oxygen species are involved in E2-induced apoptosis. We propose that these proteins are involved in the chronic inflammation caused by HCV. PMID:25317702

  9. Inflammatory response of endothelial cells to hepatitis C virus recombinant envelope glycoprotein 2 protein exposure.

    PubMed

    Urbaczek, Ana Carolina; Ribeiro, Lívia Carolina de Abreu; Ximenes, Valdecir Farias; Afonso, Ana; Nogueira, Camila Tita; Generoso, Wesley Cardoso; Alberice, Juliana Vieira; Rudnicki, Martina; Ferrer, Renila; Fonseca, Luiz Marcos da; Costa, Paulo Inácio da

    2014-09-01

    The hepatitis C virus (HCV) encodes approximately 10 different structural and non-structural proteins, including the envelope glycoprotein 2 (E2). HCV proteins, especially the envelope proteins, bind to cell receptors and can damage tissues. Endothelial inflammation is the most important determinant of fibrosis progression and, consequently, cirrhosis. The aim of this study was to evaluate and compare the inflammatory response of endothelial cells to two recombinant forms of the HCV E2 protein produced in different expression systems (Escherichia coli and Pichia pastoris). We observed the induction of cell death and the production of nitric oxide, hydrogen peroxide, interleukin-8 and vascular endothelial growth factor A in human umbilical vein endothelial cells (HUVECs) stimulated by the two recombinant E2 proteins. The E2-induced apoptosis of HUVECs was confirmed using the molecular marker PARP. The apoptosis rescue observed when the antioxidant N-acetylcysteine was used suggests that reactive oxygen species are involved in E2-induced apoptosis. We propose that these proteins are involved in the chronic inflammation caused by HCV.

  10. Transduction of human recombinant proteins into mitochondria as a protein therapeutic approach for mitochondrial disorders.

    PubMed

    Papadopoulou, Lefkothea C; Tsiftsoglou, Asterios S

    2011-11-01

    Protein therapy is considered an alternative approach to gene therapy for treatment of genetic-metabolic disorders. Human protein therapeutics (PTs), developed via recombinant DNA technology and used for the treatment of these illnesses, act upon membrane-bound receptors to achieve their pharmacological response. On the contrary, proteins that normally act inside the cells cannot be developed as PTs in the conventional way, since they are not able to "cross" the plasma membrane. Furthermore, in mitochondrial disorders, attributed either to depleted or malfunctioned mitochondrial proteins, PTs should also have to reach the subcellular mitochondria to exert their therapeutic potential. Nowadays, there is no effective therapy for mitochondrial disorders. The development of PTs, however, via the Protein Transduction Domain (PTD) technology offered new opportunities for the deliberate delivery of human recombinant proteins inside eukaryotic subcellular organelles. To this end, mitochondrial disorders could be clinically encountered with the delivery of human mitochondrial proteins (engineered via recombinant DNA and PTD technologies) at specific intramitochondrial sites to exert their function. Overall, PTD-mediated Protein Replacement Therapy emerges as a suitable model system for the therapeutic approach for mitochondrial disorders.

  11. 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.

  12. 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

  13. Purification of recombinant C-reactive protein mutants

    PubMed Central

    Thirumalai, Avinash; Singh, Sanjay K.; Hammond, David J.; Gang, Toh B.; Ngwa, Donald N.; Pathak, Asmita; Agrawal, Alok

    2017-01-01

    C-reactive protein (CRP) is an evolutionarily conserved protein, a component of the innate immune system, and an acute phase protein in humans. In addition to its raised level in blood in inflammatory states, CRP is also localized at sites of inflammation including atherosclerotic lesions, arthritic joints and amyloid plaque deposits. Results of in vivo experiments in animal models of inflammatory diseases indicate that CRP is an anti-pneumococcal, anti-atherosclerotic, anti-arthritic and an anti-amyloidogenic molecule. The mechanisms through which CRP functions in inflammatory diseases are not fully defined; however, the ligand recognition function of CRP in its native and non-native pentameric structural conformations and the complement-activating ability of ligand-complexed CRP have been suggested to play a role. One tool to understand the structure-function relationships of CRP and determine the contributions of the recognition and effector functions of CRP in host defense is to employ site-directed mutagenesis to create mutants for experimentation. For example, CRP mutants incapable of binding to phosphocholine are generated to investigate the importance of the phosphocholine-binding property of CRP in mediating host defense. Recombinant CRP mutants can be expressed in mammalian cells and, if expressed, can be purified from the cell culture media. While the methods to purify wild-type CRP are well established, different purification strategies are needed to purify various mutant forms of CRP if the mutant does not bind to either calcium or phosphocholine. In this article, we report the methods used to purify pentameric recombinant wild-type and mutant CRP expressed in and secreted by mammalian cells. PMID:1460031

  14. Production of recombinant protein by a novel oxygen-induced system in Escherichia coli.

    PubMed

    Baez, Antonino; Majdalani, Nadim; Shiloach, Joseph

    2014-04-07

    The SoxRS regulon of E. coli is activated in response to elevated dissolved oxygen concentration likely to protect the bacteria from possible oxygen damage. The soxS expression can be increased up to 16 fold, making it a possible candidate for recombinant protein expression. Compared with the existing induction approaches, oxygen induction is advantageous because it does not involve addition or depletion of growth factors or nutrients, addition of chemical inducers or temperature changes that can affect growth and metabolism of the producing bacteria. It also does not affect the composition of the growth medium simplifying the recovery and purification processes. The soxS promoter was cloned into the commercial pGFPmut3.1 plasmid creating pAB49, an expression vector that can be induced by increasing oxygen concentration. The efficiency and the regulatory properties of the soxS promoter were characterized by measuring the GFP expression when the culture dissolved oxygen concentration was increased from 30% to 300% air saturation. The expression level of recombinant GFP was proportional to the oxygen concentration, demonstrating that pAB49 is a controllable expression vector. A possible harmful effect of elevated oxygen concentration on the recombinant product was found to be negligible by determining the protein-carbonyl content and its specific fluorescence. By performing high density growth in modified LB medium, the cells were induced by increasing the oxygen concentration. After 3 hours at 300% air saturation, GFP fluorescence reached 109000 FU (494 mg of GFP/L), representing 3.4% of total protein, and the cell concentration reached 29.1 g/L (DW). Induction of recombinant protein expression by increasing the dissolved oxygen concentration was found to be a simple and efficient alternative expression strategy that excludes the use of chemical, nutrient or thermal inducers that have a potential negative effect on cell growth or the product recovery.

  15. BRCT-domain protein BRIT1 influences class switch recombination.

    PubMed

    Yen, Wei-Feng; Chaudhry, Ashutosh; Vaidyanathan, Bharat; Yewdell, William T; Pucella, Joseph N; Sharma, Rahul; Liang, Yulong; Li, Kaiyi; Rudensky, Alexander Y; Chaudhuri, Jayanta

    2017-07-19

    DNA double-strand breaks (DSBs) serve as obligatory intermediates for Ig heavy chain (Igh) class switch recombination (CSR). The mechanisms by which DSBs are resolved to promote long-range DNA end-joining while suppressing genomic instability inherently associated with DSBs are yet to be fully elucidated. Here, we use a targeted short-hairpin RNA screen in a B-cell lymphoma line to identify the BRCT-domain protein BRIT1 as an effector of CSR. We show that conditional genetic deletion of BRIT1 in mice leads to a marked increase in unrepaired Igh breaks and a significant reduction in CSR in ex vivo activated splenic B cells. We find that the C-terminal tandem BRCT domains of BRIT1 facilitate its interaction with phosphorylated H2AX and that BRIT1 is recruited to the Igh locus in an activation-induced cytidine deaminase (AID) and H2AX-dependent fashion. Finally, we demonstrate that depletion of another BRCT-domain protein, MDC1, in BRIT1-deleted B cells increases the severity of CSR defect over what is observed upon loss of either protein alone. Our results identify BRIT1 as a factor in CSR and demonstrate that multiple BRCT-domain proteins contribute to optimal resolution of AID-induced DSBs.

  16. Molecular responses of Escherichia coli caused by heat stress and recombinant protein production during temperature induction.

    PubMed

    Valdez-Cruz, Norma A; Ramírez, Octavio T; Trujillo-Roldán, Mauricio A

    2011-01-01

    In a recent review, we discussed the extensively used temperature-inducible expression system, based on the pL and/or pR phage lambda promoters that are finely regulated by the thermo-labile cI857 repressor. In this system, an increase in temperature induces the heterologous protein production and activates the heat shock response, as well as the stringent and SOS responses. The same responses are activated just by the overproduction of recombinant protein. All such responses result in a metabolic burden to the cells, a decrease in the specific growth rate, and alterations in the central carbon metabolism. Altogether, these effects can alter the quantity and quality of the produced foreign protein. Here, we compare and discuss the transcription of selected genes, and the concomitant synthesis of heat-shock proteins (hsp) soon after thermal induction, in relation to the responses that occur in other expression systems that also trigger the heat-shock response.

  17. Electron-ion recombination in laser-produced plasmas using optical interferometry

    NASA Astrophysics Data System (ADS)

    Heilmann, Nathan; Peatross, Justin; Bergeson, Scott

    2011-10-01

    We are developing methods to measure electron-ion recombination in laser-produced plasmas. A high intensity fs laser pulse is focused into a gas jet and forms a plasma. A weaker probe beam first passes through a slightly mis-aligned Michelson interferometer and is also focused into the plasma. The probe ``beam'' is actually two temporally coincident but spatially offset laser beams. One of the laser beams passes through the plasma and the other does not. These beams expand and produce interference fringes in the far field, similar to a Young's double slit experiment. The spatial position of these fringes depends on the differential phase shift in the two probe beams. This differential shift is due to the electron density in the plasma, which is probed by only one beam. By measuring the fringe shift as a function of time after the plasma is formed, we should be able to measure the time-evolving electron density. At sufficiently high densities, three-body recombination will become important. In that regime, the measured recombination rate can be used to determine the electron temperature.

  18. The transgenic rabbit as model for human diseases and as a source of biologically active recombinant proteins.

    PubMed

    Bosze, Zs; Hiripi, L; Carnwath, J W; Niemann, H

    2003-10-01

    Until recently, transgenic rabbits were produced exclusively by pronuclear microinjection which results in additive random insertional transgenesis; however, progress in somatic cell cloning based on nuclear transfer will soon make it possible to produce rabbits with modifications to specific genes by the combination of homologous recombination and subsequent prescreening of nuclear donor cells. Transgenic rabbits have been found to be excellent animal models for inherited and acquired human diseases including hypertrophic cardiomyopathy, perturbed lipoprotein metabolism and atherosclerosis. Transgenic rabbits have also proved to be suitable bioreactors for the production of recombinant protein both on an experimental and a commercial scale. This review summarizes recent research based on the transgenic rabbit model.

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

    PubMed Central

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

    2011-01-01

    Thaumatin, an intensely sweet-tasting plant protein, elicits a sweet taste at a concentration of 50 nM. The crystal structure of a recombinant form of thaumatin I produced in the yeast Pichia pastoris has been determined to a resolution of 1.1 Å. The model was refined with anisotropic B parameters and riding H atoms. A comparison of the diffraction data and refinement statistics for recombinant thaumatin I with those for plant thaumatin I revealed no significant differences in the diffraction data. The R values for recombinant thaumatin I and plant thaumatin I (F o > 4σ) were 9.11% and 9.91%, respectively, indicating the final model to be of good quality. Notably, the electron-density maps around Asn46 and Ser63, which differ between thaumatin variants, were significantly improved. Furthermore, a number of H atoms became visible in an OMIT map and could be assigned. The high-quality structure of recombinant thaumatin with H atoms should provide details about sweetness determinants in thaumatin and provide valuable insights into the mechanism of its interaction with taste receptors. PMID:21636903

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

    PubMed

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

    2011-06-01

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

  1. 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.

  2. Conformational features of tau fibrils from Alzheimer’s disease brain are faithfully propagated by unmodified recombinant protein

    PubMed Central

    Morozova, Olga A.; March, Zachary M.; Robinson, Anne S.; Colby, David W.

    2014-01-01

    Fibrils composed of tau protein are a pathological hallmark of several neurodegenerative disorders including Alzheimer’s disease (AD). Here we show that when recombinant tau protein is seeded with paired helical filaments (PHFs) isolated from AD brain, the amyloid formed shares many of the structural features of AD PHFs. In contrast, tau amyloids formed with heparin as an inducing agent—a common biochemical model of tau misfolding—are structurally distinct from brain-derived PHFs. Using ultrastructural analysis by electron microscopy, circular dichroism, and chemical denaturation, we found that AD seeded recombinant tau fibrils were not significantly different than tau fibrils isolated from AD brain tissue. Tau fibrils produced by incubating recombinant tau with heparin had significantly narrower fibrils with a longer periodicity, higher chemical stability, and distinct secondary structure compared to AD PHFs. The addition of heparin to the reaction of recombinant tau and AD PHFs also corrupted the templating process, resulting in a mixture of fibril conformations. Our results suggest that AD-isolated PHFs act as a conformational template for the formation of recombinant tau fibrils. Therefore, the use of AD PHFs as seeds to stimulate recombinant tau amyloid formation produces synthetic tau fibers that that closely resemble those associated with AD pathology and provides a biochemical model of tau misfolding that may be of improved utility for structural studies and drug screening. These results also demonstrate that posttranslational modifications such as phosphorylation are not a prerequisite for the propagation of the tau fibril conformation found in AD. PMID:24033133

  3. Production of cocktail of polyclonal antibodies using bacterial expressed recombinant protein for multiple virus detection.

    PubMed

    Kapoor, Reetika; Mandal, Bikash; Paul, Prabir Kumar; Chigurupati, Phaneendra; Jain, Rakesh Kumar

    2014-02-01

    Cocktail of polyclonal antibodies (PAb) were produced that will help in multiple virus detection and overcome the limitation of individual virus purification, protein expression and purification as well as immunization in multiple rabbits. A dual fusion construct was developed using conserved coat protein (CP) sequences of Cucumber mosaic virus (CMV) and Papaya ringspot virus (PRSV) in an expression vector, pET-28a(+). The fusion protein (∼40kDa) was expressed in Escherichia coli and purified. Likewise, a triple fusion construct was developed by fusing conserved CP sequences of CMV and PRSV with conserved nucleocapsid protein (N) sequence of Groundnut bud necrosis virus (GBNV) and expressed as a fusion protein (∼50kDa) in pET-28a(+). PAb made separately to each of these three viruses recognized the double and triple fusion proteins in Western blot indicating retention of desired epitopes for binding with target antibodies. The fusion proteins (∼40kDa and ∼50kDa) were used to produce cocktail of PAb by immunizing rabbits, which simultaneously detected natural infection of CMV and PRSV or CMV, PRSV and GBNV in Cucurbitaceous, Solanaceous and other hosts in DAC-ELISA. This is the first report on production of a cocktail of PAb to recombinant fusion protein of two or three distinct viruses.

  4. CXCR4(+)-targeted protein nanoparticles produced in the food-grade bacterium Lactococcus lactis.

    PubMed

    Cano-Garrido, Olivia; Céspedes, María Virtudes; Unzueta, Ugutz; Saccardo, Paolo; Roldán, Mònica; Sánchez-Chardi, Alejandro; Cubarsi, Rafael; Vázquez, Esther; Mangues, Ramon; García-Fruitós, Elena; Villaverde, Antonio

    2016-09-01

    Lactococcus lactis is a Gram-positive (endotoxin-free) food-grade bacteria exploited as alternative to Escherichia coli for recombinant protein production. We have explored here for the first time the ability of this platform as producer of complex, self-assembling protein materials. Biophysical properties, cell penetrability and in vivo biodistribution upon systemic administration of tumor-targeted protein nanoparticles produced in L. lactis have been compared with the equivalent material produced in E. coli. Protein nanoparticles have been efficiently produced in L. lactis, showing the desired size, internalization properties and biodistribution. In vitro and in vivo data confirm the potential and robustness of the production platform, pointing out L. lactis as a fascinating cell factory for the biofabrication of protein materials intended for therapeutic applications.

  5. Factors that contribute to the immmunogenicity of therapeutic recombinant human proteins.

    PubMed

    Mukovozov, Ilya; Sabljic, Thomas; Hortelano, Gonzalo; Ofosu, Frederick A

    2008-05-01

    Use of recombinant human proteins has revolutionized medicine by providing over 200 highly purified hormones and proteins that effectively treat many inherited and acquired peptide hormone and protein deficiencies. With the exception of therapeutic monoclonal antibodies, these biological medicines are synthesized by cultured cells using DNA sequences that would yield proteins with identical amino acid sequences as endogenous human proteins. Therefore, there was the broad expectation that recombinant human biological medicines would be non-immunogenic in patients capable of synthesizing even sub-optimal levels of these therapeutic proteins to which they are innately tolerant. However, the widespread clinical use of recombinant human proteins has demonstrated that nearly all of them are immunogenic. This observation suggests that factors additional to differences in amino acid sequences of endogenous and biotherapeutic proteins contribute to the immunogenicity of therapeutic proteins. The main aim of this review is to summarize some of the factors that are known to contribute to the immunogenicity of recombinant therapeutic proteins.

  6. 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.

  7. 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

  8. Recombinant mussel proximal thread matrix protein promotes osteoblast cell adhesion and proliferation.

    PubMed

    Yoo, Hee Young; Song, Young Hoon; Foo, Mathias; Seo, Eunseok; Hwang, Dong Soo; Seo, Jeong Hyun

    2016-02-16

    von Willebrand factor (VWF) is a key load bearing domain for mamalian cell adhesion by binding various macromolecular ligands in extracellular matrix such as, collagens, elastin, and glycosaminoglycans. Interestingly, vWF like domains are also commonly found in load bearing systems of marine organisms such as in underwater adhesive of mussel and sea star, and nacre of marine abalone, and play a critical load bearing function. Recently, Proximal Thread Matrix Protein1 (PTMP1) in mussel composed of two vWF type A like domains has characterized and it is known to bind both mussel collagens and mammalian collagens. Here, we cloned and mass produced a recombinant PTMP1 from E. coli system after switching all the minor codons to the major codons of E. coli. Recombinant PTMP1 has an ability to enhance mouse osteoblast cell adhesion, spreading, and cell proliferation. In addition, PTMP1 showed vWF-like properties as promoting collagen expression as well as binding to collagen type I, subsequently enhanced cell viability. Consequently, we found that recombinant PTMP1 acts as a vWF domain by mediating cell adhesion, spreading, proliferation, and formation of actin cytoskeleton. This study suggests that both mammalian cell adhesion and marine underwater adhesion exploits a strong vWF-collagen interaction for successful wet adhesion. In addition, vWF like domains containing proteins including PTMP1 have a great potential for tissue engineering and the development of biomedical adhesives as a component for extra-cellular matrix.

  9. Recombinant antibodies specific for the Plasmodium falciparum histidine-rich protein 2

    PubMed Central

    Ravaoarisoa, Elisabeth; Zamanka, Halima; Fusai, Thierry; Bellalou, Jacques; Bedouelle, Hugues; Mercereau-Puijalon, Odile

    2010-01-01

    Early diagnosis and appropriate treatment are key elements of malaria control programs in endemic areas. A major step forward in recent years has been the production and use of rapid diagnostic tests (RDTs) in settings where microscopy is impracticable. Many current RDTs target the Plasmodium falciparum histidine-rich protein 2 (PfHRP2) released in the plasma of infected individuals. These RDTs have had an indisputably positive effect on malaria management, but still present several limitations, including the poor characterization of the commercial monoclonal antibodies (mAbs) used for PfHRP2 detection, variable sensitivity and specificity and high costs. RDT use is further limited by impaired stability caused by temperature fluctuations during transport and uncontrolled storage in field-based facilities. To circumvent such drawbacks, an alternative could be the development of well-characterized, stabilized recombinant antibodies, with high binding affinity and specificity. Here, we report the characterization of the cDNA sequences encoding the Fab fragment of F1110 and F1546, two novel anti-PfHRP2 mAbs. FabF1546 was produced in the Escherichia coli periplasm. Its properties of binding to the parasite and to a recombinant PfHRP-2 antigen were similar to those of the parental mAb. As the affinity and stability of recombinant antibodies can be improved by protein engineering, our results open a novel approach for the development of an improved RDT for malaria diagnosis. PMID:20581462

  10. A structural requirement of zinc for the folding of recombinant link protein.

    PubMed

    Varelas, J B; Roy, C; Hering, T M

    1997-11-01

    We have cloned and ligated a full-length bovine link protein (LP) in the pMAL-c2 vector and overexpressed it in fusion with maltose-binding protein (MBP) in Escherichia coli. We have demonstrated dose-dependent binding of MBP/LP to biotinylated hyaluronan in a dot blot assay. A greater percentage of the expressed fusion protein was soluble, monomeric, and undegraded when the growth temperature was lowered, the growth medium was supplemented with zinc, and metal chelators were omitted from the lysis buffers. Similar effects were observed when we tested the effects of lower growth temperature and zinc supplementation on another construct consisting of MBP in fusion with the first proteoglycan tandem repeat of LP. Our results suggest zinc may be necessary for the folding and disulfide bond formation of recombinant LP. In addition, a greater amount of monomeric MBP/LP produced at 27 degrees C with zinc supplementation bound to biotinylated hyaluronic acid-binding region of aggrecan than MBP/LP produced at 27 or 37 degrees C without zinc. This suggests that recombinant LP may have a conformational requirement for zinc necessary for binding to aggrecan. Factor Xa cleavage of MBP/LP expressed in the presence of zinc yielded much more intact LP product than cleavage of MBP/LP expressed without zinc. These data indicate a structural role of zinc that allows MBP/LP to fold in a manner such that it is resistant to proteolytic degradation. Copyright 1997 Academic Press.

  11. A Recombinant Human Anti-Platelet scFv Antibody Produced in Pichia pastoris for Atheroma Targeting

    PubMed Central

    Vallet-Courbin, Amelie; Larivière, Mélusine; Hocquellet, Agnès; Hemadou, Audrey; Parimala, Sarjapura-Nagaraja; Laroche-Traineau, Jeanny; Santarelli, Xavier; Clofent-Sanchez, Gisèle; Jacobin-Valat, Marie-Josée; Noubhani, Abdelmajid

    2017-01-01

    Cells of the innate and adaptive immune system are key factors in the progression of atherosclerotic plaque, leading to plaque instability and rupture, potentially resulting in acute atherothrombotic events such as coronary artery disease, cerebrovascular disease and peripheral arterial disease. Here, we describe the cloning, expression, purification, and immunoreactivity assessment of a recombinant single-chain variable fragment (scFv) derived from a human anti-αIIbβ3 antibody (HuAb) selected to target atheromatous lesions for the presence of platelets. Indeed, platelets within atheroma plaques have been shown to play a role in inflammation, in platelet-leucocyte aggregates and in thrombi formation and might thus be considered relevant biomarkers of atherosclerotic progression. The DNA sequence that encodes the anti-αIIbβ3 TEG4 scFv previously obtained from a phage-display selection on activated platelets, was inserted into the eukaryote vector (pPICZαA) in fusion with a tag sequence encoding 2 cysteines useable for specific probes grafting experiments. The recombinant protein was expressed at high yields in Pichia pastoris (30 mg/L culture). The advantage of P. pastoris as an expression system is the production and secretion of recombinant proteins in the supernatant, ruling out the difficulties encountered when scFv are produced in the cytoplasm of bacteria (low yield, low solubility and reduced affinity). The improved conditions allowed for the recovery of highly purified and biologically active scFv fragments ready to be grafted in a site-directed way to nanoparticles for the imaging of atherosclerotic plaques involving inflammatory processes and thus at high risk of instability. PMID:28125612

  12. Purification and Refolding to Amyloid Fibrils of (His)6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli.

    PubMed

    Li, Qiaojing; Richard, Charles-Adrien; Moudjou, Mohammed; Vidic, Jasmina

    2015-12-19

    The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein belonging to the prion family. A chromatographic method for the purification of (His)6-tagged recombinant Shadoo expressed as inclusion bodies is described. The inclusion bodies are solubilized in 8 M urea and bound to a Ni(2+)-charged column to perform ion affinity chromatography. Bound proteins are eluted by a gradient of imidazole. Fractions containing Shadoo protein are subjected to size exclusion chromatography to obtain a highly purified protein. In the final step purified Shadoo is desalted to remove salts, urea and imidazole. Recombinant Shadoo protein is an important reagent for biophysical and biochemical studies of protein conformation disorders occurring in prion diseases. Many reports demonstrated that prion neurodegenerative diseases originate from the deposition of stable, ordered amyloid fibrils. Sample protocols describing how to fibrillate Shadoo into amyloid fibrils at acidic and neutral/basic pHs are presented. The methods on how to produce and fibrillate Shadoo can facilitate research in laboratories working on prion diseases, since it allows for production of large amounts of protein in a rapid and low cost manner.

  13. 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

  14. Triatoma virus recombinant VP4 protein induces membrane permeability through dynamic pores.

    PubMed

    Sánchez-Eugenia, Rubén; Goikolea, Julen; Gil-Cartón, David; Sánchez-Magraner, Lissete; Guérin, Diego M A

    2015-04-01

    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. 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 permeabilization in

  15. 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

  16. Functional characterization of recombinant major envelope protein (rB2L) of orf virus.

    PubMed

    Yogisharadhya, Revanaiah; Kumar, Amit; Ramappa, Raghavendra; Venkatesan, Gnanavel; Bhanuprakash, Veerakyathappa; Shivachandra, Sathish Bhadravati

    2017-04-01

    Orf, or contagious ecthyma, a highly contagious transboundary disease of sheep and goats, is caused by a double-stranded DNA virus (ORFV) belonging to the genus Parapoxvirus of the family Poxviridae. The ORFV genome encodes the major envelope proteins B2L and F1L, which have been found to be highly immunogenic and have multiple functional characteristics. In order to investigate the functional properties of the B2L protein, in this study, the B2L gene of ORFV strain 59/05, encoding recombinant mature B2L (aa 1M-D334), was produced as a fusion protein in Escherichia coli. The functional characteristics of purified rB2L fusion protein (~60 kDa) were evaluated in vivo and in vitro, showing that this protein had lipase and immunomodulatory activities. Immunization trials involving laboratory animals (mice, rabbits and guinea pigs) using either constant or graded doses of rB2L fusion protein with or without adjuvants (FCA, alum) as well as co-administration with candidate rErns-Ag protein of classical swine fever virus (CSFV) indicated that the rB2L protein is immunogenic and has immunomodulatory properties. This study shows the potential utility of the rB2L protein as a safe and novel adjuvant in veterinary vaccine formulations.

  17. 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.

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

    PubMed

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

    2016-11-01

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

  19. Recombinant capripoxvirus expressing the hemagglutinin protein gene of rinderpest virus: protection of cattle against rinderpest and lumpy skin disease viruses.

    PubMed

    Romero, C H; Barrett, T; Chamberlain, R W; Kitching, R P; Fleming, M; Black, D N

    1994-10-01

    A cDNA clone containing the complete coding sequence of the hemagglutinin (H) protein gene of the RBOK vaccine strain of rinderpest virus, under the control of the vaccinia late promoter p11, was inserted by homologous recombination into the thymidine kinase gene of the KS-1 strain of capripoxvirus. The recombinant virus produced authentic H protein as judged by its electrophoretic mobility, transport to the cell surface of infected lamb testis cells, and reactivity with monoclonal antibodies specific for the H protein of rinderpest virus. The recombinant virus induced significant levels of rinderpest virus neutralizing antibodies in vaccinated cattle and protected them from clinical rinderpest after challenge with a lethal dose of a highly virulent heterologous strain of the virus. Protection was achieved using vaccine doses lower than those used with a similar recombinant expressing the fusion protein gene of rinderpest. The parental KS-1 virus is widely used as a vaccine against capripox viruses and so the rinderpest recombinant acts as a dual vaccine to protect cattle against both rinderpest and lumpy skin disease.

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

    PubMed

    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.

  1. Production and purification of two recombinant proteins from transgenic corn.

    PubMed

    Kusnadi, A R; Hood, E E; Witcher, D R; Howard, J A; Nikolov, Z L

    1998-01-01

    This study reports the production, purification, and characterization of recombinant Escherichia coli beta-glucuronidase (GUS) and chicken egg-white avidin from transgenic corn seed. The avidin and gus genes were stably integrated in the genome and expressed over seven generations. The accumulation levels of avidin and GUS in corn kernel were 5.7% and 0.7% of extractable protein, respectively. Within the kernel, avidin and GUS accumulation was mainly localized to the germ, indicating possible tissue preference of the ubiquitin promoter. The storage-stability studies demonstrated that processed transgenic seed containing GUS or avidin can be stored at 10 degrees C for at least 3 months and at 25 degrees C for up to 2 weeks without a significant loss of activity. The heat-stability experiments indicated that GUS and avidin in the whole kernels were stable at 50 degrees C for up to 1 week. The buffer composition also had an affect on the aqueous extraction of avidin and GUS from ground kernels. Avidin was purified in one step by using 2-iminobiotin agarose, whereas GUS was purified in four steps consisting of adsorption, ion-exchange, hydrophobic interaction, and size-exclusion chromatography. Biochemical properties of purified avidin and GUS were similar to those of the respective native proteins.

  2. Transgenic birds for the production of recombinant proteins.

    PubMed

    Kamihira, Masamichi; Nishijima, Ken-ichi; Iijima, Shinji

    2004-01-01

    Transgenic birds were expected to be an excellent transgenic bioreactor for the production of recombinant pharmaceutical proteins. However, the only successful transgenic bioreactors have been based on mammals. We have developed two key techniques for obtaining transgenic birds. For bird embryo culture, we identified that the low rate of hatchability of cultured embryos is caused by limited oxygen and calcium availability. In quail embryo culture using a chicken eggshell as a culture vessel, hatchability increased to 80% by the supplement of calcium lactate in addition to oxygen aeration. A fully artificial vessel for quail embryo culture using a gas-permeable Teflon membrane was also designed. Although the hatchability was lower than that of cultures using a surrogate eggshell, we succeeded in hatching of bird embryos using a fully artificial vessel. For transgene introduction, a replication-defective pantropic retroviral vector based on Moloney murine leukemia virus (MoMLV) pseudotyped with vesicular stomatitis virus G protein (VSV-G) was injected to laid embryos at the blastodermal stage, and the embryos were hatched in vitro to generate G0 birds. The viral vector sequence was detected in the tissues of all G0 birds. The germ-line transmission efficiency was more than 80%. Plural copies of the transgene were inserted into the genome of G1 transgenic progeny.

  3. Rapid purification of recombinant proteins fused to chicken avidin.

    PubMed

    Airenne, K J; Kulomaa, M S

    1995-12-29

    A novel expression vector (pAVEX16C) has been constructed that directs the synthesis of desired polypeptides as fusions with the C terminus of chicken egg-white avidin (Avd). With this and a commercial GST gene (encoding glutathione S-transferase) fusion vector (pGEX-3X, Pharmacia), we produced Avd as fusions C- and N-terminally linked to GST in Escherichia coli. By using the Avd tail and a simple affinity purification protocol, including biotin-agarose, we were able to obtain 1-2 micrograms/ml of highly purified Avd::GST and GST::Avd from crude bacterial lysates. The produced proteins were, to a great extent, in soluble fraction when the cells were grown at 22 degrees C and disrupted with a detergent, N-laurylsarcosine. The fusion proteins could also be affinity-purified with the GST tail using glutathione-Sepharose 4B, but the yield of GST::Avd was significantly lower than when using the Avd tail. Our results therefore indicate that it is possible to produce, in E. coli, biologically active fusion proteins consisting of Avd C- or N-terminally linked with the desired protein which then can easily be purified by a simple affinity chromatography procedure.

  4. 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

  5. Effect of recombinant Lactococcus lactis producing myelin peptides on neuroimmunological changes in rats with experimental allergic encephalomyelitis.

    PubMed

    Kasarełło, K; Szczepankowska, A; Kwiatkowska-Patzer, B; Lipkowski, A W; Gadamski, R; Sulejczak, D; Łachwa, M; Biały, M; Bardowski, J

    2016-01-01

    Multiple sclerosis (MS) is a human autoimmune neurodegenerative disease with an unknown etiology. Despite various therapies, there is no effective cure for MS. Since the mechanism of the disease is based on autoreactive T-cell responses directed against myelin antigens, oral tolerance is a promising approach for the MS treatment. Here, the experiments were performed to assess the impact of oral administration of recombinant Lactococcus lactis producing encephalogenic fragments of three myelin proteins: myelin basic protein, proteolipid protein, and myelin oligodendrocyte glycoprotein, on neuroimmunological changes in rats with experimental allergic encephalomyelitis (EAE) - an animal model of MS. Lactococcus lactis whole-cell lysates were administered intragastrically at two doses (103 and 106 colony forming units) in a twenty-fold feeding regimen to Lewis rats with EAE. Spinal cord slices were subjected to histopathological analysis and morphometric evaluation, and serum levels of cytokines (IL-1b, IL-10, TNF-α and IFN-γ) were measured. Results showed that administration of the L. lactis preparations at the tested doses to rats with EAE, diminished the histopathological changes observed in EAE rats and reduced the levels of serum IL-1b, IL-10 and TNF-α, previously increased by evoking EAE. This suggests that oral delivery of L. lactis producing myelin peptide fragments could be an alternative strategy to induce oral tolerance for the treatment of MS.

  6. High-level expression and purification of recombinant human growth hormone produced in soluble form in Escherichia coli.

    PubMed

    Levarski, Zdenko; Šoltýsová, Andrea; Krahulec, Ján; Stuchlík, Stanislav; Turňa, Ján

    2014-08-01

    Human growth hormone (hGH) was one of the first recombinant proteins approved for the treatment of human growth disorders. Its small size (191 amino acids), possession of only 2 disulphide bonds and absence of posttranslational modifications make Escherichia coli the host of choice for its production on any scale. In this work, we have utilized an efficient T7 based expression system to produce high levels of soluble thioredoxin-hGH (Trx-hGH) fusion protein. We outline a relatively simple three step purification process employing two immobilized metal-affinity chromatography and one anion-exchange steps and removal of fusion partner by enterokinase cleavage yielding native hGH. The ability of cell populations to produce quantities of up to 1 g/L of the soluble Trx-hGH fusion protein has been tested in flask cultivations as well as in batch and fed-batch bioreactor runs. The sequence and structure of derived hGH were confirmed by mass spectrometry and circular dichroism and its native function, to induce cell proliferation, was confirmed by employing a Nb2 cell line proliferation assay.

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

    NASA Astrophysics Data System (ADS)

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

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

  8. Bioinformatics approaches for improved recombinant protein production in Escherichia coli: protein solubility prediction.

    PubMed

    Chang, Catherine Ching Han; Song, Jiangning; Tey, Beng Ti; Ramanan, Ramakrishnan Nagasundara

    2014-11-01

    The solubility of recombinant protein expressed in Escherichia coli often represents the production yield. However, up-to-date, instances of successful production of soluble recombinant proteins in E. coli expression system with high yield remain scarce. This is mainly due to the difficulties in improving the overall production capacity, as most of the well-established strategies usually involve a series of trial and error steps with unguaranteed success. One way to concurrently improve the production yield and minimize the production cost would be incorporating the potency of bioinformatics tools to conduct in silico studies, which forecasts the outcome before actual experimental work. In this article, we review and compare seven prediction tools available, which predict the solubility of protein expressed in E. coli, using the following criteria: prediction performance, usability, utility, prediction tool development and validation methodologies. This comprehensive review will be a valuable resource for researchers with limited prior experience in bioinformatics tools. As such, this will facilitate their choice of appropriate tools for studies related to enhancement of intracellular recombinant protein production in E. coli. © The Author 2013. Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.

  9. 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.)

  10. Synthesis of nylon 4 from gamma-aminobutyrate (GABA) produced by recombinant Escherichia coli.

    PubMed

    Park, Si Jae; Kim, Eun Young; Noh, Won; Oh, Young Hoon; Kim, Hye Young; Song, Bong Keun; Cho, Kwang Myung; Hong, Soon Ho; Lee, Seung Hwan; Jegal, Jonggeon

    2013-07-01

    In this study, we developed recombinant Escherichia coli strains expressing Lactococcus lactis subsp. lactis Il1403 glutamate decarboxylase (GadB) for the production of GABA from glutamate monosodium salt (MSG). Syntheses of GABA from MSG were examined by employing recombinant E. coli XL1-Blue as a whole cell biocatalyst in buffer solution. By increasing the concentration of E. coli XL1-Blue expressing GadB from the OD₆₀₀ of 2-10, the concentration and conversion yield of GABA produced from 10 g/L of MSG could be increased from 4.3 to 4.8 g/L and from 70 to 78 %, respectively. Furthermore, E. coli XL1-Blue expressing GadB highly concentrated to the OD₆₀₀ of 100 produced 76.2 g/L of GABA from 200 g/L of MSG with 62.4 % of GABA yield. Finally, nylon 4 could be synthesized by the bulk polymerization using 2-pyrrolidone that was prepared from microbially synthesized GABA by the reaction with Al₂O₃ as catalyst in toluene with the yield of 96 %.

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

    PubMed Central

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

    2015-01-01

    Background Insertion of T4 lysozyme (T4L) into the GPCR successfully enhanced GPCR protein stability and solubilization. However, the biological functions of the recombinant GPCR protein have not been analyzed. Methods We engineered the CCR5-T4L mutant and expressed and purified the soluble recombinant protein using an E.coli expression system. The antiviral effects of this recombinant protein in THP-1 cell lines, primary human macrophages, and PBMCs from different donors were investigated. We also explored the possible mechanisms underlying the observed antiviral effects. Results Our data showed the biphasic inhibitory and promotion effects of different concentrations of soluble recombinant CCR5-T4L protein on R5 tropic human immunodeficiency virus-1 (HIV-1) infection in THP-1 cell lines, human macrophages, and PBMCs from clinical isolates. We demonstrated that soluble recombinant CCR5-T4L acts as a HIV-1 co-receptor, interacts with wild type CCR5, down-regulates the surface CCR5 expression in human macrophages, and interacts with CCL5 to inhibit macrophage migration. Using binding assays, we further determined that recombinant CCR5-T4L and [125I]-CCL5 compete for the same binding site on wild type CCR5. Conclusions Our results suggest that recombinant CCR5-T4L protein marginally promotes HIV-1 infection at low concentrations and markedly inhibits infection at higher concentrations. This recombinant protein may be helpful in the future development of anti-HIV-1 therapeutic agents. PMID:26154172

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

    PubMed

    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.

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

    PubMed

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

    2016-11-23

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

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

    PubMed Central

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

    2016-01-01

    The production of recombinant spider silk proteins continues to be a key area of interest for a number of research groups. Several key obstacles exist in their production as well as in their formulation into useable products. The original reported method to solubilize recombinant spider silk proteins (rSSp) in an aqueous solution involved using microwaves to quickly generate heat and pressure inside of a sealed vial containing rSSp and water. Fibers produced from this system are remarkable in their mechanical ability and demonstrate the ability to be stretched and recover 100 times. The microwave method dissolves the rSSPs with dissolution time increasing with higher molecular weight constructs, increasing concentration of rSSPs, protein type, and salt concentration. It has proven successful in solvating a number of different rSSPs including native-like sequences (MaSp1, MaSp2, piriform, and aggregate) as well as chimeric sequences (FlAS) in varied concentrations that have been spun into fibers and formed into films, foams, sponges, gels, coatings, macro and micro spheres and adhesives. The system is effective but inherently unpredictable and difficult to control. Provided that the materials that can be generated from this method of dissolution are impressive, an alternative means of applying heat and pressure that is controllable and predictable has been developed. Results indicate that there are combinations of heat and pressure (135 °C and 140 psi) that result in maximal dissolution without degrading the recombinant MaSp2 protein tested, and that heat and pressure are the key elements to the method of dissolution. PMID:27886066

  15. Production and Characterization of Monoclonal Antibody against Recombinant Virus Coat Protein CP42.

    PubMed

    Shibaei, Naeimeh; Majidi, Jafar; Razavi, Khadijeh; Karkhane, Ali Asghar; Sokhandan-Bashir, Nemat; Aghebati-Maleki, Leili

    2017-02-01

    There are many studies related to the production of a ELISA kit for diagnosing virus infections. However, production of most kits depends on purification of whole virus particles, which involves the use of costly equipment and reagents. The purpose of this study was to check out if the anti-CP42 antibodies could be used as a diagnostic assay for detection of Grapevine fanleaf Virus (GFLV). In this study, recombinant GFLV coat protein gene related to selected antigenic determinants was inserted into pET-28a bacterial expression vector and the construct (pET-28a CP42) was cloned into E. coli strain (DE3). Expressed protein was verified with western blotting assay by the use of commercially available anti-GFLV antibody. The recombinant protein was purified using nickel-nitrilotriacetic acid (Ni-NTA) resin. Balb/c mice were immunized with purified protein and splenocytes of hyperimmunized mice were fused with murine myeloma Sp2/0 cells. Positive hybridomas were selected by ELISA using CP42 as coating antigen. The results showed that monoclonal antibody (MAb) specific to CP42 has been successfully generated. Efficiency of produced antibody was analyzed by ELISA and western blotting assay using some confirmed grapevine samples. The infection was confirmed previously based on morphological features and ELISA assay, performed using commercial anti-GFLV antibody. The monoclonal antibody reacted with antigen in ELISA and immunoblot method. Our results demonstrated that anti recombinant CP42 monoclonal antibodies are able to diagnose whole virus in infected grapevine sample using ELISA test.

  16. Recombination of engineered defective RNA species produces infective potyvirus in planta.

    PubMed

    Gal-On, A; Meiri, E; Raccah, B; Gaba, V

    1998-06-01

    Recombination occurred between viral genomes when squash plants were cobombarded with mixtures of engineered disabled constructs of a zucchini yellow mosaic potyvirus. Single and double recombinants were detected in the progeny. Genes involved in the recombination process and the mechanisms of recombination were studied in potyviruses for the first time.

  17. Recombination of Engineered Defective RNA Species Produces Infective Potyvirus In Planta†

    PubMed Central

    Gal-On, Amit; Meiri, Eti; Raccah, Benjamin; Gaba, Victor

    1998-01-01

    Recombination occurred between viral genomes when squash plants were cobombarded with mixtures of engineered disabled constructs of a zucchini yellow mosaic potyvirus. Single and double recombinants were detected in the progeny. Genes involved in the recombination process and the mechanisms of recombination were studied in potyviruses for the first time. PMID:9573302

  18. In vivo post-translational modifications of recombinant mussel adhesive protein in insect cells.

    PubMed

    Lim, Seonghye; Kim, Kyoung Ro; Choi, Yoo Seong; Kim, Dae-Kyum; Hwang, Daehee; Cha, Hyung Joon

    2011-01-01

    Mussel adhesive proteins (MAPs) have been suggested as promising bioadhesives for diverse application fields, including medical uses. Previously, we successfully constructed and produced a new type of functional recombinant MAP, fp-151, in a prokaryotic Escherichia coli expression system. Even though the E. coli-derived MAP showed several excellent features, such as high production yield and efficient purification, in vitro enzymatic modification is required to convert tyrosine residues to l-3,4-dihydroxyphenyl alanine (dopa) molecules for its adhesive ability, due to the intrinsic inability of E. coli to undergo post-translational modification. In this work, we produced a soluble recombinant MAP in insect Sf9 cells, which are widely used as an effective and convenient eukaryotic expression system for eukaryotic foreign proteins. Importantly, we found that insect-derived MAP contained converted dopa residues by in vivo post-translational modification. In addition, insect-derived MAP also had other post-translational modifications including phosphorylation of serine and hydroxylation of proline that originally occurred in some natural MAPs. To our knowledge, this is the first report on in vivo post-translational modifications of MAP containing dopa and other modified amino acid residues.

  19. Protein body-inducing fusions for high-level production and purification of recombinant proteins in plants.

    PubMed

    Conley, Andrew J; Joensuu, Jussi J; Richman, Alex; Menassa, Rima

    2011-05-01

    For the past two decades, therapeutic and industrially important proteins have been expressed in plants with varying levels of success. The two major challenges hindering the economical production of plant-made recombinant proteins include inadequate accumulation levels and the lack of efficient purification methods. To address these limitations, several fusion protein strategies have been recently developed to significantly enhance the production yield of plant-made recombinant proteins, while simultaneously assisting in their subsequent purification. Elastin-like polypeptides are thermally responsive biopolymers composed of a repeating pentapeptide 'VPGXG' sequence that are valuable for the purification of recombinant proteins. Hydrophobins are small fungal proteins capable of altering the hydrophobicity of their respective fusion partner, thus enabling efficient purification by surfactant-based aqueous two-phase systems. Zera, a domain of the maize seed storage protein γ-zein, can induce the formation of protein storage bodies, thus facilitating the recovery of fused proteins using density-based separation methods. These three novel protein fusion systems have also been shown to enhance the accumulation of a range of different recombinant proteins, while concurrently inducing the formation of protein bodies. The packing of these fusion proteins into protein bodies may exclude the recombinant protein from normal physiological turnover. Furthermore, these systems allow for quick, simple and inexpensive nonchromatographic purification of the recombinant protein, which can be scaled up to industrial levels of protein production. This review will focus on the similarities and differences of these artificial storage organelles, their biogenesis and their implication for the production of recombinant proteins in plants and their subsequent purification.

  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.

  1. Specificity of recombination of H and L chains from human gamma-G-myeloma proteins.

    PubMed

    Grey, H M; Mannik, M

    1965-09-01

    Dissociated H and L chains of human gammaG-myeloma proteins were recombined by removal of conditions interrupting non-covalent interactions. In the process of recombination 7S molecules were formed. It was demonstrated that the H chains from individual gammaG-myeloma proteins recombine with their own L chains but also with L chains derived from other myeloma proteins. In some instances, however, the L chains from other myeloma proteins did not recombine as avidly with the H chains as the autologous L chains. The specificity of the non-covalent interactions of H and L chains was particularly well brought out by competitive recombination experiments where an individual H chain had a choice of recombining with its own L chain or with the L chain obtained from another myeloma protein. In this manner a spectrum of affinities between individual H chains and several L chains was demonstrated. In the vast majority of recombinations there was a clearcut preference for recombination to take place between the H and L chains derived from the same protein. It is postulated that this specificity is related to differences in the primary structure, which cause differences in configuration of these homogeneous H and L chains and that these configurations then dictate on thermodynamic grounds the pairing of H chains with particular L chains.

  2. Production and characterization of monoclonal antibody specific to recombinant dengue multi-epitope protein.

    PubMed

    Abhyankar, Ajay Vinayak; Bhargava, Rakesh; Jana, Asha Mukul; Sahni, Ajay Kumar; Rao, P V Lakshmana

    2008-06-01

    Monoclonal antibodies against novel dengue recombinant protein were produced following immunization of Balb/c mice with recombinant dengue multi-epitope protein (r-DMEP) expressed in Escherichia coli vector and purified in a single-step chromatography system. Antigenicity of r-DMEP was evaluated by dot enzyme immunoassay. Mice were immunized intraperitoneally with five doses each of 100 microg of this novel antigen at 1-week intervals and a final intravenous booster dose prior to the fusion. Hybridomas resulted from fusion of myeloma cells and splenocytes using PEG-1500 as an additive. Selection of the hybrids was done using HAT medium, and the hybrids thus selected were finally screened qualitatively and quantitatively by dot and plate immunoassays, respectively. Five antibody secretory hybrid clones exhibited specific reactivity against r-DMEP by dot-ELISA, whereas a lone clone was found to be cross-reactive with Japanese encephalitis virus (JEV). Monoclonal antibodies (MAbs) specific to r-DME protein recognized the envelope and non-structural epitopes by Western blot analysis. These MAbs were further checked for their diagnostic efficacy using dengue suspected clinical samples and found overall sensitivity and specificity for DRDE dipstick ELISA. MAb-based dipstick ELISA results were 85%, 75% and 85%, 90%, respectively.

  3. pGreen-S: a clone vector bearing absence of enhanced green fluorescent protein for screening recombinants.

    PubMed

    Tang, Jinbao; Liang, Shujuan; Zhang, Jinbao; Gao, Zhiqin; Zhang, Suhua

    2009-05-01

    The bacterial cloning vector, pGreen-S, was constructed by inserting the enhanced green fluorescent protein (EGFP) gene at the XbaI restriction site of pUC18 plasmid. When expressed in Escherichia coli DH5alpha produced colonies that were an absinthe green color under daylight and strongly fluorescent green under longwave ultraviolet light. The pGreen-S vector was used to select for directional insert based on the loss of green fluorescence in recombinant colonies that was caused by the absence of EGFP. The EGFP reporter system differs from the conventional complementation of lacZ, making screening recombinants simpler, less expensive, and more effective.

  4. The S-layer protein from Campylobacter rectus: sequence determination and function of the recombinant protein.

    PubMed

    Miyamoto, M; Maeda, H; Kitanaka, M; Kokeguchi, S; Takashiba, S; Murayama, Y

    1998-09-15

    The gene encoding the crystalline surface layer (S-layer) protein from Campylobacter rectus, designated slp, was sequenced and the recombinant gene product was expressed in Escherichia coli. The gene consisted of 4086 nucleotides encoding a protein with 1361 amino acids. The N-terminal amino acid sequence revealed that Slp did not contain a signal sequence, but that the initial methionine residue was processed. The deduced amino acid sequence displayed some common characteristic features of S-layer proteins previously reported. A homology search showed a high similarity to the Campylobacter fetus S-layer proteins, especially in their N-terminus. The C-terminal third of Slp exhibited homology with the RTX toxins from Gram-negative bacteria via the region including the glycine-rich repeats. The Slp protein had the same N-terminal sequence as a 104-kDa cytotoxin isolated from the culture supernatants of C. rectus. However, neither native nor recombinant Slp showed cytotoxicity against HL-60 cells or human peripheral white blood cells. These data support the idea that the N-terminus acts as an anchor to the cell surface components and that the C-terminus is involved in the assembly and/or transport of the protein.

  5. Generating Functional Recombinant NRPS Enzymes in the Laboratory Setting via Peptidyl Carrier Protein Engineering.

    PubMed

    Owen, Jeremy G; Calcott, Mark J; Robins, Katherine J; Ackerley, David F

    2016-11-17

    Non-ribosomal peptide synthetases (NRPSs) are modular enzymatic assembly lines where substrates and intermediates undergo rounds of transformation catalyzed by adenylation (A), condensation (C), and thioesterase (TE) domains. Central to the NRPS biosynthesis are peptidyl carrier protein (PCP) domains, small, catalytically inactive domains that shuttle substrates and intermediates between the catalytic modules and govern product release from TE domains. There is strong interest in recombination of NRPS systems to generate new chemical entities. However, the intrinsic complexity of these systems has been a major challenge. Here, we employ domain substitution and random mutagenesis to recapitulate NRPS evolution, focusing on PCP domains. Using NRPS model systems that produce two different pigmented molecules, pyoverdine and indigoidine, we found that only evolutionarily specialized recombinant PCP domains could interact effectively with the native TE domain for product release. Overall, we highlight that substituted PCP domains require very minor changes to result in functional NRPSs, and infer that positive selection pressure may improve recombinant NRPS outcomes.

  6. Recombinant Reg3β protein protects against streptozotocin-induced β-cell damage and diabetes

    PubMed Central

    Luo, Chen; Yu, Lu-Ting; Yang, Meng-Qi; Li, Xiang; Zhang, Zhi-Yuan; Alfred, Martin O; Liu, Jun-Li; Wang, Min

    2016-01-01

    Regenerating genes (Reg) have been found during the search for factors involved in pancreatic islet regeneration. Our recent study discovered that pancreatic β-cell-specific overexpression of Reg3β protects against streptozotocin (Stz) -induced diabetes in mice. To investigate its potential roles in the treatment of diabetes, we produced a recombinant Reg3β protein and provided evidence that it is active in promoting islet β-cell survival against Stz- triggered cell death. Though ineffective in alleviating preexisting diabetes, pretreatment of recombinant Reg3β was capable of minimizing the Stz-induced hyperglycemia and weight loss, by preserving serum and pancreatic insulin levels, and islet β-cell mass. No obvious changes were observed in the rate of cell proliferation and hypertrophy in α- or acinar-cells after treatment with recombinant Reg3β. The underlying mechanism of Reg3β-mediated protection seems to involve Akt activation which upregulates Bcl-2 and Bcl-xL levels and consequently promotes cell survival. PMID:27767186

  7. Detection, characterization and quantitation of coxsackievirus A16 using polyclonal antibodies against recombinant capsid subunit proteins.

    PubMed

    Liu, Qingwei; Ku, Zhiqiang; Cai, Yicun; Sun, Bing; Leng, Qibin; Huang, Zhong

    2011-04-01

    Coxsackievirus A16 (CVA16), together with enterovirus type 71 (EV71), is responsible for most cases of hand, foot and mouth disease (HFMD) worldwide. Recent findings suggest that the recombination between CVA16 and EV71, and co-circulation of these two viruses may have contributed to the increase of HFMD cases in China over the past few years. Thus, for CVA16, further understanding of its virology, epidemiology and development of diagnostic tests and vaccines are of importance. The present study aimed to develop reagents and protocols for the detection, characterization and quantitation of CVA16. Recombinant CVA16 capsid subunit proteins VP0, VP3 and truncated VP1, were produced in Escherichia coli and used to immunize guinea pigs to generate polyclonal antibodies. The resultant three antisera detected specifically CVA16 propagated in Vero cells by immunostaining, ELISA and Western blotting. The antisera was used to show that CVA16 capsids were composed of correctly processed VP0, VP1 and VP3 subunits, and were present in the form of efficiently assembled particles. A method for the quantitation of the yield of CVA16 in Vero cells was established based on a Western blotting protocol using the recombinant VP0 as a reference standard and anti-VP0 as the detection antibody. This study shows the development and validation of reagents and methods, for qualitative and quantitative determination of CVA16, which are essential for the development of vaccines.

  8. Development of recombinant nucleocapsid protein based IgM-ELISA for the early detection of distemper infection in dogs.

    PubMed

    Latha, D; Geetha, M; Ramadass, P; Narayanan, R B

    2007-10-15

    An IgM-ELISA based on a 16-kDa recombinant protein produced for the conserved and functional middle region of nucleocapsid protein of Canine distemper virus was developed. Out of 70 serum samples from distemper-suspected and vaccinated dogs analyzed, 34 serum samples (49%) were positive. The specificity of this ELISA was confirmed by blocking and adsorption experiments. The IgM-ELISA based on the recombinant nucleocapsid protein showed a strong correlation (r=0.857, p<0.0001 at 95% CI) and good agreement (kappa=0.714) with the conventional Vero cell culture distemper antigen based IgM-ELISA. The percent positivity was more in dogs with systemic signs (62%) by recombinant nucleocapsid protein IgM-ELISA. Out of 70 clinical serum samples, 69 samples were used along with 4 control sera used in the IgM-ELISA for the detection of viral RNA by Slot blot hybridization and 26 of them (36%) were positive. Fifty-one percent agreement was observed between the recombinant nucleocapsid protein IgM-ELISA and Slot blot hybridization. The analysis of clinical history of the dogs with systemic signs supported the application of IgM-ELISA over Slot blot hybridization in the early detection of distemper infection.

  9. Specific CEA-producing colorectal carcinoma cell killing with recombinant adenoviral vector containing cytosine deaminase gene

    PubMed Central

    Shen, Li-Zong; Wu, Wen-Xi; Xu, De-Hua; Zheng, Zhong-Cheng; Liu, Xin-Yuan; Ding, Qiang; Hua, Yi-Bing; Yao, Kun

    2002-01-01

    AIM: To kill CEA positive colorectal carcinoma cells specifically using the E coli cytosine deaminase (CD) suicide gene, a new replication-deficient recombinant adenoviral vector was constructed in which CD gene was controlled under CEA promoter and its in vitro cytotoxic effects were evaluated. METHODS: Shuttle plasmid containing CD gene and regulatory sequence of the CEA gene was constructed and recombined with the right arm of adenovirus genome DNA in 293 cell strain. Dot blotting and PCR were used to identify positive plaques. The purification of adenovirus was performed with ultra-concentration in CsCl step gradients and the titration was measured with plaque formation assay. Cytotoxic effects were assayed with MTT method, The fifty percent inhibition concentration (IC50) of 5-FC was calculated using a curve-fitting parameter. The human colorectal carcinoma cell line, which was CEA-producing, and the CEA-nonproducing Hela cell line were applied in cytological tests. An established recombinant adenovirus vector AdCMVCD, in which the CD gene was controlled under CMV promoter, was used as virus control. Quantitative results were expressed as the mean ± SD of the mean. Statistical analysis was performed using ANOVA test. RESULTS: The desired recombinant adenovirus vector was named AdCEACD. The results of dot blotting and PCR showed that the recombinant adenovirus contained CEA promoter and CD gene. Virus titer was about 5.0 × 1014 pfu/L-1 after purification. The CEA-producing Lovo cells were sensitive to 5-FC and had the same cytotoxic effect after infection with AdCEACD and AdCMVCD (The IC50 values of 5-FC in parent Lovo cells, Lovo cells infected with 100 M.O.I AdCEACD and Lovo cells infected with 10 M.O.I AdCMVCD were > 15000, 216.5 ± 38.1 and 128.8 ± 25.4 μmol•L⁻¹, P < 0.001, respectively), and the cytotoxicity of 5-FC increased accordingly when the M.O.I of adenoviruses were enhanced (The value of IC50 of 5-FC was reduced to 27.9 ± 4.2 μmol•L-1

  10. Novel insect cell line capable of complex N-glycosylation and sialylation of recombinant proteins.

    PubMed

    Palomares, Laura A; Joosten, Christoph E; Hughes, Patrick R; Granados, Robert R; Shuler, Michael L

    2003-01-01

    Paucimannose or oligomannose structures are usually attached to glycoproteins produced by insect cells, while mammalian glycoproteins usually have complex glycans. The lack of complex glycosylation has limited the use of the insect cell baculovirus expression vector system (BEVS), despite its high productivity and versatility. The availability of cell lines capable of complex glycosylation can overcome such a problem and potentially increase the utility of BEVS. In this work the capability of two novel cell lines, one from Pseudaletia unipuncta (A7S) and one from Danaus plexippus (DpN1), to produce and glycosylate a recombinant protein (secreted human placental alkaline phosphatase, SeAP) was assessed. SeAP produced by Tn5B1-4 cells at a low passage number (<200) was utilized for comparison. The optimal conditions for the production of SeAP by DpN1 cells were defined, and the glycosylation profiles of SeAP produced by the cell lines were quantitatively determined. Both the A7S and the DpN1 cells produced lower concentrations of SeAP than the Tn5B1-4 cells. Less than 5% of the glycans attached to SeAP produced by the Tn5B1-4 cells had complex forms. Glycans attached to SeAP from A7S cells contained 4% hybrid and 8% complex forms. Galactosylated biantennary structures were identified. Glycans attached to SeAP produced by the DpN1 cell line had 6% hybrid and 26% complex forms. Of the complex forms in SeAP from DpN1, 13% were identified as sialylated glycans. The galactosyltransferase activity of the three cell lines was measured and correlated to their ability to produce complex forms. Even though neither novel cell line produced as much recombinant protein as the Tn5B1-4 cells, the glycosylation of SeAP expressed by both cell lines was more complete. These novel cell lines represent interesting alternatives for the production of complex glycosylated proteins utilizing the BEVS.

  11. Highly specific confirmatory western blot test for African swine fever virus antibody detection using the recombinant virus protein p54.

    PubMed

    Alcaraz, C; Rodriguez, F; Oviedo, J M; Eiras, A; De Diego, M; Alonso, C; Escribano, J M

    1995-03-01

    A Western blot technique using a recombinant protein has been developed to confirm positive results obtained in African swine fever (ASF)-specific antibody detection by ELISA. The new confirmatory Western blot is based on the use of protein p54, one of the most antigenic ASF virus structural proteins, expressed in Escherichia coli fused to the N-terminus of MS2 polymerase. The recombinant Western blot assay was highly specific and equally sensitive for ASF virus-infected pigs detection as the conventional Western blot, which uses virus-induced proteins ranging in molecular weight between 23 and 35 kDa. The novel Western blot assay provides a simpler interpretation of the test, eliminates the possibility of false-positive reactions produced by cellular compounds that contaminate the antigen employed in the conventional technique, and avoids the use of live virus in antigen production.

  12. Rational Engineering of Recombinant Picornavirus Capsids to Produce Safe, Protective Vaccine Antigen

    PubMed Central

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

    2013-01-01

    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

  13. 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

    2016-05-03

    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.

  14. 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

  15. Purification and basic biochemical characterization of 19 recombinant plant peroxidase isoenzymes produced in Pichia pastoris☆

    PubMed Central

    Krainer, Florian W.; Pletzenauer, Robert; Rossetti, Laura; Herwig, Christoph; Glieder, Anton; Spadiut, Oliver

    2014-01-01

    The plant enzyme horseradish peroxidase (HRP) is used in several important industrial and medical applications, of which especially biosensors and diagnostic kits describe an emerging field. Although there is an increasing demand for high amounts of pure enzyme preparations, HRP is still isolated from the plant as a mixture of different isoenzymes with different biochemical properties. Based on a recent next generation sequencing approach of the horseradish transcriptome, we produced 19 individual HRP isoenzymes recombinantly in the yeast Pichia pastoris. After optimizing a previously reported 2-step purification strategy for the recombinant isoenzyme HRP C1A by substituting an unfavorable size exclusion chromatography step with an anion exchange step using a monolithic column, we purified the 19 HRP isoenzymes with varying success. Subsequent basic biochemical characterization revealed differences in catalytic activity, substrate specificity and thermal stability of the purified HRP preparations. The preparations of the isoenzymes HRP A2A and HRP A2B were found to be highly interesting candidates for future applications in diagnostic kits with increased sensitivity. PMID:24342173

  16. A recombinant Eimeria protein inducing interferon-gamma production: comparison of different gene expression systems and immunization strategies for vaccination against coccidiosis.

    PubMed

    Lillehoj, H S; Choi, K D; Jenkins, M C; Vakharia, V N; Song, K D; Han, J Y; Lillehoj, E P

    2000-01-01

    A rabbit antiserum against an 18- to 27-kD native protein fraction (F3) from Eimeria acervulina merozoites identified a cDNA (3-1E) containing a 1086-base pair insertion with an open reading frame of 170 amino acids (predicted molecular weight, 18,523). The recombinant 3-1E cDNA expressed in Escherichia coli produced a 60-kD fusion protein and a 23-kD protein after factor Xa treatment of the fusion protein. Both proteins were reactive with the F3 antiserum by western blot analysis. A rabbit antiserum against a synthetic peptide deduced from the amino acid sequence of the 3-1E cDNA reacted with a 27-kD recombinant 3-1E protein expressed in Sf9 insect cells and a 20-kD native protein expressed by E. acervulina sporozoites and Eimeria tenella sporozoites and merozoites. By immunofluorescence staining, a monoclonal antibody produced against the recombinant 3-1E protein reacted with sporozoites and merozoites of E. acervulina, E. tenella, and Eimeria maxima. Spleen lymphocytes from E. acervulina-immune chickens showed antigen-specific proliferation and interferon (IFN)-gamma production upon stimulation with the recombinant 3-1E protein, indicating that the protein activates cell-mediated immunity during coccidiosis. Immunization of chickens with either the E. coli- or Sf9-expressed recombinant 3-1E protein with adjuvant, or direct injection of the 3-1E cDNA, induced protective immunity against live E. acervulina. Simultaneous injection of the recombinant 3-1E protein, or the 3-1E cDNA, with cDNAs encoding chicken IFN-gamma or interleukin (IL)-2/15 further enhanced protective immunity. These results indicate that the recombinant E. acervulina 3-1E cDNA or its polypeptide product may prove useful as vaccines against avian coccidiosis.

  17. Antigenic and immunosuppressive properties of a trimeric recombinant transmembrane envelope protein gp41 of HIV-1

    PubMed Central

    Mühle, Michael; Lehmann, Melissa; Hoffmann, Kerstin; Stern, Daniel; Kroniger, Tobias; Luttmann, Werner

    2017-01-01

    The transmembrane envelope (TM) protein gp41 of the human immunodeficiency virus—1 (HIV-1) plays an important role during virus infection inducing the fusion of the viral and cellular membranes. In addition, there are indications that the TM protein plays a role in the immunopathogenesis leading to the acquired immunodeficiency syndrome (AIDS). Inactivated virus particles and recombinant gp41 have been reported to inhibit lymphocyte proliferation, as well as to alter cytokine release and gene expression. The same was shown for a peptide corresponding to a highly conserved domain of all retroviral TM proteins, the immunosuppressive domain. Due to its propensity to aggregate and to be expressed at low levels, studies comprising authentic gp41 produced in eukaryotic cells are extremely rare. Here we describe the production of a secreted, soluble recombinant gp41 in 293 cells. The antigen was purified to homogeneity and characterised thoroughly by various biochemical and immunological methods. It was shown that the protein was glycosylated and assembled into trimers. Binding studies by ELISA and surface plasmon resonance using conformation-specific monoclonal antibodies implied a six-helix bundle conformation. The low binding of broadly neutralising antibodies (bnAb) directed against the membrane proximal external region (MPER) suggested that this gp41 is probably not suited as vaccine to induce such bnAb. Purified gp41 bound to monocytes and to a lesser extent to lymphocytes and triggered the production of specific cytokines when added to normal peripheral blood mononuclear cells. In addition, gp41 expressed on target cells inhibited the antigen-specific response of murine CD8+ T cells by drastically impairing their IFNγ production. To our knowledge, this is the first comprehensive analysis of a gp41 produced in eukaryotic cells including its immunosuppressive properties. Our data provide another line of evidence that gp41 might be directly involved in HIV-1

  18. Antigenic and immunosuppressive properties of a trimeric recombinant transmembrane envelope protein gp41 of HIV-1.

    PubMed

    Mühle, Michael; Lehmann, Melissa; Hoffmann, Kerstin; Stern, Daniel; Kroniger, Tobias; Luttmann, Werner; Denner, Joachim

    2017-01-01

    The transmembrane envelope (TM) protein gp41 of the human immunodeficiency virus-1 (HIV-1) plays an important role during virus infection inducing the fusion of the viral and cellular membranes. In addition, there are indications that the TM protein plays a role in the immunopathogenesis leading to the acquired immunodeficiency syndrome (AIDS). Inactivated virus particles and recombinant gp41 have been reported to inhibit lymphocyte proliferation, as well as to alter cytokine release and gene expression. The same was shown for a peptide corresponding to a highly conserved domain of all retroviral TM proteins, the immunosuppressive domain. Due to its propensity to aggregate and to be expressed at low levels, studies comprising authentic gp41 produced in eukaryotic cells are extremely rare. Here we describe the production of a secreted, soluble recombinant gp41 in 293 cells. The antigen was purified to homogeneity and characterised thoroughly by various biochemical and immunological methods. It was shown that the protein was glycosylated and assembled into trimers. Binding studies by ELISA and surface plasmon resonance using conformation-specific monoclonal antibodies implied a six-helix bundle conformation. The low binding of broadly neutralising antibodies (bnAb) directed against the membrane proximal external region (MPER) suggested that this gp41 is probably not suited as vaccine to induce such bnAb. Purified gp41 bound to monocytes and to a lesser extent to lymphocytes and triggered the production of specific cytokines when added to normal peripheral blood mononuclear cells. In addition, gp41 expressed on target cells inhibited the antigen-specific response of murine CD8+ T cells by drastically impairing their IFNγ production. To our knowledge, this is the first comprehensive analysis of a gp41 produced in eukaryotic cells including its immunosuppressive properties. Our data provide another line of evidence that gp41 might be directly involved in HIV-1

  19. Recombinant bovine dihydrofolate reductase produced by mutagenesis and nested PCR of murine dihydrofolate reductase cDNA.

    PubMed

    Cody, Vivian; Mao, Qilong; Queener, Sherry F

    2008-11-01

    Recent reports of the slow-tight binding inhibition of bovine liver dihydrofolate reductase (bDHFR) in the presence of polyphenols isolated from green tea leaves has spurred renewed interest in the biochemical properties of bDHFR. Earlier studies were done with native bDHFR but in order to validate models of polyphenol binding to bDHFR, larger quantities of bDHFR are necessary to support structural studies. Bovine DHFR differs from its closest sequence homologue, murine DHFR, by 19 amino acids. To obtain the bDHFR cDNA, murineDHFR cDNA was transformed by a series of nested PCRs to reproduce the amino acid coding sequence for bovine DHFR. The bovine liver DHFR cDNA has an open reading frame of 561 base pairs encoding a protein of 187 amino acids that has a high level of conservation at the primary sequence level with other DHFR enzymes, and more so for the amino acid residues in the active site of the mammalian DHFR enzymes. Expression of the bovine DHFR cDNA in bacterial cells produced a stable recombinant protein with high enzymatic activity and kinetic properties similar to those previously reported for the native protein.

  20. Biochemical characterization of LR769, a new recombinant factor VIIa bypassing agent produced in the milk of transgenic rabbits.

    PubMed

    Chevreux, G; Tilly, N; Leblanc, Y; Ramon, C; Faid, V; Martin, M; Dhainaut, F; Bihoreau, N

    2017-07-01

    The bypassing agent factor VII (FVIIa) is a first-line therapy for the treatment of acute bleeding episodes in patients with haemophilia and high-titre inhibitors. FVIIa is a highly post-translationally modified protein that requires eukaryotic expression systems to produce a fully active molecule. A recombinant FVIIa was produced in the milk of transgenic rabbits to increase expression and provide an efficient, safe and affordable product after purification to homogeneity (LR769). To present the biochemical and functional in vitro characteristics of LR769. Mass spectrometric analyses of the intact protein and of heavy and light chains revealed a fully activated, mature and properly post-translationally modified protein notably regarding N/O-glycosylations and γ-carboxylation. Primary structure analysis, performed by peptide mapping, confirmed 100% of the sequence and the low level or absence of product-derived impurities such as oxidized, deamidated and glycated forms. Low levels of aggregates and fragments were observed by different chromatographic methods. Higher order structure investigated by circular dichroism showed appropriate secondary/tertiary structures and conformational change in the presence of Ca(2+) ions. Finally, activated partial thromboplastin time and thrombin generation assays showed the ability of LR769 to decrease coagulation time and to generate thrombin in haemophiliac-A-plasmas, even in the presence of inhibitors. The innovative expression system used to produce LR769 yields a new safe and effective rhFVIIa for the treatment of haemophilia A or B patients with inhibitors. © 2017 LFB Biotechnologies. Haemophilia Published by John Wiley & Sons Ltd.

  1. Biological Activity of Recombinant Bovine Interferon τ Produced by a Silkworm-Baculovirus Gene Expression System

    PubMed Central

    TAKAHASHI, Hitomi; TSUNAZAKI, Makoto; HAMANO, Takashi; TAKAHASHI, Masashi; OKUDA, Kiyoshi; INUMARU, Shigeki; OKANO, Akira; GESHI, Masaya; HIRAKO, Makoto

    2013-01-01

    ABSTRACT Bovine interferon (bIFN) τ plays a crucial role in maternal-fetal recognition and was expressed using a Bombyx mori (Bm) nuclear polyhedrosis virus (silkworm baculovirus) gene expression system. The biological effects of Bm-recombinant bIFNτ (rbIFNτ) on prostaglandin (PG) F2α synthesis were investigated in cultured bovine endometrial epithelial cells with oxytocin (OT, 100 nM) and on the in vitro development of bovine embryos. Bm-rbIFNτ and OT were shown to suppress PGF2α production in a dose-dependent manner. When in vitro produced morula stage embryos were cultured for 72 hr in modified CR1aa medium supplemented with or without rbIFNτ, Bm-rbIFNτ (10 ng/ml) significantly promoted development to the expanded blastocyst stage. In conclusion, Bm-rbIFNτ was suggested to have the same bioactivity as native IFNτ. PMID:24212505

  2. 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

  3. Recombinant human milk fat globule-EGF factor 8 produces dose-dependent benefits in sepsis.

    PubMed

    Shah, Kavin G; Wu, Rongqian; Jacob, Asha; Molmenti, Ernesto P; Nicastro, Jeffrey; Coppa, Gene F; Wang, Ping

    2012-01-01

    Animal milk fat globule-EGF factor 8 (MFG-E8) has been shown to be beneficial in attenuating the inflammatory response in sepsis. In this study, we examined the effect of recombinant human MFG-E8 (rhMFG-E8) in an animal model of sepsis in an effort to develop it as a potential therapy against sepsis in humans. Rats were subjected to sepsis by cecal ligation and puncture (CLP), and at 5 h post-CLP, they were given different doses of rhMFG-E8 (20, 40, 80, 160 μg/kg BW) in normal saline. At 20 h post-CLP, samples were collected for further analysis. A 10-day survival study was also performed. At 20 h after CLP, organ injury indicators, serum IL-6 and TNF-α, and plasma HMGB-1 levels were significantly increased as compared to sham-operated animals. Treatment with 20 μg/kg rhMFG-E8 significantly reduced these levels. With higher doses, further reductions in AST and ALT (59-62%), creatinine (65-68%), and lactate (46-57%), and serum IL-6 and TNF-α were obtained. The 160 μg/kg dose produced the greatest reduction in serum TNF-α. With treatment with 20 μg/kg rhMFG-E8, HMGB-1 levels decreased by 80%, returning back to sham values. In a 10-day survival study, vehicle-treated animals produced a 36% survival rate, while rhMFG-E8 significantly improved the survival rate to 68-72%. Treatment with increasing doses of rhMFG-E8 significantly reduced the number of apoptotic cells detected and markedly attenuated the tissue damages observed in the lungs. These data suggest that recombinant human MFG-E8 is beneficial in ameliorating sepsis in an animal model of sepsis.

  4. Fusion proteins useful for producing pinene

    DOEpatents

    Peralta-Yahya, Pamela P.; Keasling, Jay D

    2016-06-28

    The present invention provides for a modified host cell comprising a heterologous pinene synthase (PS), or enzymatically active fragment or variant thereof, and optionally a geranyl pyrophosphate synthase (GPPS), or enzymatically active fragment or variant thereof, or a fusion protein comprising: (a) a PS and (b) a GPPS linked by a linker.

  5. 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

  6. The MRE4 gene encodes a novel protein kinase homologue required for meiotic recombination in Saccharomyces cerevisiae.

    PubMed Central

    Leem, S H; Ogawa, H

    1992-01-01

    The MRE4 gene was cloned by complementation of the defects of meiotic recombination and haploidization in an mre4-1 mutant. Disruption of MRE4 resulted in reduced meiotic recombination and spore inviability. The mre4 spore lethality can be suppressed by spo13, a mutation that causes cells to bypass the reductional division. Analysis of meiotic DNA extracted from the mre4 mutant cells revealed that double-strand breaks occurred at the two sites of the HIS4-LEU2 recombination hot spot, but at a frequency of about 10-20% of the wild type. Northern blot analysis indicated that the MRE4 gene produces four transcripts of 1.63, 3.2, 4.0 and 6.2 kb. All of these transcripts are absent from mitotic cells and are meiotically induced. The DNA sequence of the MRE4 open reading frame predicts a 497-amino acids protein with a molecular mass of 56.8 kDa. The Mre4 protein contains highly conserved amino acid sequences found specifically in serine-threonine protein kinases. These results suggest that protein phosphorylation is required directly or indirectly for meiotic recombination. Images PMID:1741279

  7. 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.

  8. 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.

  9. Comparison of two codon optimization strategies to enhance recombinant protein production in Escherichia coli

    PubMed Central

    2011-01-01

    Background Variations in codon usage between species are one of the major causes affecting recombinant protein expression levels, with a significant impact on the economy of industrial enzyme production processes. The use of codon-optimized genes may overcome this problem. However, designing a gene for optimal expression requires choosing from a vast number of possible DNA sequences and different codon optimization methods have been used in the past decade. Here, a comparative study of the two most common methods is presented using calf prochymosin as a model. Results Seven sequences encoding calf prochymosin have been designed, two using the "one amino acid-one codon" method and five using a "codon randomization" strategy. When expressed in Escherichia coli, the variants optimized by the codon randomization approach produced significantly more proteins than the native sequence including one gene that produced an increase of 70% in the amount of prochymosin accumulated. On the other hand, no significant improvement in protein expression was observed for the variants designed with the one amino acid-one codon method. The use of codon-optimized sequences did not affect the quality of the recovered inclusion bodies. Conclusions The results obtained in this study indicate that the codon randomization method is a superior strategy for codon optimization. A significant improvement in protein expression was obtained for the largely established process of chymosin production, showing the power of this strategy to reduce production costs of industrial enzymes in microbial hosts. PMID:21371320

  10. Cloning of habutobin cDNA and antithrombotic activity of recombinant protein

    SciTech Connect

    Sunagawa, Masanori Nakamura, Mariko; Kosugi, Tadayoshi

    2007-11-03

    The habutobin cDNA was cloned from total RNA extracted from venom glands of Trimeresurus flavoviridis (the habu snake). The conceptual translation of 1539 bp of habutobin cDNA consists of 236 amino acids and its molecular weight is 25.7 kDa. Histidine (His)-tagged recombinant habutobin fusion protein, pET-r-habutobin and AcNPV-r-habutobin, was purified by bacterial system and baculoviral system, respectively. After refolding pET-r-habutobin, there were two protein bands at about 32 kDa and 65 kDa, indicating that habutobin might be produced as a monomer protein and processed to form two concatenated protein. Purified AcNPV-r-habutobin dose-dependently increased fibrin forming activity and inhibited collagen-induced aggregation of rabbit washed platelets. Thus, AcNPV-r-habutobin produced by baculoviral system is very useful for study on structure-function relationship, which is necessary for developing an antithrombotic drug from habutobin.

  11. Homologous recombination with linear DNA to insert antigenic protein in the flagellin: improvement of the Th1 immune response.

    PubMed

    Le Moigne, Vincent; Robreau, Georges; Mahana, Wahib

    2006-01-01

    Bacterial flagellin is a surface protein with numerous advantages for the presentation of exogenous peptides. However, the production of recombinant bacteria and the expression of fusion proteins is laborious and time consuming. Here, we present a simple way to produce modified bacteria. Partially deleted, non-functional, chromosomal flagellin gene (fliC ) was changed using homologous recombination by a functional linear fliC gene in which we introduced an exogenous oligonucleotide encoding for the peptide of interest. The modified fliC gene was produced by polymerase chain amplification. Linear amplicons were introduced into the non-motile E. coli by electroporation. The formation of functional flagellar filaments allowed the discrimination of motile transformants from non-motile, non-transformed cells. Thus antibiotic selection and gene expression inductors are not required since transformed bacteria can be easily isolated and used as a vector and adjuvant for immunization. To validate this hypothesis, we studied the immune response against the N-terminal peptide of Clostridium tyrobutyricum flagellin fragment. BALB/c mice were immunized either with the protein displayed as flagellin fusion protein on the surface of E. coli, with the recombinant protein in Freund's adjuvant (FA), or with the pcDNA3 vector bearing the DNA fragment encoding this protein. Immunization with the flagellin recombinant bacteria induced a strong Th1 response as measured by high level of IFN-gamma production and the lack of IL-4 production. The results indicate that the flagellar filament protein carrying a specific epitope can be a potent inducer of the Th1 cellular response.

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

    PubMed

    Carmona, Lina Marcela; Schatz, David G

    2017-06-01

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

  13. Induction of immune responses by two recombinant proteins of brucella abortus, outer membrane proteins 2b porin and Cu/Zn superoxide dismutase, in mouse model.

    PubMed

    Sung, Kyung Yong; Jung, Myunghwan; Shin, Min-Kyoung; Park, Hyun-Eui; Lee, Jin Ju; Kim, Suk; Yoo, Han Sang

    2014-06-28

    The diagnosis of Brucella abortus is mainly based on serological methods using antibody against LPS, which has diagnostic problems. Therefore, to solve this problem, we evaluated two proteins of B. abortus, Cu/Zn superoxide dismutase (SodC) and outer membrane proteins 2b porin (Omp2b). The genes were cloned and expressed in a pMAL system, and the recombinant proteins, rOmp2b and rSodC, were purified as fusion forms with maltosebinding protein. The identity of the proteins was confirmed by SDS-PAGE and Western blot analysis with sera of mice infected with B. abortus. Production of cytokines and nitric oxide (NO) was investigated in RAW 264.7 cells and mouse splenocytes after stimulation with the proteins. Moreover, cellular and humoral immune responses were investigated in BALB/c mice after immunization with the proteins. TNF-α, IL-6, and NO were significantly inducible in RAW 264.7 cells. Splenocytes of naive mice produced IFN-γ and IL-4 significantly by stimulation. Moreover, number of IgG, IFN-γ, and IL-4 producing cells were increased in immunized mice with the two proteins. Production of IgG and IgM with rOmp2b was higher than those with rSodC in immunized mice. These results suggest that the two recombinant proteins of B. abortus may be potential LPS-free proteins for diagnosis.

  14. One-step affinity purification of recombinant TATA binding proteins utilizing a modular protein interaction partner.

    PubMed

    Shooltz, Dean D; Alberts, Glen L; Triezenberg, Steven J

    2008-06-01

    We describe a rapid and effective procedure for purifying recombinant eukaryotic TATA binding protein (TBP) from Escherichia coli. The method employs an affinity ligand comprising glutathione-S-transferase fused to the carboxyl-terminal activation domain of the transcriptional activator VP16 and an amino-terminal domain (TAND2) of the yeast TBP-associated factor TAF1. TBP can be purified without the need for extrinsic affinity tags, subsequent proteolysis, or downstream clean-up steps. This TBP purification process is rapid (requiring about 4h after bacterial harvest) and does not require sophisticated chromatographic equipment. The resulting material is monodisperse, structured, and functionally active. We demonstrate the efficacy of this method for purifying recombinant full-length or TBP core fragments encoded by yeast, humans and Arabidopsis.

  15. 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.

  16. Preparation of chain-end clickable recombinant protein and its bio-orthogonal modification.

    PubMed

    Wang, Lin; Jiang, Rui; Wang, Lin; Liu, Yang; Sun, Xue-Long

    2016-04-01

    Introducing unique functional group into protein is an attractive approach for site-selective protein modification applications. In this report, we systemically investigated four site-selective strategies to introduce azide functionality into recombinant thrombomodulin (TM456), via direct recombinant expression with unnatural amino acid, chemical, and enzymatic modification for its bio-orthogonal modification application. First, a straightforward recombinant method to express TM456 with azide functionality near C-terminus by replacing methionine with azidohomoanlanine from methionine auxotroph Escherichia coli cell was investigated. Next, a sortase-mediated ligation (SML) method to incorporate azide functionality into the C-terminus of recombinant TM456 was demonstrated. The third is to add azide functionality to the N-terminal amine of recombinant TM456via amidation chemistry, and the fourth is tyrosine selective three-component Mannich reaction to introduce azide functionality to recombinant TM456. Overall, SML of recombinant protein affords the highest overall yield for incorporating azide functionality into the C-terminus recombinant TM456 since the key protein expression step uses natural amino acids. Also, single site modification facilitates the highest TM456 activity.

  17. Expression, delivery and function of insecticidal proteins expressed by recombinant baculoviruses

    USDA-ARS?s Scientific Manuscript database

    Since the development of methods for inserting and expressing genes in baculoviruses, a line of research has focused on developing recombinant baculoviruses that express insecticidal peptides and proteins. These recombinant viruses have been engineered with the goal of improving their pesticidal po...

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

    PubMed Central

    2011-01-01

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

  19. 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.

  20. Secretory delivery of recombinant proteins in attenuated Salmonella strains: potential and limitations of Type I protein transporters.

    PubMed

    Hahn, Heinz P; von Specht, Bernd Ulrich

    2003-07-15

    Live attenuated Salmonella strains have been extensively explored as oral delivery systems for recombinant vaccine antigens and effector proteins with immunoadjuvant and immunomodulatory potential. The feasibility of this approach was demonstrated in human vaccination trials for various antigens. However, immunization efficiencies with live vaccines are generally significantly lower compared to those monitored in parenteral immunizations with the same vaccine antigen. This is, at least partly, due to the lack of secretory expression systems, enabling large-scale extracellular delivery of vaccine and effector proteins by these strains. Because of their low complexity and the terminal location of the secretion signal in the secreted protein, Type I (ATP-binding cassette) secretion systems appear to be particularly suited for development of such recombinant extracellular expression systems. So far, the Escherichia coli hemolysin system is the only Type I secretion system, which has been adapted to recombinant protein secretion in Salmonella. However, this system has a number of disadvantages, including low secretion capacity, complex genetic regulation, and structural restriction to the secreted protein, which eventually hinder high-level in vivo delivery of recombinant vaccines and effector proteins. Thus, the development of more efficient recombinant protein secretion systems, based on Type I exporters can help to improve efficacies of live recombinant Salmonella vaccines. Type I secretion systems, mediating secretion of bacterial surface layer proteins, such as RsaA in Caulobacter crescentus, are discussed as promising candidates for improved secretory delivery systems.

  1. Intraclonal Protein Expression Heterogeneity in Recombinant CHO Cells

    PubMed Central

    Pilbrough, Warren; Munro, Trent P.; Gray, Peter

    2009-01-01

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

  2. Recombinant lipidated dengue-3 envelope protein domain III stimulates broad immune responses in mice.

    PubMed

    Chiang, Chen-Yi; Liu, Shih-Jen; Hsieh, Chun-Hsiang; Chen, Mei-Yu; Tsai, Jy-Ping; Liu, Hsueh-Hung; Chen, I-Hua; Chong, Pele; Leng, Chih-Hsiang; Chen, Hsin-Wei

    2016-02-17

    The linkage of an immunogen with a toll-like receptor ligand has great potential to induce highly potent immune responses with the initial features of antigen-presenting cell activation. In the current study, we expressed recombinant dengue-3 envelope protein domain III (D3ED III) in lipidated form using an Escherichia coli-based system. The recombinant lipidated dengue-3 envelope protein domain III (LD3ED III) augments the expression levels of IL-12 family cytokines. LD3ED III-immunized mice enhance wide ranges of T cell responses as indicated by IFN-γ, IL-17, IL-21 production. Additionally, LD3ED III-immunized mice increase the frequencies of anti-D3ED III antibody producing cells. The boosted antibody titers cover various IgG isotypes, including IgG1, IgG2a, IgG2b, and IgG3. Importantly, LD3ED III-immunized mice induce neutralizing antibody capacity associated with a reduction of viremia levels after challenges. In contrast, mice that are immunized with D3ED III formulated with aluminum phosphate (D3ED III/Alum) only enhance Th2 responses and boost IgG1 antibody titers. Neither neutralizing antibody responses nor the inhibition of viremia levels after challenge is observed in mice that are immunized with D3ED III/Alum. These results suggest that LD3ED III can induce broad profiles of cellular and humoral immune responses.

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

    PubMed

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

    2017-09-01

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

  4. Immune response to recombinant Escherichia coli Iss protein in poultry.

    PubMed

    Lynne, Aaron M; Foley, Steven L; Nolan, Lisa K

    2006-06-01

    Colibacillosis accounts for significant losses to the poultry industry, and control efforts are hampered by limited understanding of the mechanisms used by avian pathogenic Escherichia coli (APEC) to cause disease. We have found that the presence of the increased serum survival gene (iss) is strongly associated with APEC but not with commensal E. coli, making iss, and the protein it encodes (Iss), candidate targets of colibacillosis control procedures. To assess the potential of Iss to elicit a protective response in chickens against APEC challenge, Iss fusion proteins were produced and administered subcutaneously to four groups of 2-wk-old specific-pathogen-free leghorn chickens. At 4 wk postimmunization, birds were challenged with APEC from serogroups 02 and 078 via intramuscular injection. At 2 wk postchallenge, birds were necropsied, and lesions consistent with colibacillosis were scored. Also, sera were collected from the birds pre- and postimmunization, and antibody titers to Iss were determined. Immunized birds produced a humoral response to Iss, and they had significantly lower lesion scores than the unimmunized control birds following challenge with both APEC strains. Birds that received the smallest amount of immunogen had the lowest lesion scores. Although further study will be needed to confirm the value of Iss as an immunoprotective antigen, these preliminary data suggest that Iss may have the potential to elicit significant protection in birds against heterologous E. coli challenge.

  5. [Detection and antigenic characteristics of the recombinant nucleocapsid proteins of Lassa and Marburg viruses].

    PubMed

    Vladyko, A S; Scheslenok, E P; Fomina, E G; Semizhon, P A; Ignat'ev, G M; Shkolina, T V; Kras'ko, A G; Semenov, S F; Vinokurov, N V

    2012-01-01

    Two plasmid vectors, which allow the recombinant polypeptides of Lassa and Marburg viruses to be expressed in prokaryotic cells E. coli strain BL21 (DE3), were produced. The two recombinant polypeptides are able to bind specific antibodies. This provides an opportunity to use them as antigenic components of immunoassay diagnostic test kits.

  6. Engineering the cellular protein secretory pathway for enhancement of recombinant tissue plasminogen activator expression in Chinese hamster ovary cells: effects of CERT and XBP1s genes.

    PubMed

    Rahimpour, Azam; Vaziri, Behrouz; Moazzami, Reza; Nematollahi, Leila; Barkhordari, Farzaneh; Kokabee, Leila; Adeli, Ahmad; Mahboudi, Fereidoun

    2013-08-01

    Cell line development is the most critical and also the most time-consuming step in the production of recombinant therapeutic proteins. In this regard, a variety of vector and cell engineering strategies have been developed for generating high-producing mammalian cells; however, the cell line engineering approach seems to show various results on different recombinant protein producer cells. In order to improve the secretory capacity of a recombinant tissue plasminogen activator (t-PA)-producing Chinese hamster ovary (CHO) cell line, we developed cell line engineering approaches based on the ceramide transfer protein (CERT) and X-box binding protein 1 (XBP1) genes. For this purpose, CERT S132A, a mutant form of CERT that is resistant to phosphorylation, and XBP1s were overexpressed in a recombinant t-PA-producing CHO cell line. Overexpression of CERT S132A increased the specific productivity of t-PA-producing CHO cells up to 35%. In contrast, the heterologous expression of XBP1s did not affect the t-PA expression rate. Our results suggest that CERTS132A- based secretion engineering could be an effective strategy for enhancing recombinant t- PA production in CHO cells.

  7. Novel Feruloyl Esterase from Lactobacillus fermentum NRRL B-1932 and Analysis of the Recombinant Enzyme Produced in Escherichia coli

    PubMed Central

    Bischoff, Kenneth M.; Anderson, Amber M.; Rich, Joseph O.

    2016-01-01

    ABSTRACT A total of 33 Lactobacillus strains were screened for feruloyl esterase (FE) activity using agar plates containing ethyl ferulate as the sole carbon source, and Lactobacillus fermentum NRRL B-1932 demonstrated the strongest FE activity among a dozen species showing a clearing zone on the opaque plate containing ethyl ferulate. FE activities were monitored using high-performance liquid chromatography with an acetonitrile-trifluoroacetic acid gradient. To produce sufficient purified FE from L. fermentum strain NRRL B-1932 (LfFE), the cDNA encoding LfFE (Lffae) was amplified and cloned by using available closely related genome sequences and overexpressed in Escherichia coli. A 29.6-kDa LfFE protein was detected from the protein extract of E. coli BL21(pLysS) carrying pET28bLffae upon IPTG (isopropyl-β-d-thiogalactopyranoside) induction. The recombinant LfFE containing a polyhistidine tag was purified by nickel-nitrilotriacetic acid affinity resin. The purified LfFE showed strong activities against several artificial substrates, including p-nitrophenyl acetate and 4-methylumbelliferyl p-trimethylammoniocinnamate chloride. The optimum pH and temperature of the recombinant LfFE were around 6.5 and 37°C, respectively, as determined using either crude or purified recombinant LfFE. This study will be essential for the production of the LfFE in E. coli on a larger scale that could not be readily achieved by L. fermentum fermentation. IMPORTANCE The production of feruloyl esterase (FE) from Lactobacillus fermentum NRRL B-1932 reported in this study will have immense potential commercial applications not only in biofuel production but also in pharmaceutical, polymer, oleo chemical, cosmetic additive, and detergent industries, as well as human health-related applications, including food flavoring, functional foods, probiotic agents, preventive medicine, and animal feed. Given the essential role FE plays in the production of hydroxycinnamic acids and ferulic acid

  8. 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

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

    SciTech Connect

    Douette, Pierre; Navet, Rachel; Gerkens, Pascal; Galleni, Moreno; Levy, Daniel; Sluse, Francis E. . E-mail: F.Sluse@ulg.ac.be

    2005-08-05

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

  10. 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.

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

    PubMed Central

    Sørensen, Hans Peter; Mortensen, Kim Kusk

    2005-01-01

    Pure, soluble and functional proteins are of high demand in modern biotechnology. Natural protein sources rarely meet the requirements for quantity, ease of isolation or price and hence recombinant technology is often the method of choice. Recombinant cell factories are constantly employed for the production of protein preparations bound for downstream purification and processing. Eschericia coli is a frequently used host, since it facilitates protein expression by its relative simplicity, its inexpensive and fast high density cultivation, the well known genetics and the large number of compatible molecular tools available. In spite of all these qualities, expression of recombinant proteins with E. coli as the host often results in insoluble and/or nonfunctional proteins. Here we review new approaches to overcome these obstacles by strategies that focus on either controlled expression of target protein in an unmodified form or by applying modifications using expressivity and solubility tags. PMID:15629064

  12. Low-Tech, Pilot Scale Purification of a Recombinant Spider Silk Protein Analog from Tobacco Leaves

    PubMed Central

    Heppner, René; Weichert, Nicola; Schierhorn, Angelika; Conrad, Udo; Pietzsch, Markus

    2016-01-01

    Spider dragline is used by many members of the Araneae family not only as a proteinogenic safety thread but also for web construction. Spider dragline has been shown to possess high tensile strength in combination with elastic behavior. This high tensile strength can be attributed to the presence of antiparallel β-sheets within the thread; these antiparallel β-sheets are why the protein is classified as a silk. Due to the properties of spider silk and its technical and medical uses, including its use as a suture material and as a scaffold for tissue regeneration, spider dragline is a focus of the biotechnology industry. The production of sufficient amounts of spider silk is challenging, as it is difficult to produce large quantities of fibers because of the cannibalistic behavior of spiders and their large spatial requirements. In recent years, the heterologous expression of genes coding for spider silk analogs in various hosts, including plants such as Nicotiana tabacum, has been established. We developed a simple and scalable method for the purification of a recombinant spider silk protein elastin-like peptide fusion protein (Q-/K-MaSp1-100× ELP) after heterologous production in tobacco leaves involving heat and acetone precipitation. Further purification was performed using centrifugal Inverse Transition Cycling (cITC). Up to 400 mg of highly pure spider silk protein derivatives can be isolated from six kilograms of tobacco leaves, which is the highest amount of silk protein derivatives purified from plants thus far. PMID:27735843

  13. Low-Tech, Pilot Scale Purification of a Recombinant Spider Silk Protein Analog from Tobacco Leaves.

    PubMed

    Heppner, René; Weichert, Nicola; Schierhorn, Angelika; Conrad, Udo; Pietzsch, Markus

    2016-10-09

    Spider dragline is used by many members of the Araneae family not only as a proteinogenic safety thread but also for web construction. Spider dragline has been shown to possess high tensile strength in combination with elastic behavior. This high tensile strength can be attributed to the presence of antiparallel β-sheets within the thread; these antiparallel β-sheets are why the protein is classified as a silk. Due to the properties of spider silk and its technical and medical uses, including its use as a suture material and as a scaffold for tissue regeneration, spider dragline is a focus of the biotechnology industry. The production of sufficient amounts of spider silk is challenging, as it is difficult to produce large quantities of fibers because of the cannibalistic behavior of spiders and their large spatial requirements. In recent years, the heterologous expression of genes coding for spider silk analogs in various hosts, including plants such as Nicotiana tabacum, has been established. We developed a simple and scalable method for the purification of a recombinant spider silk protein elastin-like peptide fusion protein (Q-/K-MaSp1-100× ELP) after heterologous production in tobacco leaves involving heat and acetone precipitation. Further purification was performed using centrifugal Inverse Transition Cycling (cITC). Up to 400 mg of highly pure spider silk protein derivatives can be isolated from six kilograms of tobacco leaves, which is the highest amount of silk protein derivatives purified from plants thus far.

  14. Nanofibrillar hydrogel scaffolds from recombinant protein-based polymers with integrin- and proteoglycan-binding domains.

    PubMed

    Włodarczyk-Biegun, Małgorzata K; Werten, Marc W T; Posadowska, Urszula; Storm, Ingeborg M; de Wolf, Frits A; van den Beucken, Jeroen J J P; Leeuwenburgh, Sander C G; Cohen Stuart, Martien A; Kamperman, Marleen

    2016-12-01

    This study describes the design, production, and testing of functionalized variants of a recombinant protein-based polymer that forms nanofibrillar hydrogels with self-healing properties. With a view to bone tissue engineering applications, we equipped these variants with N-terminal extensions containing either (1) integrin-binding (RGD) or (2) less commonly studied proteoglycan-binding (KRSR) cell-adhesive motifs. The polymers were efficiently produced as secreted proteins using the yeast Pichia pastoris and were essentially monodisperse. The pH-responsive protein-based polymers are soluble at low pH and self-assemble into supramolecular fibrils and hydrogels at physiological pH. By mixing functionalized and nonfunctionalized proteins in different ratios, and adjusting pH, hydrogel scaffolds with the same protein concentration but varying content of the two types of cell-adhesive motifs were readily obtained. The scaffolds were used for the two-dimensional culture of MG-63 osteoblastic cells. RGD domains had a slightly stronger effect than KRSR domains on adhesion, activity, and spreading. However, scaffolds featuring both functional domains revealed a clear synergistic effect on cell metabolic activity and spreading, and provided the highest final degree of cell confluency. The mixed functionalized hydrogels presented here thus allowed to tailor the osteoblastic cell response, offering prospects for their further development as scaffolds for bone regeneration. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 3082-3092, 2016.

  15. 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

  16. Recombinant L-Asparaginase from Zymomonas mobilis: A Potential New Antileukemic Agent Produced in Escherichia coli

    PubMed Central

    Pereira, Juliana Christina Castanheira Vicente; Costa-Amaral, Isabele Campos; da Costa, Elaine Sobral; Ribeiro, Maria Cecília Menks; Land, Marcelo Gerardin Poirot; Alves, Tito Lívio Moitinho; Larentis, Ariane Leites; Almeida, Rodrigo Volcan

    2016-01-01

    L-asparaginase is an enzyme used as a chemotherapeutic agent, mainly for treating acute lymphoblastic leukemia. In this study, the gene of L-asparaginase from Zymomonas mobilis was cloned in pET vectors, fused to a histidine tag, and had its codons optimized. The L-asparaginase was expressed extracellularly and intracellularly (cytoplasmically) in Escherichia coli in far larger quantities than obtained from the microorganism of origin, and sufficient for initial cytotoxicity tests on leukemic cells. The in silico analysis of the protein from Z. mobilis indicated the presence of a signal peptide in the sequence, as well as high identity to other sequences of L-asparaginases with antileukemic activity. The protein was expressed in a bioreactor with a complex culture medium, yielding 0.13 IU/mL extracellular L-asparaginase and 3.6 IU/mL intracellular L-asparaginase after 4 h of induction with IPTG. The cytotoxicity results suggest that recombinant L-asparaginase from Z. mobilis expressed extracellularly in E.coli has a cytotoxic and cytostatic effect on leukemic cells. PMID:27253887

  17. Recombinant L-Asparaginase from Zymomonas mobilis: A Potential New Antileukemic Agent Produced in Escherichia coli.

    PubMed

    Einsfeldt, Karen; Baptista, Isis Cavalcante; Pereira, Juliana Christina Castanheira Vicente; Costa-Amaral, Isabele Campos; Costa, Elaine Sobral da; Ribeiro, Maria Cecília Menks; Land, Marcelo Gerardin Poirot; Alves, Tito Lívio Moitinho; Larentis, Ariane Leites; Almeida, Rodrigo Volcan

    2016-01-01

    L-asparaginase is an enzyme used as a chemotherapeutic agent, mainly for treating acute lymphoblastic leukemia. In this study, the gene of L-asparaginase from Zymomonas mobilis was cloned in pET vectors, fused to a histidine tag, and had its codons optimized. The L-asparaginase was expressed extracellularly and intracellularly (cytoplasmically) in Escherichia coli in far larger quantities than obtained from the microorganism of origin, and sufficient for initial cytotoxicity tests on leukemic cells. The in silico analysis of the protein from Z. mobilis indicated the presence of a signal peptide in the sequence, as well as high identity to other sequences of L-asparaginases with antileukemic activity. The protein was expressed in a bioreactor with a complex culture medium, yielding 0.13 IU/mL extracellular L-asparaginase and 3.6 IU/mL intracellular L-asparaginase after 4 h of induction with IPTG. The cytotoxicity results suggest that recombinant L-asparaginase from Z. mobilis expressed extracellularly in E.coli has a cytotoxic and cytostatic effect on leukemic cells.

  18. Fractionation of transgenic corn seed by dry and wet milling to recover recombinant collagen-related proteins.

    PubMed

    Zhang, Cheng; Glatz, Charles E; Fox, Steven R; Johnson, Lawrence A

    2009-01-01

    Corn continues to be considered an attractive transgenic host for producing recombinant therapeutic and industrial proteins because of its potential for producing recombinant proteins at large volume and low cost as coproducts of corn seed-based biorefining. Efforts to reduce production costs have been primarily devoted to increasing accumulation level, optimizing protein extraction conditions, and simplifying the purification. In the present work, we evaluated two grain fractionation methods, dry milling and wet milling, to enrich two recombinant collagen-related proteins; thereby, reducing the amount and type of corn-derived impurities in subsequent protein extraction and purification steps. The two proteins were a full-length human recombinant collagen type I alpha 1(rCIalpha1) chain with telopeptides and peptide foldon to effect triple helix formation and a 44-kDa rCIalpha1 fragment. For each, approximately 60% of the rCIalpha1s in the seed was recovered in the dry-milled germ-rich fractions making up ca. 25% of the total kernel mass. For wet milling, approximately 60% of each was recovered in three fractions accounting for 20-25% of the total kernel mass. The rCIalpha1s in the dry-milled germ-rich fractions were enriched three to six times compared with the whole corn kernel, whereas the rCIalpha1s were enriched 4-10 times in selected wet-milled fractions. The recovered starch from wet milling was almost free of rCIalpha1. Therefore, it was possible to generate rCIalpha1-enriched fractions by both dry and wet milling along with rCIalpha1-free starch using wet milling. Because of its simplicity, the dry milling procedure could be accomplished on-farm thus minimizing the risk of inadvertent release of viable transgenic seeds.

  19. Transplantation of genetically engineered cardiac fibroblasts producing recombinant human erythropoietin to repair the infarcted myocardium

    PubMed Central

    Ruvinov, Emil; Sharabani-Yosef, Orna; Nagler, Arnon; Einbinder, Tom; Feinberg, Micha S; Holbova, Radka; Douvdevani, Amos; Leor, Jonathan

    2008-01-01

    Background Erythropoietin possesses cellular protection properties. The aim of the present study was to test the hypothesis that in situ expression of recombinant human erythropoietin (rhEPO) would improve tissue repair in rat after myocardial infarction (MI). Methods and results RhEPO-producing cardiac fibroblasts were generated ex vivo by transduction with retroviral vector. The anti-apoptotic effect of rhEPO-producing fibroblasts was evaluated by co-culture with rat neonatal cardiomyocytes exposed to H2O2-induced oxidative stress. Annexin V/PI assay and DAPI staining showed that compared with control, rhEPO forced expression markedly attenuated apoptosis and improved survival of cultured cardiomyocytes. To test the effect of rhEPO on the infarcted myocardium, Sprague-Dawley rats were subjected to permanent coronary artery occlusion, and rhEPO-producing fibroblasts, non-transduced fibroblasts, or saline, were injected into the scar tissue seven days after infarction. One month later, immunostaining identified rhEPO expression in the implanted engineered cells but not in controls. Compared with non-transduced fibroblasts or saline injection, implanted rhEPO-producing fibroblasts promoted vascularization in the scar, and prevented cell apoptosis. By two-dimensional echocardiography and postmortem morphometry, transplanted EPO-engineered fibroblasts did not prevent left ventricular (LV) dysfunction and adverse LV remodeling 5 and 9 weeks after MI. Conclusion In situ expression of rhEPO enhances vascularization and reduces cell apoptosis in the infarcted myocardium. However, local EPO therapy is insufficient for functional improvement after MI in rat. PMID:19014419

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

    PubMed Central

    Yamaguchi, Hiroshi; Miyazaki, Masaya

    2014-01-01

    Biologically active proteins are useful for studying the biological functions of genes and for the development of therapeutic drugs and biomaterials in a biotechnology industry. Overexpression of recombinant proteins in bacteria, such as Escherichia coli, often results in the formation of inclusion bodies, which are protein aggregates with non-native conformations. As inclusion bodies contain relatively pure and intact proteins, protein refolding is an important process to obtain active recombinant proteins from inclusion bodies. However, conventional refolding methods, such as dialysis and dilution, are time consuming and, often, recovered yields of active proteins are low, and a trial-and-error process is required to achieve success. Recently, several approaches have been reported to refold these aggregated proteins into an active form. The strategies largely aim at reducing protein aggregation during the refolding procedure. This review focuses on protein refolding techniques using chemical additives and laminar flow in microfluidic chips for the efficient recovery of active proteins from inclusion bodies. PMID:24970214

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

    PubMed

    Yamaguchi, Hiroshi; Miyazaki, Masaya

    2014-02-20

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

  2. Differential humoral and cellular immunity induced by vaccination using plasmid DNA and protein recombinant expressing the NS3 protein of dengue virus type 3.

    PubMed

    Hurtado-Melgoza, M L; Ramos-Ligonio, A; Álvarez-Rodríguez, L M; Meza-Menchaca, T; López-Monteon, A

    2016-12-01

    The dengue non-structural 3 (NS3) is a multifunctional protein, containing a serine-protease domain, located at the N-terminal portion, and helicase, NTPase and RTPase domains present in the C-terminal region. This protein is considered the main target for CD4+ and CD8+ T cell responses during dengue infection, which may be involved in protection. However, few studies have been undertaken evaluating the use of this protein as a protective antigen against dengue, as well as other flavivirus. In the present work we evaluated the potential of the NS3 (protease domain) as a protective antigen by comparing the administration of a recombinant protein versus a DNA vaccine in the mouse model. BALB/c mice were immunized with the recombinant protein NS3-DEN3 via intraperitoneal and with plasmid pcDNA3/NS3-DEN3 intramuscularly and the immune response was evaluated. The activity of T lymphocytes was analyzed by the MTT assay, and cells of mice immunized with the recombinant protein showed no activity when stimulated with the homologous protein. However, cells from mice immunized with DNA, responded to stimulation with the recombinant protein. When the expression (RT-PCR) and cytokine production (ELISA) was evaluated in the splenocytes, different behavior depending on the type of immunization was observed, splenocytes of mice immunized with the recombinant protein expressed cytokines such as IL-4, IL-10 and produced high concentrations of IL-1, IL-6 and TNFα. Splenocytes from mice immunized with DNA expressed IL-2 and IFNγ and did not produce IL-6. In addition, immunization with the recombinant protein induced the production of antibodies that are detected up to a dilution 1:3200 by ELISA and Western blot assays, however, the serum of mice immunized with DNA presented no detectable antibody titers. The results obtained in this study show that administration of pcDNA3/NS3-DEN3 induces a favorable response in the activation of T lymphocytes with low production of specific

  3. Artificially designed recombinant protein composed of multiple epitopes of foot-and-mouth disease virus as a vaccine candidate.

    PubMed

    Lee, Ho-Bin; Piao, Da-Chuan; Lee, Jun-Yeong; Choi, Jae-Yun; Bok, Jin-Duck; Cho, Chong-Su; Kang, Sang-Kee; Choi, Yun-Jaie

    2017-02-22

    Concerns regarding the safety of inactivated foot-and-mouth disease (FMD) vaccine have been raised since it is produced from cultured live FMD virus (FMDV). To overcome this issue, recombinant protein has been studied as an alternative vaccine. We designed a chimerical multi-epitope recombinant protein (5BT), which is comprised of tandem repeats of five B cell epitopes (residue of VP1 136-162) derived from different FMDV variants and one T-cell epitope (residue of 3A 21-35). To increase solubility and stability of 5BT, it was conjugated with BmpB, the membrane protein B of Brachyspira hyodysenteriae (B5BT). Our results indicated that 5BT was susceptible to degradation by host protease and produced with substantial fraction of inclusion body. The stability and solubility of 5BT was greatly increased by conjugating to BmpB. FMDV specific antibodies were observed in the serum of mice immunized with 5BT and B5BT comparable to inactivated FMD vaccine. Sera from 5BT and B5BT groups also exhibited high epitope-specific antibody titers in peptide specific ELISA, indicating that all five epitopes are exposed to the B cell receptor for the antibody reaction. Thus the multi-epitope recombinant protein designed in this study may be a potential candidate as an alternative vaccine against FMDV epidemic variants.

  4. Lifetime and quenching of CO /a super 3 pi/ produced by recombination of CO2 ions in a helium afterglow.

    NASA Technical Reports Server (NTRS)

    Wauchop, T. S.; Broida, H. P.

    1972-01-01

    Demonstration that rapid dissociative recombination of CO2(+) in a flowing, helium afterglow is an efficient source of CO in the a super 3 pi metastable state. Ions produced by mixing CO2 with He(2 super 3 S) recombine to produce a CO metastable afterglow with a number density as great as 10 to the 9th per sq cm. Monitoring of the (a super 3 pi-X super 1 sigma) Cameron transition in CO was used to study the lifetime and quenching of CO (a super 3 pi) by CO2, N2, NO, and He. Recombination of CO2(+) also produces CO in the d super 3 delta and a' super 3 sigma states.

  5. Growth of recombinant Drosophila melanogaster Schneider 2 cells producing rabies virus glycoprotein in bioreactor employing serum-free medium

    PubMed Central

    Galesi, Adriana L. L.; Aguiar, Marcelo A.; Astray, Renato M.; Augusto, Elisabeth F. P.

    2008-01-01

    Drosophila melanogaster Schneider 2 (S2) cells have been increasingly used as a suitable expression system for the production of different recombinant proteins, and the employment of bioreactors for large-scale culture is an important tool for this purpose. In this work, Drosophila S2 cells producing the rabies virus glycoprotein RVGP were cultivated in bioreactor, employing a serum-free medium, aiming an improvement in cell growth and in glycoprotein production. To overcome cell growth limitation commonly observed in stirred flasks, different experiments in bioreactor were performed, in which some system modifications were carried out to attain the desired goal. The study showed that this cell line is considerably sensitive to hydrodynamic forces, and a high cell density (about 16.0 × 106 cells mL−1) was only obtained when Pluronic F68® percentage was increased to 0.6% (w/v). Despite ammonium concentration affected RVGP production, and also cell growth, an elevated amount of the target protein was obtained, attaining 563 ng 10−7 cells. PMID:19003175

  6. Development of rhizosecretion as a production system for recombinant proteins from hydroponic cultivated tobacco.

    PubMed

    Drake, Pascal M W; Barbi, Tommaso; Sexton, Amy; McGowan, Edward; Stadlmann, Johannes; Navarre, Catherine; Paul, Matthew J; Ma, Julian K-C

    2009-10-01

    a contained environment for pharmaceutical-producing plants, this study demonstrates advantages with respect to the quality and downstream purification of recombinant proteins.

  7. 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.

  8. Escherichia coli can produce recombinant chitinase in the soil to control the pathogenesis by Fusarium oxysporum without colonization.

    PubMed

    Chung, Soohee; Kim, Sang-Dal

    2007-03-01

    Fusarium wilt of cucumbers was effectively controlled by Escherichia coli expressing an endochitinase gene (chiA), and the rate was as effective (60.0%) as the wildtype strain S. proteamaculans 3095 (55.0%) where the gene was cloned. However, live cells of soil inoculated E. coli host harboring the chiA gene did not proliferate but declined 100-fold from 108 CFU during the first week and showed less than 10 cells after day 14, suggesting that E. coli was able to express and produce the chitinase enzyme to the soil even as the population was gradually decreasing. Because the majority of the strains was alive for only a short period of time and the Fusarium-affected seedlings showed symptoms of wilting within 7-10 days, it seems that the pathogen control was decided early after the introduction of the biocontrol agent, eliminating the survival of the antagonist. These results indicated that soil inoculated E. coli could sufficiently express and produce the recombinant protein to control the pathogen, and root or soil colonization of the antagonist might not be a significant factor in determining the efficacy of biological control.

  9. Bacterial-based Systems for Expression and Purification of Recombinant Lassa Virus Proteins of Immunological Relevance

    DTIC Science & Technology

    2008-06-06

    Biowarfare potential further jus- tifies the development of countermeasures against this highly virulent class of viruses . Methods Virus , cells, plasmids...BioMed CentralVirology Journal ssOpen AcceResearch Bacterial-based systems for expression and purification of recombinant Lassa virus proteins of...recombinant Lassa virus (LASV) proteins may serve as valuable tools in diverse antiviral applications. Bacterial-based systems were engineered for

  10. Development of a Recombinant Protein Vaccine Based on Cell-Free Protein Synthesis for Sevenband Grouper Epinephelus septemfasciatus Against Viral Nervous Necrosis.

    PubMed

    Kim, Jong-Oh; Kim, Jae-Ok; Kim, Wi-Sik; Oh, Myung-Joo

    2015-10-01

    Sevenband grouper, Epinephelus septemfasciatus, is becoming an important aquaculture species in Korea. However, viral nervous necrosis disease is a large problem causing mass mortality in sevenband grouper aquaculture. Recombinant protein vaccines are one of the best methods to reduce these economic losses. However, the cell-based expression method mainly produces inclusion bodies and requires additional procedures. In this study, we expressed a recombinant viral coat protein of sevenband grouper nervous necrosis virus (NNV) using a cell-free protein synthesis system. The purified recombinant NNV coat protein (rNNV-CP) was injected into sevenband grouper at different doses followed by a NNV challenge. Nonimmunized fish in the first trial (20 μg/fish) began to die 5 days post-challenge and reached 70% cumulative mortality. In contrast, immunized fish also starting dying 5 days postchallenge but lower cumulative mortality (10%) was observed. Cumulative morality in the second trial with different doses (20, 4, and 0.8 μg/fish) was 10%, 40%, and 50%, respectively. These results suggest that rNNV-CP can effectively immunize sevenband grouper depending on the dose administered. This study provides a new approach to develop a recombinant vaccine against NNV infection for sevenband grouper.

  11. A novel model system for design of biomaterials based on recombinant analogs of spider silk proteins.

    PubMed

    Bogush, Vladimir G; Sokolova, Olga S; Davydova, Lyubov I; Klinov, Dmitri V; Sidoruk, Konstantin V; Esipova, Natalya G; Neretina, Tatyana V; Orchanskyi, Igor A; Makeev, Vsevolod Yu; Tumanyan, Vladimir G; Shaitan, Konstantin V; Debabov, Vladimir G; Kirpichnikov, Mikhail P

    2009-03-01

    Spider dragline silk possesses impressive mechanical and biochemical properties. It is synthesized by a couple of major ampullate glands in spiders and comprises of two major structural proteins--spidroins 1 and 2. The relationship between structure and mechanical properties of spider silk is not well understood. Here, we modeled the complete process of the spider silk assembly using two new recombinant analogs of spidroins 1 and 2. The artificial genes sequence of the hydrophobic core regions of spidroin 1 and 2 have been designed using computer analysis of existing databases and mathematical modeling. Both proteins were expressed in Pichia pastoris and purified using a cation exchange chromatography. Despite the absence of hydrophilic N- and C-termini, both purified proteins spontaneously formed the nanofibrils and round micelles of about 1 microm in aqueous solutions. The electron microscopy study has revealed the helical structure of a nanofibril with a repeating motif of 40 nm. Using the electrospinning, the thin films with an antiparallel beta-sheet structure were produced. In summary, we were able to obtain artificial structures with characteristics that are perspective for further biomedical applications, such as producing three-dimensional matrices for tissue engineering and drug delivery.

  12. Scaled-up manufacturing of recombinant antibodies produced by plant cells in a 200-L orbitally-shaken disposable bioreactor.

    PubMed

    Raven, Nicole; Rasche, Stefan; Kuehn, Christoph; Anderlei, Tibor; Klöckner, Wolf; Schuster, Flora; Henquet, Maurice; Bosch, Dirk; Büchs, Jochen; Fischer, Rainer; Schillberg, Stefan

    2015-02-01

    Tobacco BY-2 cells have emerged as a promising platform for the manufacture of biopharmaceutical proteins, offering efficient protein secretion, favourable growth characteristics and cultivation in containment under a controlled environment. The cultivation of BY-2 cells in disposable bioreactors is a useful alternative to conventional stainless steel stirred-tank reactors, and orbitally-shaken bioreactors could provide further advantages such as simple bag geometry, scalability and predictable process settings. We carried out a scale-up study, using a 200-L orbitally-shaken bioreactor holding disposable bags, and BY-2 cells producing the human monoclonal antibody M12. We found that cell growth and recombinant protein accumulation were comparable to standard shake flask cultivation, despite a 200-fold difference in cultivation volume. Final cell fresh weights of 300-387 g/L and M12 yields of ∼20 mg/L were achieved with both cultivation methods. Furthermore, we established an efficient downstream process for the recovery of M12 from the culture broth. The viscous spent medium prevented clarification using filtration devices, but we used expanded bed adsorption (EBA) chromatography with SP Sepharose as an alternative for the efficient capture of the M12 antibody. EBA was introduced as an initial purification step prior to protein A affinity chromatography, resulting in an overall M12 recovery of 75-85% and a purity of >95%. Our results demonstrate the suitability of orbitally-shaken bioreactors for the scaled-up cultivation of plant cell suspension cultures and provide a strategy for the efficient purification of antibodies from the BY-2 culture medium.

  13. Selenomethionyl proteins produced for analysis by multiwavelength anomalous diffraction (MAD): a vehicle for direct determination of three-dimensional structure.

    PubMed Central

    Hendrickson, W A; Horton, J R; LeMaster, D M

    1990-01-01

    An expression system has been established for the incorporation of selenomethionine into recombinant proteins produced from plasmids in Escherichia coli. Replacement of methionine by selenomethionine is demonstrated at the level of 100% for both T4 and E. coli thioredoxins. The natural recombinant proteins and the selenomethionyl variants of both thioredoxins crystallize isomorphously. Anomalous scattering factors were deduced from synchrotron X-ray absorption measurements of crystals of the selenomethionyl proteins. Taken with reference to experience in the structural analysis of selenobiotinyl streptavidin by the method of multiwavelength anomalous diffraction (MAD), these data indicate that recombinant selenomethionyl proteins analyzed by MAD phasing offer a rather general means for the elucidation of atomic structures. Images Fig. 3. PMID:2184035

  14. Specific and efficient cleavage of fusion proteins by recombinant plum pox virus NIa protease.

    PubMed

    Zheng, Nuoyan; Pérez, José de Jesús; Zhang, Zhonghui; Domínguez, Elvira; Garcia, Juan Antonio; Xie, Qi

    2008-02-01

    Site-specific proteases are the most popular kind of enzymes for removing the fusion tags from fused target proteins. Nuclear inclusion protein a (NIa) proteases obtained from the family Potyviridae have become promising due to their high activities and stringencies of sequences recognition. NIa proteases from tobacco etch virus (TEV) and tomato vein mottling virus (TVMV) have been shown to process recombinant proteins successfully in vitro. In this report, recombinant PPV (plum pox virus) NIa protease was employed to process fusion proteins with artificial cleavage site in vitro. Characteristics such as catalytic ability and affecting factors (salt, temperature, protease inhibitors, detergents, and denaturing reagents) were investigated. Recombinant PPV NIa protease expressed and purified from Escherichia coli demonstrated efficient and specific processing of recombinant GFP and SARS-CoV nucleocapsid protein, with site F (N V V V H Q black triangle down A) for PPV NIa protease artificially inserted between the fusion tags and the target proteins. Its catalytic capability is similar to those of TVMV and TEV NIa protease. Recombinant PPV NIa protease reached its maximal proteolytic activity at approximately 30 degrees C. Salt concentration and only one of the tested protease inhibitors had minor influences on the proteolytic activity of PPV NIa protease. Recombinant PPV NIa protease was resistant to self-lysis for at least five days.

  15. Recombinant chymosin used for exact and complete removal of a prochymosin derived fusion tag releasing intact native target protein

    PubMed Central

    Justesen, Sune F L; Lamberth, Kasper; Nielsen, Lise-Lotte B; Schafer-Nielsen, Claus; Buus, Søren

    2009-01-01

    Fusion tags add desirable properties to recombinant proteins, but they are not necessarily acceptable in the final products. Ideally, fusion tags should be removed releasing the intact native protein with no trace of the tag. Unique endoproteinases with the ability to cleave outside their own recognition sequence can potentially cleave at the boundary of any native protein. Chymosin was recently shown to cleave a pro-chymosin derived fusion tag releasing native target proteins. In our hands, however, not all proteins are chymosin-resistant under the acidic cleavage conditions (pH 4.5) used in this system. Here, we have modified the pro-chymosin fusion tag and demonstrated that chymosin can remove this tag at more neutral pH (pH 6.2); conditions, that are less prone to compromise the integrity of target proteins. Chymosin was successfully used to produce intact native target protein both at the level of small and large-scale preparations. Using short peptide substrates, we further examined the influence of P1′ amino acid (the N-terminus of the native target protein) and found that chymosin accepts many different, although not all, amino acids. We conclude that chymosin has several appealing characteristics for the exact removal of fusion tags. It is readily available in highly purified recombinant versions approved by the FDA for preparation of food for human consumption. We suggest that one should consider extending the use of chymosin to the preparation of pharmaceutical proteins. PMID:19388053

  16. 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

  17. 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.

  18. Recombinants proteins for industrial uses: utilization of Pichia pastoris expression system.

    PubMed

    Rabert, Claudia; Weinacker, Daniel; Pessoa, Adalberto; Farías, Jorge G

    2013-01-01

    The innovation in industrial process with impact in the efficient production is the major challenge for actual industry. A high numerous of enzymes are utilized in at different level of process; the search for new alternatives with better characteristic has become a field of study of great interest, the recombinant protein achievement in a different host system is an alternative widely assessed for production of this. The microorganism Pichia pastoris has been used like a successful expression system in diverse areas, improved the yield and extraction-recovery of the product expressed. The reported of diverse authors in the production of enzymes with different application in industry is varied, in this review the different industry areas and the characteristic of the enzymes produced are detailed.

  19. Recombinants proteins for industrial uses: utilization of Pichia pastoris expression system

    PubMed Central

    Rabert, Claudia; Weinacker, Daniel; Pessoa, Adalberto; Farías, Jorge G.

    2013-01-01

    The innovation in industrial process with impact in the efficient production is the major challenge for actual industry. A high numerous of enzymes are utilized in at different level of process; the search for new alternatives with better characteristic has become a field of study of great interest, the recombinant protein achievement in a different host system is an alternative widely assessed for production of this. The microorganism Pichia pastoris has been used like a successful expression system in diverse areas, improved the yield and extraction-recovery of the product expressed. The reported of diverse authors in the production of enzymes with different application in industry is varied, in this review the different industry areas and the characteristic of the enzymes produced are detailed. PMID:24294221

  20. Involvement of a periplasmic protein kinase in DNA strand break repair and homologous recombination in Escherichia coli.

    PubMed

    Khairnar, Nivedita P; Kamble, Vidya A; Mangoli, Suhas H; Apte, Shree K; Misra, Hari S

    2007-07-01

    The involvement of signal transduction in the repair of radiation-induced damage to DNA has been known in eukaryotes but remains understudied in bacteria. This article for the first time demonstrates a role for the periplasmic lipoprotein (YfgL) with protein kinase activity transducing a signal for DNA strand break repair in Escherichia coli. Purified YfgL protein showed physical as well as functional interaction with pyrroloquinoline-quinone in solution and the protein kinase activity of YfgL was strongly stimulated in the presence of pyrroloquinoline-quinone. Transgenic E. coli cells producing Deinococcus radiodurans pyrroloquinoline-quinone synthase showed nearly four log cycle improvement in UVC dark survival and 10-fold increases in gamma radiation resistance as compared with untransformed cells. Pyrroloquinoline-quinone enhanced the UV resistance of E. coli through the YfgL protein and required the active recombination repair proteins. The yfgL mutant showed higher sensitivity to UVC, mitomycin C and gamma radiation as compared with wild-type cells and showed a strong impairment in homologous DNA recombination. The mutant expressing an active YfgL in trans recovered the lost phenotypes to nearly wild-type levels. The results strongly suggest that the periplasmic phosphoquinolipoprotein kinase YfgL plays an important role in radiation-induced DNA strand break repair and homologous recombination in E. coli.

  1. G protein modulation of recombinant P/Q-type calcium channels by regulators of G protein signalling proteins.

    PubMed

    Mark, M D; Wittemann, S; Herlitze, S

    2000-10-01

    1. Fast synaptic transmission is triggered by the activation of presynaptic Ca2+ channels which can be inhibited by Gbetagamma subunits via G protein-coupled receptors (GPCR). Regulators of G protein signalling (RGS) proteins are GTPase-accelerating proteins (GAPs), which are responsible for >100-fold increases in the GTPase activity of G proteins and might be involved in the regulation of presynaptic Ca2+ channels. In this study we investigated the effects of RGS2 on G protein modulation of recombinant P/Q-type channels expressed in a human embryonic kidney (HEK293) cell line using whole-cell recordings. 2. RGS2 markedly accelerates transmitter-mediated inhibition and recovery from inhibition of Ba2+ currents (IBa) through P/Q-type channels heterologously expressed with the muscarinic acetylcholine receptor M2 (mAChR M2). 3. Both RGS2 and RGS4 modulate the prepulse facilitation properties of P/Q-type Ca2+ channels. G protein reinhibition is accelerated, while release from inhibition is slowed. These kinetics depend on the availability of G protein alpha and betagamma subunits which is altered by RGS proteins. 4. RGS proteins unmask the Ca2+ channel beta subunit modulation of Ca2+ channel G protein inhibition. In the presence of RGS2, P/Q-type channels containing the beta2a and beta3 subunits reveal significantly altered kinetics of G protein modulation and increased facilitation compared to Ca2+ channels coexpressed with the beta1b or beta4 subunit.

  2. Vacuolar deposition of recombinant proteins in plant vegetative organs as a strategy to increase yields.

    PubMed

    Marin Viegas, Vanesa Soledad; Ocampo, Carolina Gabriela; Petruccelli, Silvana

    2017-05-04

    Delivery of recombinant proteins to vegetative tissue vacuoles was considered inconvenient since this compartment was expected to be hydrolytic; nevertheless there is growing evidence that certain foreign proteins accumulate at high yields in vacuoles. For example avidin, cellulolytic enzymes, endolysin, and transglutaminases were produced at high yields when were sorted to leaf central vacuole avoiding the detrimental effect of these proteins on plant growth. Also, several secretory mammalian proteins such as collagen, α1-proteinase inhibitor, complement-5a, interleukin-6 and immunoglobulins accumulated at higher yields in leaf vacuoles than in the apoplast or cytosol. To reach this final destination, fusions to sequence specific vacuolar sorting signals (ssVSS) typical of proteases or proteinase inhibitors and/or Ct-VSS representative of storage proteins or plant lectins were used and both types of motifs were capable to increase accumulation. Importantly, the type of VSSs or position, either the N or C-terminus, did not alter protein stability, levels or pos-translational modifications. Vacuolar sorted glycoproteins had different type of oligosaccharides indicating that foreign proteins reached the vacuole by 2 different pathways: direct transport from the ER, bypassing the Golgi (high mannose oligosaccharides decorated proteins) or trafficking through the Golgi (Complex oligosaccharide containing proteins). In addition, some glycoproteins lacked of paucimannosidic oligosaccharides suggesting that vacuolar trimming of glycans did not occur. Enhanced accumulation of foreign proteins fused to VSS occurred in several plant species such as tobacco, Nicotiana benthamiana, sugarcane, tomato and in carrot and the obtained results were influenced by plant physiological state. Ten different foreign proteins fused to vacuolar sorting accumulated at higher levels than their apoplastic or cytosolic counterparts. For proteins with cytotoxic effects vacuolar sorted forms

  3. The Slx5-Slx8 complex affects sumoylation of DNA repair proteins and negatively regulates recombination.

    PubMed

    Burgess, Rebecca C; Rahman, Sadia; Lisby, Michael; Rothstein, Rodney; Zhao, Xiaolan

    2007-09-01

    Recombination is important for repairing DNA lesions, yet it can also lead to genomic rearrangements. This process must be regulated, and recently, sumoylation-mediated mechanisms were found to inhibit Rad51-dependent recombination. Here, we report that the absence of the Slx5-Slx8 complex, a newly identified player in the SUMO (small ubiquitin-like modifier) pathway, led to increased Rad51-dependent and Rad51-independent recombination. The increases were most striking during S phase, suggesting an accumulation of DNA lesions during replication. Consistent with this view, Slx8 protein localized to replication centers. In addition, like SUMO E2 mutants, slx8Delta mutants exhibited clonal lethality, which was due to the overamplification of 2 microm, an extrachromosomal plasmid. Interestingly, in both SUMO E2 and slx8Delta mutants, clonal lethality was rescued by deleting genes required for Rad51-independent recombination but not those involved in Rad51-dependent events. These results suggest that sumoylation negatively regulates Rad51-independent recombination, and indeed, the Slx5-Slx8 complex affected the sumoylation of several enzymes involved in early steps of Rad51-independent recombination. We propose that, during replication, the Slx5-Slx8 complex helps prevent DNA lesions that are acted upon by recombination. In addition, the complex inhibits Rad51-independent recombination via modulating the sumoylation of DNA repair proteins.

  4. Recombinant proteinase 3 produced in different expression systems: recognition by anti-PR3 antibodies.

    PubMed

    van der Geld, Y M; Oost-Kort, W; Limburg, P C; Specks, U; Kallenberg, C G

    2000-10-20

    Anti-neutrophil cytoplasm autoantibodies (ANCA) directed against proteinase 3 (PR3) are highly sensitive and specific markers for Wegener's granulomatosis (WG). Consequently, antigen-specific assays for detection of PR3-ANCA are helpful for the diagnosis and follow-up of patients with WG. Purification of PR3 is laborious and requires large amounts of granulocytes. Therefore, several attempts have been made to produce recombinant PR3 that is recognized by PR3-ANCA. The purpose of this study was to compare the recognition of different recombinant forms of PR3 (rPR3) by anti-PR3 antibodies. Recombinant PR3 produced in E. coli (rcPR3), P. pastoris (rpPR3), insect cells using the baculovirus system (rbPR3), the human mast cell line, HMC-1 (HMC-1/PR3-S176A), or the human epithelial cell line, 293 (Delta-rPR3-S176A) as well as purified neutrophil PR3 (nPR3) were used. Recognition of these rPR3s by anti-PR3 antibodies was determined by direct and capture ELISA with 19 PR3-ANCA sera, 13 anti-PR3 mAbs and a rabbit serum raised against human PR3. In the capture ELISA rabbit anti-PR3 strongly bound nPR3 and all rPR3 products. By capture ELISA rcPR3 and rpPR3 were recognized by 11 (57%) and 13 (68%) of the 19 PR3-ANCA sera, respectively, whereas rbPR3, HMC-1/PR3-S176A, Delta-rPR3-S176A and nPR3 were recognized by all PR3-ANCA sera. By direct ELISA rabbit anti-PR3 strongly bound nPR3 and all tested rPR3 products. Using the direct ELISA none of the PR3-ANCA sera recognized rcPR3, whereas rpPR3 and rbPR3 were recognized by two (11%) and 17 (89%) of the 19 PR3-ANCA sera, respectively. All 13 anti-PR3 mAbs recognized nPR3 in the direct as well as in the capture ELISA. The rcPR3 was recognized by two mAbs in the capture ELISA but by none of the mAbs in the direct ELISA. The rpPR3 was recognized by seven mAbs in the capture ELISA and only by two mAbs in the direct ELISA. All but one of the anti-PR3 mAbs recognized rbPR3, whereas HMC-1/PR3-S176A and Delta-rPR3-S176A were recognized by

  5. Production of Recombinant CCN Proteins by Brevibacillus choshinensis.

    PubMed

    Hanagata, Hiroshi; Mizukami, Makoto

    2017-01-01

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

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

    SciTech Connect

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

    2012-10-26

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

  7. Mgm101 is a Rad52-related protein required for mitochondrial DNA recombination.

    PubMed

    Mbantenkhu, MacMillan; Wang, Xiaowen; Nardozzi, Jonathan D; Wilkens, Stephan; Hoffman, Elizabeth; Patel, Anamika; Cosgrove, Michael S; Chen, Xin Jie

    2011-12-09

    Homologous recombination is a conserved molecular process that has primarily evolved for the repair of double-stranded DNA breaks and stalled replication forks. However, the recombination machinery in mitochondria is poorly understood. Here, we show that the yeast mitochondrial nucleoid protein, Mgm101, is related to the Rad52-type recombination proteins that are widespread in organisms from bacteriophage to humans. Mgm101 is required for repeat-mediated recombination and suppression of mtDNA fragmentation in vivo. It preferentially binds to single-stranded DNA and catalyzes the annealing of ssDNA precomplexed with the mitochondrial ssDNA-binding protein, Rim1. Transmission electron microscopy showed that Mgm101 forms large oligomeric rings of ∼14-fold symmetry and highly compressed helical filaments. Specific mutations affecting ring formation reduce protein stability in vitro. The data suggest that the ring structure may provide a scaffold for stabilization of Mgm101 by preventing the aggregation of the otherwise unstable monomeric conformation. Upon binding to ssDNA, Mgm101 is remobilized from the rings to form distinct nucleoprotein filaments. These studies reveal a recombination protein of likely bacteriophage origin in mitochondria and support the notion that recombination is indispensable for mtDNA integrity.

  8. Expression and purification of recombinant proteins based on human prostate stem cell antigen and heat shock protein-70

    PubMed Central

    DONG, LEI; ZHANG, XIAOPENG; YU, CHANGMING; REN, JUN; HOU, LIHUA; FU, LING; YI, SHAOQIONG; CHEN, WEI

    2013-01-01

    The aim of this study was to express and purify recombinant proteins based on human prostate stem cell antigen (PSCA) and heat shock protein-70 (HSP70). The PSCA gene and various structural domains of HSP70 were amplified by polymerase chain reaction (PCR) with the respective primers. Then, the PSCA was cloned into the prokaryotic expression vector pET21a(+) with the amino-terminus, carboxyl-terminus and overall length of HSP70, by enzyme digestion to construct the recombinant plasmids pET21-PSCA-HSPN, pET21-PSCA-HSPC and pET21-PSCA-HSP, respectively. After being expressed in Escherichia coli (E. coli) by isopropyl β-D-1-thiogalactopyranoside (IPTG) induction, recombinant fusion proteins were purified. Western blotting was performed to confirm the expression of the recombinant proteins. The results revealed that recombinant plasmids were successfully constructed. The PSCA-HSPC and PSCA-HSP expressed in E. coli existed in soluble form, as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purity of the recombinant proteins PSCA-HSPC and PSCA-HSP reached >95% following purification with the nickel-nitrilotriacetic acid (Ni-NTA) resin, Phenyl-Sepharose Fast Flow and Superdex 75, which lays a foundation for the development of vaccines for prostate cancer. PMID:23596484

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

    PubMed

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

    2016-02-15

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

  10. Recombinant mussel adhesive protein fp-5 (MAP fp-5) as a bulk bioadhesive and surface coating material.

    PubMed

    Choi, Yoo Seong; Kang, Dong Gyun; Lim, Seonghye; Yang, Yun Jung; Kim, Chang Sup; Cha, Hyung Joon

    2011-08-01

    Mussel adhesive proteins (MAPs) attach to all types of inorganic and organic surfaces, even in wet environments. MAP of type 5 (fp-5), in particular, has been considered as a key adhesive material. However, the low availability of fp-5 has hampered its biochemical characterization and practical applications. Here, soluble recombinant fp-5 is mass-produced in Escherichia coli. Tyrosinase-modified recombinant fp-5 showed ∼1.11 MPa adhesive shear strength, which is the first report of a bulk-scale adhesive force measurement for purified recombinant of natural MAP type. Surface coatings were also performed through simple dip-coating of various objects. In addition, complex coacervate using recombinant fp-5 and hyaluronic acid was prepared as an efficient adhesive formulation, which greatly improved the bulk adhesive strength. Collectively, it is expected that this work will enhance basic understanding of mussel adhesion and that recombinant fp-5 can be successfully used as a realistic bulk-scale bioadhesive and an efficient surface coating material.

  11. Recombinant Scorpine Produced Using SUMO Fusion Partner in Escherichia coli Has the Activities against Clinically Isolated Bacteria and Inhibits the Plasmodium falciparum Parasitemia In Vitro

    PubMed Central

    Gu, Yaping; Zhou, Huayun; Cao, Jun; Gao, Qi

    2014-01-01

    Scorpine, a small cationic peptide from the venom of Pandinus imperator, which has been shown to have anti-bacterial and anti-plasmodial activities, has potential important applications in the pharmaceutical industries. However, the isolation of scorpine from natural sources is inefficient and time-consuming. Here, we first report the expression and purification of recombinant scorpine in Escherichia coli, using small ubiquitin-related modifier (SUMO) fusion partner. The fusion protein was expressed in soluble form in E. coli, and expression was verified by SDS-PAGE and western blotting analysis. The fusion protein was purified to 90% purity by nickel–nitrilotriacetic acid (Ni2+–NTA) resin chromatography. After the SUMO-scorpine fusion protein was cleaved by the SUMO protease, the cleaved sample was reapplied to a Ni2+–NTA column. Tricine/SDS-PAGE gel results indicated that Scorpine had been purified successfully to more than 95% purity. The recombinantly expressed Scorpine showed anti-bacterial activity against two standard bacteria including Staphylococcus aureus ATCC 29213 and Acinetobacter baumannii ATCC 19606, and clinically isolated bacteria including S. aureus S, S. aureus R, A. baumannii S, and A. baumannii R. It also produced 100% reduction in Plasmodium falciparum parasitemia in vitro. Thus, the expression strategy presented in this study allowed convenient high yield and easy purification of recombinant Scorpine for pharmaceutical applications in the future. PMID:25068263

  12. Recombinant Adenovirus Delivery of Calreticulin–ESAT-6 Produces an Antigen-Specific Immune Response but no Protection Against a Mycobacterium Tuberculosis Challenge

    PubMed Central

    Esparza-González, S. C.; Troy, A.; Troudt, J.; Loera-Arias, M. J.; Villatoro-Hernández, J.; Torres-López, E.; Ancer-Rodríguez, J.; Gutiérrez-Puente, Y.; Muñoz-Maldonado, G.; Saucedo-Cárdenas, O.; Montes-de-Oca-Luna, R.; Izzo, A.

    2015-01-01

    Bacillus Calmette–Guerin (BCG) has failed to efficaciously control the worldwide spread of the disease. New vaccine development targets virulence antigens of Mycobacterium tuberculosis that are deleted in Mycobacterium bovis BCG. Immunization with ESAT-6 and CFP10 provides protection against M. tuberculosis in a murine infection model. Further, previous studies have shown that calreticulin increases the cell-mediated immune responses to antigens. Therefore, to test whether calreticulin enhances the immune response against M. tuberculosis antigens, we fused ESAT-6 to calreticulin and constructed a recombinant replication-deficient adenovirus to express the resulting fusion protein (AdCRT–ESAT-6). The adjuvant effect of calreticulin was assayed by measuring cytokine responses specific to ESAT-6. Recombinant adenovirus expressing the fusion protein produced higher levels of interferon-γ and tumour necrosis factor-α in response to ESAT-6. This immune response was not improved by the addition of CFP-10 to the CRT-ESAT-6 fusion protein (AdCRT–ESAT-6–CFP10). Mice immunized with these recombinant adenoviruses did not decrease the mycobacterial burden after low-dose aerosol infection with M. tuberculosis. We conclude that calreticulin can be used as an adjuvant to enhance the immune response against mycobacterial antigens, but it is not enough to protect against tuberculosis. PMID:22010821

  13. Use of the 27-Kilodalton Recombinant Protein from Paracoccidioides brasiliensis in Serodiagnosis of Paracoccidioidomycosis

    PubMed Central

    Ortiz, B. L.; Díez, S.; Urán, M. E.; Rivas, J. M.; Romero, M.; Caicedo, V.; Restrepo, A.; McEwen, J. G.

    1998-01-01

    Paracoccidioidomycosis (PCM) is one of the most important endemic mycoses in Latin America; it is usually diagnosed by observation and/or isolation of the etiologic agent, Paracoccidioides brasiliensis, as well as by a variety of immunological methods. Although the latter are effective, two circumstances, cross-reactions with other mycotic agents and antigen preparation that is marked by extreme variability among lots, hinder proper standardization of the procedures. To circumvent this lack of reproducibility, molecular biology tools were used to produce a recombinant 27-kDa-molecular-mass antigen from this fungus; a sizable quantity of this antigen was obtained through fermentation of Escherichia coli DH5α, which is capable of expressing the fungal protein. The latter was purified by the Prep-Cell System (Bio-Rad); the recovery rate of the pure protein was approximately 6%. A battery of 160 human serum samples, consisting of 64 specimens taken at the time of diagnosis from patients with PCM representing the various clinical forms plus 15 serum specimens each from patients with histoplasmosis and aspergillosis, 10 each from patients with cryptococcosis and tuberculosis, 6 from patients with coccidioidomycosis, and 40 from healthy subjects, were all tested by an indirect enzyme-linked immunosorbent assay with the purified 27-kDa recombinant protein. The latter was used at a concentration of 1.0 μg/well; there were three serum dilutions (1:1,000, 1:2,000, and 1:4,000). The experiment was repeated at least twice. The average sensitivity for both experiments was 73.4%; in comparison with the healthy subjects, the specificity for PCM patients was 87.5% while for patients with other mycoses, it was 58.7%. Important cross-reactions with sera from patients with aspergillosis and histoplasmosis were detected. The positive predictive value of the test was 90.4%. These results indicate that it is possible to employ recombinant antigenic proteins for the immunologic

  14. Use of the 27-kilodalton recombinant protein from Paracoccidioides brasiliensis in serodiagnosis of paracoccidioidomycosis.

    PubMed

    Ortiz, B L; Díez, S; Urán, M E; Rivas, J M; Romero, M; Caicedo, V; Restrepo, A; McEwen, J G

    1998-11-01

    Paracoccidioidomycosis (PCM) is one of the most important endemic mycoses in Latin America; it is usually diagnosed by observation and/or isolation of the etiologic agent, Paracoccidioides brasiliensis, as well as by a variety of immunological methods. Although the latter are effective, two circumstances, cross-reactions with other mycotic agents and antigen preparation that is marked by extreme variability among lots, hinder proper standardization of the procedures. To circumvent this lack of reproducibility, molecular biology tools were used to produce a recombinant 27-kDa-molecular-mass antigen from this fungus; a sizable quantity of this antigen was obtained through fermentation of Escherichia coli DH5alpha, which is capable of expressing the fungal protein. The latter was purified by the Prep-Cell System (Bio-Rad); the recovery rate of the pure protein was approximately 6%. A battery of 160 human serum samples, consisting of 64 specimens taken at the time of diagnosis from patients with PCM representing the various clinical forms plus 15 serum specimens each from patients with histoplasmosis and aspergillosis, 10 each from patients with cryptococcosis and tuberculosis, 6 from patients with coccidioidomycosis, and 40 from healthy subjects, were all tested by an indirect enzyme-linked immunosorbent assay with the purified 27-kDa recombinant protein. The latter was used at a concentration of 1.0 microgram/well; there were three serum dilutions (1:1,000, 1:2,000, and 1:4,000). The experiment was repeated at least twice. The average sensitivity for both experiments was 73.4%; in comparison with the healthy subjects, the specificity for PCM patients was 87.5% while for patients with other mycoses, it was 58.7%. Important cross-reactions with sera from patients with aspergillosis and histoplasmosis were detected. The positive predictive value of the test was 90.4%. These results indicate that it is possible to employ recombinant antigenic proteins for the immunologic

  15. Increased Understanding of the Biochemistry and Biosynthesis of MUC2 and Other Gel-Forming Mucins Through the Recombinant Expression of Their Protein Domains

    PubMed Central

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

    2016-01-01

    The gel-forming mucins are large and heavily O-glycosylated proteins which build up mucus gels. The recombinant production of full-length gel-forming mucins has not been possible to date. In order to study mucin biosynthesis and biochemistry, we and others have taken the alternative approach of constructing different recombinant proteins consisting of one or several domains of these large proteins and expressing them separately in different cell lines. Using this approach, we have determined that MUC2, the intestinal gel-forming mucin, dimerizes via its C-terminal cysteine-knot domain and also trimerizes via one of the N-terminal von Willebrand D domains. Both of these interactions are disulfide bond mediated. Via this assembly, a molecular network is built by which the mucus gel is formed. Here we discuss not only the functional understanding obtained from studies of the recombinant proteins, but also highlight the difficulties encountered when these proteins were produced recombinantly. We often found an accumulation of the proteins in the ER and consequently no secretion. This was especially apparent when the cysteine-rich domains of the N- and C-terminal parts of the mucins were expressed. Other proteins that we constructed were either not secreted or not expressed at all. Despite these problems, the knowledge of mucin biosynthesis and assembly has advanced considerably through the studies of these recombinant proteins. PMID:23359125

  16. Recombination produces coherent bacterial species clusters in both core and accessory genomes

    PubMed Central

    Croucher, Nicholas J.; Gutmann, Michael U.; Corander, Jukka; Hanage, William P.

    2015-01-01

    Background: Population samples show bacterial genomes can be divided into a core of ubiquitous genes and accessory genes that are present in a fraction of isolates. The ecological significance of this variation in gene content remains unclear. However, microbiologists agree that a bacterial species should be ‘genomically coherent’, even though there is no consensus on how this should be determined. Results: We use a parsimonious model combining diversification in both the core and accessory genome, including mutation, homologous recombination (HR) and horizontal gene transfer (HGT) introducing new loci, to produce a population of interacting clusters of strains with varying genome content. New loci introduced by HGT may then be transferred on by HR. The model fits well to a systematic population sample of 616 pneumococcal genomes, capturing the major features of the population structure with parameter values that agree well with empirical estimates. Conclusions: The model does not include explicit selection on individual genes, suggesting that crude comparisons of gene content may be a poor predictor of ecological function. We identify a clearly divergent subpopulation of pneumococci that are inconsistent with the model and may be considered genomically incoherent with the rest of the population. These strains have a distinct disease tropism and may be rationally defined as a separate species. We also find deviations from the model that may be explained by recent population bottlenecks or spatial structure.

  17. Cytotoxic and Apoptotic Effects of Recombinant Subtilase Cytotoxin Variants of Shiga Toxin-Producing Escherichia coli

    PubMed Central

    Funk, J.; Biber, N.; Schneider, M.; Hauser, E.; Enzenmüller, S.; Förtsch, C.

    2015-01-01

    In this study, the cytotoxicity of the recently described subtilase variant SubAB2-2 of Shiga toxin-producing Escherichia coli was determined and compared to the plasmid-encoded SubAB1 and the chromosome-encoded SubAB2-1 variant. The genes for the respective enzymatic active (A) subunits and binding (B) subunits of the subtilase toxins were amplified and cloned. The recombinant toxin subunits were expressed and purified. Their cytotoxicity on Vero cells was measured for the single A and B subunits, as well as for mixtures of both, to analyze whether hybrids with toxic activity can be identified. The results demonstrated that all three SubAB variants are toxic for Vero cells. However, the values for the 50% cytotoxic dose (CD50) differ for the individual variants. Highest cytotoxicity was shown for SubAB1. Moreover, hybrids of subunits from different subtilase toxins can be obtained which cause substantial cytotoxicity to Vero cells after mixing the A and B subunits prior to application to the cells, which is characteristic for binary toxins. Furthermore, higher concentrations of the enzymatic subunit SubA1 exhibited cytotoxic effects in the absence of the respective B1 subunit. A more detailed investigation in the human HeLa cell line revealed that SubA1 alone induced apoptosis, while the B1 subunit alone did not induce cell death. PMID:25824835

  18. CHO-gmt5, a novel CHO glycosylation mutant for producing afucosylated and asialylated recombinant antibodies.

    PubMed

    Haryadi, Ryan; Zhang, Peiqing; Chan, Kah Fai; Song, Zhiwei

    2013-01-01

    Engineered zinc-finger nucleases (ZFNs) are powerful tools for creating double-stranded-breaks (DSBs) in genomic DNA in a site-specific manner. These DSBs generated by ZFNs can be repaired by homology-directed repair or nonhomologous end joining, in which the latter can be exploited to generate insertion or deletion mutants. Based on published literature, we designed a pair of zinc-finger nucleases and inactivated the GDP-fucose transporter gene (Slc35c1) in a previously reported CHO mutant that has a dysfunctional CMP-sialic acid transporter gene (Slc35a1). The resulting mutant cell line, CHO-gmt5, lacks functional GDP-fucose transporter and CMP-sialic acid transporter. As a result, these cells can only produce asialylated and afucosylated glycoproteins. It is now widely recognized that removal of the core fucose from the N-glycans attached to Asn(297) of human IgG1 significantly enhances its binding to its receptor, FcγRIIIa, and thereby dramatically improves antibody-dependent cellular cytotoxicity (ADCC). Recent reports showed that removal of sialic acid from IgG1 also enhances ADCC. Therefore, CHO-gmt5 may represent a more advantageous cell line for the production of recombinant antibodies with enhanced ADCC. These cells show comparable growth rate to wild type CHO-K1 cells and uncompromised transfection efficiency, which make them desirable for use as a production line.

  19. Detection and typing of herpes simplex viruses by using recombinant immunoglobulin fragments produced in bacteria.

    PubMed Central

    Cattani, P; Rossolini, G M; Cresti, S; Santangelo, R; Burton, D R; Williamson, R A; Sanna, P P; Fadda, G

    1997-01-01

    Thirty-seven bacterial clones producing human recombinant monoclonal antibody Fab fragments (rFabs) reactive to herpes simplex virus (HSV) antigens were selected from a human combinatorial antibody library constructed in a phage-display vector by a panning procedure against an HSV lysate. Thirty-four of the HSV-specific rFabs were able to specifically recognize HSV-infected cells in indirect immunofluorescence (IF) assays; of these, 25 recognized cells infected by either HSV type 1 (HSV-1) or HSV-2, while 9 recognized only HSV-1-infected cells. One HSV type-common rFab (rFab H37) and one HSV-1-specific rFab (rFab H85) were further evaluated as reagents for viral detection and typing by IF staining in 134 HSV-positive (72 HSV-1 and 62 HSV-2) viral cultures from clinical specimens. The results obtained with these two rFabs were fully consistent with those obtained with a commercial preparation of fluorescein-labeled anti-HSV type-specific murine monoclonal antibodies. The detection sensitivity with the type-common rFab in indirect IF assays was higher overall than that provided by the type-specific murine monoclonal antibodies. Preparations of rFabs suitable for IF staining can be easily and inexpensively obtained in a clinical microbiology laboratory from Escherichia coli cultures. Similar HSV-specific rFabs, therefore, could be advantageous for in vitro diagnostic purposes. PMID:9163470

  20. Aspergillus fumigatus Produces Two Arabinofuranosidases From Glycosyl Hydrolase Family 62: Comparative Properties of the Recombinant Enzymes.

    PubMed

    Pérez, Rodrigo; Eyzaguirre, Jaime

    2016-04-01

    The genes of two α-L-arabinofuranosidases (AbfI and II) from family GH 62 have been identified in the genome of Aspergillus fumigatus wmo. Both genes have been expressed in Pichia pastoris and the enzymes have been purified and characterized. AbfI is composed of 999 bp, does not contain introns and codes for a protein (ABFI) of 332 amino acid residues. abfII has 1246 bp, including an intron of 51 bp; the protein ABFII has 396 amino acid residues; it includes a family 1 carbohydrate-binding module (CBM) in the N-terminal region, followed by a catalytic module. The sequence of ABFI and the catalytic module of ABFII show a 79 % identity. Both enzymes are active on p-nitrophenyl α-L-arabinofuranoside (pNPAra) with KM of 94.2 and 3.9 mM for ABFI and II, respectively. Optimal temperature for ABFI is 37 °C and for ABFII 42 °C, while the pH optimum is about 4.5 to 5 for both enzymes. ABFII shows a higher thermostability. When assayed using natural substrates, both show higher activity over rye arabinoxylan as compared to wheat arabinoxylan. ABFII only is active on sugar beet pulp arabinan and both are inactive towards debranched arabinan. The higher thermostability, higher affinity for pNPAra and wider activity over natural substrates shown by ABFII may be related to the presence of a CBM. The availability of the recombinant enzymes may be useful in biotechnological applications for the production of arabinose.

  1. A recombinant inwardly rectifying potassium channel coupled to GTP- binding proteins

    PubMed Central

    1996-01-01

    GTP-binding (G) proteins have been shown to mediate activation of inwardly rectifying potassium (K+) channels in cardiac, neuronal and neuroendocrine cells. Here, we report functional expression of a recombinant inwardly rectifying channel which we call KGP (or hpKir3.4), to signify that it is K+ selective, G-protein-gated and isolated from human pancreas. KGP expression in Xenopus oocytes resulted in sizeable basal (or agonist-independent) currents while coexpression with a G-protein-linked receptor, yielded additional agonist-induced currents. Coexpression of KGP and hGIRK1 (a human brain homolog of GIRK1/Kir3.1) produced much larger basal currents than those observed with KGP or hGIRK1 alone, and upon coexpression with receptor, similarly large agonist-induced currents could be obtained. Pertussis toxin treatment significantly diminished agonist-dependent currents due to either KGP or KGP/hGIRK1 expression. Interestingly, PTX also significantly reduced basal KGP or KGP/hGIRK1 currents, suggesting that basal activity is largely the result of G-protein gating as well. When the two channels were coexpressed with receptor, the relative increase in current elicited by agonist was similar whether KGP and hGIRK1 were expressed alone or together. When in vitro translated or when expressed in Xenopus oocytes or CHO mammalian cells, KGP gave rise to a nonglycosylated 45-kD protein. Antibodies directed against either KGP or hGIRK1 coprecipitated both proteins coexpressed in oocytes, providing evidence for the heteromeric assembly of the two channels and suggesting that the current potentiation seen with coexpression of the two channel subunits is due to specific interactions between them. An endogenous oocyte protein similar in size to KGP was also coprecipitated with hGIRK1. PMID:8868049

  2. [Prokaryotic expression, purification and antigenicity identification of recombinant human survivin protein].

    PubMed

    Yin, Xiaotao; Wang, Wei; Tian, Renli; Xu, Yuanji; Yan, Jinqi; Zhang, Wei; Gao, Jiangping; Yu, Jiyun

    2013-08-01

    To construct a prokaryotic expression plasmid pET28a-survivin, optimize the recombinant protein expression conditions in E.coli, and purify the survivin recombinant protein and identify its antigenicity. Survivin cDNA segment was amplified by PCR and cloned into prokaryotic expression vector pET28a(+) to construct the recombinant expression vector pET28a-survivin. The expression vector was transformed into BL21 (DE3) and the fusion protein survivin/His was induced by IPTG. The fusion protein was purified through Ni affinity chromatography. The antigenicity of the purified survivin protein was identified by Western blotting and ELISA. The recombinant expression vector was verified successfully by BamHI and HindIII. The fusion protein induced by IPTG was obtained with Mr; about 24 000. The purity of the purified protein reached 90% by SDS-PAGE analysis. And the antigenicity of the survivin protein was validated by Western blotting and ELISA. The prokaryotic expression plasmid pET28a-survivin was successfully constructed and the survivin protein was expressed and purified in E.coli. The antigenicity of the purified survivin protein was demonstrated desirable.

  3. Spo11-accessory proteins link double-strand break sites to the chromosome axis in early meiotic recombination.

    PubMed

    Panizza, Silvia; Mendoza, Marco A; Berlinger, Marc; Huang, Lingzhi; Nicolas, Alain; Shirahige, Katsuhiko; Klein, Franz

    2011-08-05

    Meiotic recombination between homologous chromosomes initiates via programmed DNA double-strand breaks (DSBs), generated by complexes comprising Spo11 transesterase plus accessory proteins. DSBs arise concomitantly with the development of axial chromosome structures, where the coalescence of axis sites produces linear arrays of chromatin loops. Recombining DNA sequences map to loops, but are ultimately tethered to the underlying axis. How and when such tethering occurs is currently unclear. Using ChIPchip in yeast, we show that Spo11-accessory proteins Rec114, Mer2, and Mei4 stably interact with chromosome axis sequences, upon phosphorylation of Mer2 by S phase Cdk. This axis tethering requires meiotic axis components (Red1/Hop1) and is modulated in a domain-specific fashion by cohesin. Loss of Rec114, Mer2, and Mei4 binding correlates with loss of DSBs. Our results strongly suggest that hotspot sequences become tethered to axis sites by the DSB machinery prior to DSB formation.

  4. Treatment of Burn and Surgical Wounds With Recombinant Human Tropoelastin Produces New Elastin Fibers in Scars.

    PubMed

    Xie, Hua; Lucchesi, Lisa; Zheng, Bo; Ladich, Elena; Pineda, Teresa; Merten, Rose; Gregory, Cynthia; Rutten, Michael; Gregory, Kenton

    2017-02-15

    Tropoelastin (TE), the soluble precursor of insoluble elastin fibers, is produced in minimal amounts in adults. Burn injuries result in inflexible collagen-rich scars because of lack of elastin fiber formation. We studied the feasibility of using recombinant human tropoelastin to enable elastin fiber production in burn and surgical scars to improve skin flexibility. In a swine hypertrophic burn scar model, normal skin and 3 × 3-cm partial thickness thermal burns underwent dermatome resection at 1 week post burn and randomized to four subcutaneous injections of saline or TE (either 0.5, 5, or 10 mg/ml) spaced 3 days apart. Two burn sites received TE injections after wound closure (0.5 or 10 mg/ml). At 90 days, skin hardness, flexibility, and histology were evaluated. All injury sites developed hypertrophic scars. New elastin fibers were found in burn scars in all injuries injected after skin closure with low (5/5) and high (6/6) TE doses (P < .05). No elastin fibers were observed without TE treatment. No significant differences in skin hardness, flexibility, or inflammation were observed. This is the first report demonstrating that subcutaneous injections of TE into surgical and burn injuries can safely produce new elastin fibers in scars. Despite the development of new elastin fibers, skin flexibility was not improved, possibly because of insufficient elastin fiber maturation or the hypertrophic model used. The ability to restore elastin fiber formation in adult skin after burns, trauma, and surgery may improve skin regeneration and reduce disabling complications of scar formation.

  5. Thermostable tag (TST) protein expression system: engineering thermotolerant recombinant proteins and vaccines.

    PubMed

    Luke, Jeremy M; Carnes, Aaron E; Sun, Ping; Hodgson, Clague P; Waugh, David S; Williams, James A

    2011-02-10

    Methods to increase temperature stability of vaccines and adjuvants are needed to reduce dependence on cold chain storage. We report herein creation and application of pVEX expression vectors to improve vaccine and adjuvant manufacture and thermostability. Defined media fermentation yields of 6g/L thermostable toll-like receptor 5 agonist flagellin were obtained using an IPTG inducible pVEX-flagellin expression vector. Alternative pVEX vectors encoding Pyrococcus furiosus maltodextrin-binding protein (pfMBP) as a fusion partner improved Influenza hemagglutinin antigen vaccine solubility and thermostability. A pfMBP hemagglutinin HA2 domain fusion protein was a potent immunogen. Manufacturing processes that combined up to 5 g/L defined media fermentation yields with rapid, selective, thermostable pfMBP fusion protein purification were developed. The pVEX pfMBP-based thermostable tag (TST) platform is a generic protein engineering approach to enable high yield manufacture of thermostable recombinant protein vaccine components. Copyright © 2010 Elsevier B.V. All rights reserved.

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

    PubMed

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

    2016-02-01

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

  7. Recombinant production of native human α-1-antitrypsin protein in the liver HepG2 cells.

    PubMed

    Jaberie, Hajar; Naghibalhossaini, Fakhraddin

    2016-10-01

    Alpha-1 antitrypsin (A1AT) deficiency is associated with emphysema and liver disease. Only plasma-derived A1AT protein is available for augmentation therapy. Recombinant A1AT (recA1AT) protein expressed in various types of available hosts are either non-glycosylated or aberrantly glycosylated resulting into reduced stability and biological activity. To overcome these limitations, we have used the human liver HepG2 cell line to produce recA1AT protein. HepG2 cells were transfected by A1AT cDNA and cell populations were generated that stably overexpressed A1AT protein. Real-time RT-PCR and rocket immunoelectrophoresis of cell culture supernatants indicated that the transfection resulted more than two-fold increase in A1AT production compared to that of control parental cells. Immunoblot analysis showed that both plasma and HepG2-produced A1AT proteins have identical molecular weight in either glycosylated or deglycosylated form. Partial digestion with PNGase F indicated that the three N-glycosylation sites of recA1AT, like the native A1AT protein in plasma, are occupied. Recombinant A1AT also like the native A1AT was thermostable and could efficiently inhibit trypsin proteolytic activity against BSA and BAPNA chromogenic substrate. The recombinant HepG2 cells cultured in media containing B27 serum free supplement released recA1AT at the same level as in the serum containing media. RecA1AT production in HepG2 cells grown under serum free condition at a large scale could provide a reliable source of the native protein suitable for therapeutic use in human.

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

    PubMed

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

    2015-11-01

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

  9. Induction of homologous recombination between sequence repeats by the activation induced cytidine deaminase (AID) protein.

    PubMed

    Buerstedde, Jean-Marie; Lowndes, Noel; Schatz, David G

    2014-07-08

    The activation induced cytidine deaminase (AID) protein is known to initiate somatic hypermutation, gene conversion or switch recombination by cytidine deamination within the immunoglobulin loci. Using chromosomally integrated fluorescence reporter transgenes, we demonstrate a new recombinogenic activity of AID leading to intra- and intergenic deletions via homologous recombination of sequence repeats. Repeat recombination occurs at high frequencies even when the homologous sequences are hundreds of bases away from the positions of AID-mediated cytidine deamination, suggesting DNA end resection before strand invasion. Analysis of