Science.gov

Sample records for recombinant hybrid protein

  1. 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.

  2. Production and purification of recombinant human oxytocin overexpressed as a hybrid protein in Escherichia coli.

    PubMed

    Esipov, Roman S; Chupova, Larisa A; Shvets, Sergey V; Chuvikovsky, Dmitry V; Gurevich, Alexandr I; Muravyova, Tatyana I; Miroshnikov, Anatoly I

    2003-08-01

    The plasmid DNA pERilox4 containing the gene of the recombinant protein, which included the leader sequence and the oxytocinoyl lysine tetramer, was constructed. The high level of gene expression in E. coli was achieved. The method for purification of the recombinant protein and its isolation in the soluble form was developed. The conditions for digestion of the hybrid protein by trypsin and carboxypeptidase B were matched. The effective method for transformation of oxytocinic acid to oxytocin was worked out. The scheme suggested allowed obtaining oxytocin in high yield.

  3. 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.

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

    PubMed

    Hong, Minsun; Yoon, Sung-Il; Wilson, Ian A

    2012-10-12

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

  5. Production of a recombinant hybrid molecule of cholera toxin-B-subunit and proteolipid-protein-peptide for the treatment of experimental encephalomyelitis.

    PubMed

    Yuki, Y; Byun, Y; Fujita, M; Izutani, W; Suzuki, T; Udaka, S; Fujihashi, K; McGhee, J R; Kiyono, H

    2001-07-05

    Mucosal administration of experimental autoimmune encephalomyelitis (EAE)-specific autoantigens can reduce the onset of disease. To examine whether cholera toxin-B-subunit (CTB)-conjugated EAE-specific T-cell epitope can reduce development of the autoimmune disease in mice, we produced a recombinant hybrid molecule of CTB fusion protein linked with proteolipid-protein (PLP)-peptide139-151(C140S) at levels up to 0.1 gram per liter culture media in Bacillus brevis as a secretion-expression system. Amino acid sequencing and GM1-receptor binding assay showed that this expression system produced a uniformed recombinant hybrid protein. EAE was induced in SJL/J mice by systemic administration with the PLP-peptide. When nasally immunized 5 times with 70 microg rCTB PLP-peptide hybrid protein, mice showed a significantly suppressed development of ongoing EAE and an inhibition of both the PLP-peptide-specific delayed-type hypersensitivity (DTH) responses and leukocyte infiltration into the spinal cord. In contrast, all mice given the PLP-peptide alone or the PLP-peptide with the free form of CTB did not suppress the development of EAE and DTH responses. These results suggest that nasal treatment with the recombinant B. brevis-derived hybrid protein of CTB and autoantigen peptide could prove useful in the control of multiple sclerosis.

  6. 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.

  7. 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.

  8. Hybrid metabolic flux analysis and recombinant protein prediction in Pichia pastoris X-33 cultures expressing a single-chain antibody fragment.

    PubMed

    Isidro, Inês A; Portela, Rui M; Clemente, João J; Cunha, António E; Oliveira, Rui

    2016-09-01

    Despite the growing importance of the Pichia pastoris expression system as industrial workhorse, the literature is almost absent in systematic studies on how culture medium composition affects central carbon fluxes and heterologous protein expression. In this study we investigate how 26 variations of the BSM+PTM1 medium impact central carbon fluxes and protein expression in a P. pastoris X-33 strain expressing a single-chain antibody fragment. To achieve this goal, we adopted a hybrid metabolic flux analysis (MFA) methodology, which is a modification of standard MFA to predict the rate of synthesis of recombinant proteins. Hybrid MFA combines the traditional parametric estimation of central carbon fluxes with non-parametric statistical modeling of product-related quantitative or qualitative measurements as a function of central carbon fluxes. It was observed that protein yield variability was 53.6 % (relative standard deviation) among the different experiments. Protein yield is much more sensitive to medium composition than biomass growth, which is mainly determined by the carbon source availability and main salts. Hybrid MFA was able to describe accurately the protein yield with normalized RMSE of 6.3 % over 5 independent experiments. The metabolic state that promotes high protein yields is characterized by high overall metabolic rates through main central carbon pathways concomitantly with a relative shift of carbon flux from biosynthetic towards energy generating pathways.

  9. The use of hybrid phage displaying antigen epitope and recombinant protein in the diagnosis of systemic Candida albicans infection in rabbits and cancer patients.

    PubMed

    Quanping, Su; Yanyan, Huai; Yicun, Wang; Zhigang, Ju; Yuling, Geng; Li, Wang

    2010-12-01

    Hsp90 and Sap2 are 2 immunodominant antigens of Candida albicans. Both of them can induce the production of antibody. In this article, systemically infected rabbits were used to study the Hsp90 and Sap2 antibody production. Also, pET28a-Hsp90 protein, pET28a-Sap2 protein, hybrid phage displaying LKVIRK epitope, and hybrid phage displaying VKYTS epitope were used for diagnosis of the antibody in cancer patients. The results showed that the Sap2 antibody appeared earlier than Hsp90 antibody in systemically infected rabbits. Meanwhile, both of the antibodies can perform protection in rabbits. The conclusion is that Sap2 antibody, which appears at early stage in systemic candidiasis, may be better than Hsp90 antibody for the diagnosis of invasive candidiasis. For 141 sera of cancer patients, 52 sera were detected Sap2 antibody and 57 sera were detected Hsp90 antibody. Only 14 sera contained both the 2 antibodies. Although recombinant protein was slightly more sensitive than hybrid phage, there was no significant difference between them. For its easy preparation, less expensive hybrid phage displaying antigen epitope may be a better agent for diagnosis of candidiasis.

  10. 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.

  11. Production of a recombinant cholera toxin B subunit-insulin B chain peptide hybrid protein by Brevibacillus choshinensis expression system as a nasal vaccine against autoimmune diabetes.

    PubMed

    Yuki, Yoshikazu; Hara-Yakoyama, Chisato; Guadiz, Abigail A E; Udaka, Shigezo; Kiyono, Hiroshi; Chatterjee, Subhendra

    2005-12-30

    Mucosally induced tolerance is an attractive strategy for preventing or reducing autoimmune diseases. Here, we produced a recombinant CTB fusion protein linked with autoantigen T cell epitope of insulin B chain peptide 9-23 (C19S) at levels up to 200 mg/L culture media in Brevibacillus choshinensis secretion-expression system. Receptor-competitive assay showed that the CTB-insulin peptide binds to GM1 receptor almost equivalent degree as the native form of CTB. Non-obese diabetes (NOD) mice that spontaneously develop an insulin-dependent diabetes were nasally immunized with CTB-insulin peptide (5 microg) for three times. The nasal treatment significantly reduced the development of insulin-dependent diabetes and peptide specific DTH responses after systemic immunization with the insulin peptide B 9-23(C19S) in CFA. Nasal administration of as high as 50 microg of the peptide alone demonstrated a similar level of the disease inhibition. In contrast, all mice given 5 microg of the insulin peptide alone or 5 microg of insulin peptide with 25 microg of the free form of CTB did not lead to the suppression of diabetes development and DTH responses. Because molecular weight of the insulin peptide is about one tenth of that of the CTB-insulin peptide, the results demonstrate that the recombinant hybrid of autoantigen and CTB increased its tolerogenic potential for nasal administration by up 100-fold on molar base of autoantigen peptide. Taken together, nasally-induced tolerance by administration of the recombinant B. choshinensis-derived hybrid protein of CTB and autoantigen T cell-epitope peptide could be useful mucosal immunetherapy for the control of T cell-mediated autoimmune diseases.

  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. 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.

  14. 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...

  15. 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.

  16. 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.

  17. Hybrid Sterility Locus on Chromosome X Controls Meiotic Recombination Rate in Mouse

    PubMed Central

    Balcova, Maria; Faltusova, Barbora; Gergelits, Vaclav; Bhattacharyya, Tanmoy; Mihola, Ondrej; Trachtulec, Zdenek; Knopf, Corinna; Fotopulosova, Vladana; Chvatalova, Irena; Gregorova, Sona; Forejt, Jiri

    2016-01-01

    Meiotic recombination safeguards proper segregation of homologous chromosomes into gametes, affects genetic variation within species, and contributes to meiotic chromosome recognition, pairing and synapsis. The Prdm9 gene has a dual role, it controls meiotic recombination by determining the genomic position of crossover hotspots and, in infertile hybrids of house mouse subspecies Mus m. musculus (Mmm) and Mus m. domesticus (Mmd), it further functions as the major hybrid sterility gene. In the latter role Prdm9 interacts with the hybrid sterility X 2 (Hstx2) genomic locus on Chromosome X (Chr X) by a still unknown mechanism. Here we investigated the meiotic recombination rate at the genome-wide level and its possible relation to hybrid sterility. Using immunofluorescence microscopy we quantified the foci of MLH1 DNA mismatch repair protein, the cytological counterparts of reciprocal crossovers, in a panel of inter-subspecific chromosome substitution strains. Two autosomes, Chr 7 and Chr 11, significantly modified the meiotic recombination rate, yet the strongest modifier, designated meiotic recombination 1, Meir1, emerged in the 4.7 Mb Hstx2 genomic locus on Chr X. The male-limited transgressive effect of Meir1 on recombination rate parallels the male-limited transgressive role of Hstx2 in hybrid male sterility. Thus, both genetic factors, the Prdm9 gene and the Hstx2/Meir1 genomic locus, indicate a link between meiotic recombination and hybrid sterility. A strong female-specific modifier of meiotic recombination rate with the effect opposite to Meir1 was localized on Chr X, distally to Meir1. Mapping Meir1 to a narrow candidate interval on Chr X is an important first step towards positional cloning of the respective gene(s) responsible for variation in the global recombination rate between closely related mouse subspecies. PMID:27104744

  18. Hybrid Sterility Locus on Chromosome X Controls Meiotic Recombination Rate in Mouse.

    PubMed

    Balcova, Maria; Faltusova, Barbora; Gergelits, Vaclav; Bhattacharyya, Tanmoy; Mihola, Ondrej; Trachtulec, Zdenek; Knopf, Corinna; Fotopulosova, Vladana; Chvatalova, Irena; Gregorova, Sona; Forejt, Jiri

    2016-04-01

    Meiotic recombination safeguards proper segregation of homologous chromosomes into gametes, affects genetic variation within species, and contributes to meiotic chromosome recognition, pairing and synapsis. The Prdm9 gene has a dual role, it controls meiotic recombination by determining the genomic position of crossover hotspots and, in infertile hybrids of house mouse subspecies Mus m. musculus (Mmm) and Mus m. domesticus (Mmd), it further functions as the major hybrid sterility gene. In the latter role Prdm9 interacts with the hybrid sterility X 2 (Hstx2) genomic locus on Chromosome X (Chr X) by a still unknown mechanism. Here we investigated the meiotic recombination rate at the genome-wide level and its possible relation to hybrid sterility. Using immunofluorescence microscopy we quantified the foci of MLH1 DNA mismatch repair protein, the cytological counterparts of reciprocal crossovers, in a panel of inter-subspecific chromosome substitution strains. Two autosomes, Chr 7 and Chr 11, significantly modified the meiotic recombination rate, yet the strongest modifier, designated meiotic recombination 1, Meir1, emerged in the 4.7 Mb Hstx2 genomic locus on Chr X. The male-limited transgressive effect of Meir1 on recombination rate parallels the male-limited transgressive role of Hstx2 in hybrid male sterility. Thus, both genetic factors, the Prdm9 gene and the Hstx2/Meir1 genomic locus, indicate a link between meiotic recombination and hybrid sterility. A strong female-specific modifier of meiotic recombination rate with the effect opposite to Meir1 was localized on Chr X, distally to Meir1. Mapping Meir1 to a narrow candidate interval on Chr X is an important first step towards positional cloning of the respective gene(s) responsible for variation in the global recombination rate between closely related mouse subspecies.

  19. 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.

  20. Recombination in interpopulation hybrids of the copepod Tigriopus californicus: release of beneficial variation despite hybrid breakdown.

    PubMed

    Edmands, Suzanne

    2008-01-01

    Crosses between divergent populations of the copepod Tigriopus californicus typically result in fitness reductions for both F2 and backcross hybrids. Because females in this species lack chiasmatic meiosis, both recombinant and nonrecombinant backcross hybrids can be created. Recombinant hybrids were found to have significantly faster development time for both males and females in 2 pairs of crosses, indicating the creation of favorable gene combinations by disrupting parental linkage groups.

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. Interspecies hybridization and recombination in Saccharomyces wine yeasts.

    PubMed

    Sipiczki, Matthias

    2008-11-01

    The ascomycetous yeasts traditionally referred to as the Saccharomyces sensu stricto complex are a group of closely related species that are isolated by a postzygotic barrier. They can easily hybridize; and their allodiploid hybrids propagate by mitotic divisions as efficiently as the parental strains, but can barely divide by meiosis, and thus rarely produce viable spores (sterile interspecies hybrids). The postzygotic isolation is not effective in allotetraploids that are able to carry out meiosis and produce viable spores (fertile interspecies hybrids). By application of molecular identification methods, double (Saccharomyces cerevisiae x Saccharomyces uvarum and S. cerevisiae x Saccharomyces kudriavzevii) and triple (S. cerevisiae x S. uvarum x S. kudriavzevii) hybrids were recently identified in yeast populations of fermenting grape must and cider in geographically distinct regions. The genetic analysis of these isolates and laboratory-bred hybrids revealed great variability of hybrid genome structures and demonstrated that the alloploid genome of the zygote can undergo drastic changes during mitotic and meiotic divisions of the hybrid cells. This genome-stabilization process involves loss of chromosomes and genes and recombination between the partner genomes. This article briefly reviews the results of the analysis of interspecies hybrids, proposes a model for the mechanism of genome stabilization and highlights the potential of interspecies hybridization in winemaking.

  6. Generation of hybrid human immunodeficiency virus by homologous recombination.

    PubMed Central

    Srinivasan, A; York, D; Jannoun-Nasr, R; Kalyanaraman, S; Swan, D; Benson, J; Bohan, C; Luciw, P A; Schnoll, S; Robinson, R A

    1989-01-01

    Human immunodeficiency virus (HIV) type 1, isolated from diverse sources, exhibits genomic diversity. The mechanisms by which the genomic diversity takes place in individuals exposed to multiple virus isolates is yet to be elucidated. Genetic variation, in general, might result from mutagenic events such as point mutations, rearrangements (insertions and deletions), and recombination. In an attempt to evaluate the process of genetic diversity, we designed experiments to analyze recombination between HIV DNAs by using DNA transfection in cell cultures. Here we report the successful recombination between truncated HIV proviral DNAs with an overlap homology of 53 base pairs that leads to the formation of viable hybrid virus. Recombination was also seen between exogenous DNA introduced into cells and homologous HIV sequences resident in the cells. These results indicate that recombination among various HIV isolates may play a significant role in the generation of genetic diversity of HIV. Further, the method used here enables the construction of hybrid HIV genomes to identify the viral determinants responsible for tropism, replication, and cytopathic effects. Images PMID:2474834

  7. 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.

  8. 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.

  9. Hybrid proteins between Pseudomonas exotoxin A and poliovirus protease 2Apro.

    PubMed

    Novoa, I; Feduchi, E; Carrasco, L

    1994-11-21

    Two hybrid proteins between Pseudomonas aeruginosa exotoxin A (PE) and poliovirus protease 2Apro have been generated. One hybrid protein contains the poliovirus 2Apro sequence replacing the region of PE corresponding to amino acids 413-607. The other hybrid contains in addition the transforming growth factor sequence. The two hybrid proteins were efficiently synthesized in E. coli cells using the inducible pET vectors. Both hybrid toxins cleaved p220 (eIF-4 gamma) when the recombinant plasmids were transfected in COS cells infected with recombinant vaccinia virus bearing the T7 RNA polymerase gene.

  10. 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.

  11. 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).

  12. 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).

  13. 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.

  14. 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.

  15. 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.

  16. Protein-inorganic hybrid nanoflowers

    NASA Astrophysics Data System (ADS)

    Ge, Jun; Lei, Jiandu; Zare, Richard N.

    2012-07-01

    Flower-shaped inorganic nanocrystals have been used for applications in catalysis and analytical science, but so far there have been no reports of `nanoflowers' made of organic components. Here, we report a method for creating hybrid organic-inorganic nanoflowers using copper (II) ions as the inorganic component and various proteins as the organic component. The protein molecules form complexes with the copper ions, and these complexes become nucleation sites for primary crystals of copper phosphate. Interaction between the protein and copper ions then leads to the growth of micrometre-sized particles that have nanoscale features and that are shaped like flower petals. When an enzyme is used as the protein component of the hybrid nanoflower, it exhibits enhanced enzymatic activity and stability compared with the free enzyme. This is attributed to the high surface area and confinement of the enzymes in the nanoflowers.

  17. Expression of recombinant EARLI1, a hybrid proline-rich protein of Arabidopsis, in Escherichia coli and its inhibition effect to the growth of fungal pathogens and Saccharomyces cerevisiae.

    PubMed

    Li, Lan; Zhang, Chen; Xu, Dan; Schläppi, Michael; Xu, Zi-Qin

    2012-09-10

    EARLI1 is an Arabidopsis gene with pleiotropic effects previously shown to have auxiliary functions in protecting plants against freezing-induced cellular damage and promoting germinability under low-temperature and salinity stresses. Here we determined whether recombinant EARLI1 protein has anti-fungal activity. Recombinant EARLI1 protein lacking its signal peptide was produced in Escherichia coli BL21(DE3) using isopropyl β-d-1-thiogalactopyranoside (IPTG) induction and the prokaryotic expression vector pET28a. Expression of EARLI1 was analyzed by Western blotting and the protein was purified using affinity chromatography. Recombinant EARLI1 protein was applied to fungal cultures of Saccharomyces cerevisiae, Botrytis cinerea and Fusarium oxysporum, and membrane permeability was determined using SYTOX green. Full-length EARLI1 was expressed in S. cerevisiae from the GAL1 promoter using 2% galactose and yeast cell viability was compared to control cells. Our results indicated that application of recombinant EARLI1 protein to B. cinerea and F. oxysporum could inhibit the growth of the necrotrophic fungi. Besides, addition of the recombinant protein to liquid cultures of S. cerevisiae significantly suppressed yeast growth and cell viability by increasing membrane permeability, and in vivo expression of the secreted form of EARLI1 in S. cerevisiae also had a remarkable inhibition effect on the growth of yeast cells. Copyright © 2012 Elsevier B.V. All rights reserved.

  18. Vidas UP-enzyme-linked fluorescent immunoassay based on recombinant phage protein and fluorescence in situ hybridization as alternative methods for detection of Salmonella enterica serovars in meat.

    PubMed

    Zadernowska, Anna; Chajęcka-Wierzchowska, Wioleta; Kłębukowska, Lucyna

    2014-09-01

    Several methods for the rapid and specific detection of Salmonella spp. in meat have been described. This study was conducted to evaluate the capability of the VIDAS(®) UP (SPT [Salmonella Phage Technology]), an enzyme-linked fluorescent immunoassay method, and fluorescence in situ hybridization (FISH) to complement the International Organization for Standardization Method 6579 (ISO) in detecting Salmonella spp. from beef, pork, and poultry meat samples. The meat was inoculated with a mixture of Salmonella spp. on three levels of contamination. It was also checked that the tests did not produce cross-reactions with other Enterobacteriaceae rods. On the basis of the results, the relative specificity, relative accordance, and relative sensitivity of the method were determined. In meat samples, Vidas UP and FISH detection results were in substantial agreement with ISO, with relative specificity, accordance, and sensitivity rates of 90%, 96.3%, and 100%, respectively, for Vidas UP and 100%, 100%, and 99.4%, respectively, for FISH. This is the first report on the evaluation of both Vidas UP and FISH compared to ISO for the rapid detection of Salmonella enterica serovars in meat.

  19. Transgene expression in Penaeus monodon cells: evaluation of recombinant baculoviral vectors with shrimp specific hybrid promoters.

    PubMed

    Puthumana, Jayesh; Philip, Rosamma; Bright Singh, I S

    2016-08-01

    It has been realized that shrimp cell immortalization may not be accomplished without in vitro transformation by expressing immortalizing gene in cells. In this process, efficiency of transgene expression is confined to the ability of vectors to transmit gene of interests to the genome. Over the years, unavailability of such vectors has been hampering application of such a strategy in shrimp cells. We report the use of recombinant baculovirus mediated transduction using hybrid promoter system for transgene expression in lymphoid cells of Penaeus monodon. Two recombinant baculovirus vectors with shrimp viral promoters (WSSV-Ie1 and IHHNV-P2) were constructed (BacIe1-GFP and BacP2-GFP) and green fluorescent protein (GFP) used as the transgene. The GFP expression in cells under the control of hybrid promoters, PH-Ie1 or PH-P2, were analyzed and confirmed in shrimp cells. The results indicate that the recombinant baculovirus with shrimp specific viral promoters (hybrid) can be employed for delivery of foreign genes to shrimp cells for in vitro transformation.

  20. 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.

  1. Extensive Recombination of a Yeast Diploid Hybrid through Meiotic Reversion

    PubMed Central

    Laureau, Raphaëlle; Loeillet, Sophie; Salinas, Francisco; Bergström, Anders; Legoix-Né, Patricia; Liti, Gianni; Nicolas, Alain

    2016-01-01

    In somatic cells, recombination between the homologous chromosomes followed by equational segregation leads to loss of heterozygosity events (LOH), allowing the expression of recessive alleles and the production of novel allele combinations that are potentially beneficial upon Darwinian selection. However, inter-homolog recombination in somatic cells is rare, thus reducing potential genetic variation. Here, we explored the property of S. cerevisiae to enter the meiotic developmental program, induce meiotic Spo11-dependent double-strand breaks genome-wide and return to mitotic growth, a process known as Return To Growth (RTG). Whole genome sequencing of 36 RTG strains derived from the hybrid S288c/SK1 diploid strain demonstrates that the RTGs are bona fide diploids with mosaic recombined genome, derived from either parental origin. Individual RTG genome-wide genotypes are comprised of 5 to 87 homozygous regions due to the loss of heterozygous (LOH) events of various lengths, varying between a few nucleotides up to several hundred kilobases. Furthermore, we show that reiteration of the RTG process shows incremental increases of homozygosity. Phenotype/genotype analysis of the RTG strains for the auxotrophic and arsenate resistance traits validates the potential of this procedure of genome diversification to rapidly map complex traits loci (QTLs) in diploid strains without undergoing sexual reproduction. PMID:26828862

  2. Interspecific chloroplast recombination in a Nicotiana somatic hybrid.

    PubMed

    Medgyesy, P; Fejes, E; Maliga, P

    1985-10-01

    Genetic recombination between chloroplasts of two flowering plant species, Nicotiana tabacum and Nicotiana plumbaginifolia, after somatic cell fusion is described. The parental lines differed in three cytoplasmic genetic markers. The N. tabacum mutant SR1-A15 was streptomycin-resistant, defective in chloroplast greening, and lincomycin-sensitive. The N. plumbaginifolia mutant LR400 was streptomycin-sensitive, normal green, and lincomycin-resistant. Streptomycin-resistant clones in cell culture are identified by their ability to form a green callus on a selective medium. Streptomycin resistance in the SR1-A15 mutant could not be expressed due to defective chloroplasts. Protoplasts of the two species were fused, and calli grown from the fused population were screened for the expression of streptomycin resistance from the SR1-A15 line as the result of interspecific chloroplast recombination. A somatic hybrid, pt14, expressed a new combination of the cytoplasmic genetic markers. In the pt14 chloroplast genome three N. tabacum and four N. plumbaginifolia parent specific restriction sites have been identified, indicating that the pt14 chloroplast genome contains at least six recombination sites.

  3. Exploring the Association of Surface Plasmon Resonance with Recombinant MHC:Ig Hybrid Protein as a Tool for Detecting T Lymphocytes in Mice Infected with Leishmania (Leishmania) amazonensis.

    PubMed

    da Silveira-Júnior, Lenilton Silva; Souza-Silva, Franklin; Pereira, Bernardo Acácio Santini; Cysne-Finkelstein, Léa; Cavalcanti Júnior, Geraldo Barroso; Alves, Carlos Roberto

    2017-01-01

    A surface plasmon resonance- (SPR-) based recognition method applying H-2 L(d):Ig/peptides complexes for ex vivo monitoring cellular immune responses during murine infection with Leishmania (Leishmania) amazonensis is described. Lymphocytes from lesion-draining popliteal lymph nodes were captured on a carboxylated sensor chip surface previously functionalized with H-2 L(d):Ig (DimerX) protein bound to synthetic peptides derived from the COOH-terminal region of cysteine proteinase B of L. (L.) amazonensis. In computational analysis, these peptides presented values of kinetic constants favorable to form complexes with H-2 L(d) at neutral pH, with a Gibbs free energy ΔG° < 0. The assayed DimerX:peptide complexes presented the property of attaching to distinct T lymphocytes subsets, obtained from experimentally infected BALB/c mice, in each week of infection, thus indicating a temporal variation in specific T lymphocytes populations, each directed to a different COOH-terminal region-derived peptide. The experimental design proposed herein is an innovative approach for cellular immunology studies of a neglected disease, providing a useful tool for the analysis of specific T lymphocytes subsets.

  4. Exploring the Association of Surface Plasmon Resonance with Recombinant MHC:Ig Hybrid Protein as a Tool for Detecting T Lymphocytes in Mice Infected with Leishmania (Leishmania) amazonensis

    PubMed Central

    da Silveira-Júnior, Lenilton Silva; Souza-Silva, Franklin; Pereira, Bernardo Acácio Santini; Cysne-Finkelstein, Léa; Cavalcanti Júnior, Geraldo Barroso

    2017-01-01

    A surface plasmon resonance- (SPR-) based recognition method applying H-2 Ld:Ig/peptides complexes for ex vivo monitoring cellular immune responses during murine infection with Leishmania (Leishmania) amazonensis is described. Lymphocytes from lesion-draining popliteal lymph nodes were captured on a carboxylated sensor chip surface previously functionalized with H-2 Ld:Ig (DimerX) protein bound to synthetic peptides derived from the COOH-terminal region of cysteine proteinase B of L. (L.) amazonensis. In computational analysis, these peptides presented values of kinetic constants favorable to form complexes with H-2 Ld at neutral pH, with a Gibbs free energy ΔG° < 0. The assayed DimerX:peptide complexes presented the property of attaching to distinct T lymphocytes subsets, obtained from experimentally infected BALB/c mice, in each week of infection, thus indicating a temporal variation in specific T lymphocytes populations, each directed to a different COOH-terminal region-derived peptide. The experimental design proposed herein is an innovative approach for cellular immunology studies of a neglected disease, providing a useful tool for the analysis of specific T lymphocytes subsets. PMID:28373990

  5. 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

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. Direct measurement of recombination frequency in interspecific hybrids between Hordeum vulgare and H. bulbosum using genomic in situ hybridization.

    PubMed

    Zhang, L; Pickering, R; Murray, B

    1999-09-01

    Two different genotypes of diploid Hordeum vulgare x H. bulbosum hybrids, which differ in their pattern of meiotic metaphase pairing behaviour, were investigated at MI and AI by genomic in situ hybridization (GISH). One hybrid, 102C2, showed a high frequency of bivalents at metaphase I whereas the other, 103K5, showed a high frequency of univalents. The GISH analysis of both hybrids established that pairing occurred only between chromosomes of different parental genomes and revealed that pairing frequency greatly exceeded recombination. Hybrid 102C2 had a significantly higher recombination frequency than 103K5, but in both hybrids recombination involved only distal chromosome regions. However, an interesting finding is that the ratio of recombination to pairing frequency in 103K5 (1:8.9) is twice as high compared with 102C2 (1:17). The hybrids also differed in chromosome stability; little chromosome elimination occurred in 102C2 but 103K5 showed extensive chromosome loss. It appears that the high frequency of bound arms at MI favours retention of H. bulbosum chromosomes and maintains stability of chromosome numbers in PMCs. Various ideas are put forward to explain the discrepancy between meiotic pairing frequency and recombination in these hybrids.

  17. Immunogenic Domains and Secondary Structure of Escherichia coli Recombinant Secreted Protein Escherichia coli-Secreted Protein B

    PubMed Central

    Caetano, Bruna Alves; Rocha, Letícia Barboza; Carvalho, Eneas; Piazza, Roxane Maria Fontes; Luz, Daniela

    2017-01-01

    Several pathogenic bacteria are able to induce the attaching and effacing (A/E) lesion. The A/E lesion is caused by effector proteins, such as Escherichia coli-secreted protein B (EspB), responsible together with Escherichia coli-secreted protein D for forming a pore structure on the host cell, which allows the translocation of effector proteins. Different variants of this protein can be found in E. coli strains, and during natural infection or when this protein is injected, this leads to variant-specific production of antibodies, which may not be able to recognize other variants of this bacterial protein. Herein, we describe the production of a hybrid recombinant EspB toxin that comprises all known variants of this protein. This recombinant protein could be useful as an antigen for the production of antibodies with broad-range detection of EspB-bearing bacteria, or as an antigen that could be used in vaccine formulation to generate antibodies against different EspB variants, thereby increasing immunization potential. In addition, the recombinant protein allowed us to analyze its secondary structure, to propose the immunogenic regions of EspB variants, and also to characterize anti-EspB antibodies. Our results suggest that this hybrid protein or a protein composed of the conserved immunogenic regions could be used for a variety of clinical applications. PMID:28484467

  18. Immunogenic Domains and Secondary Structure of Escherichia coli Recombinant Secreted Protein Escherichia coli-Secreted Protein B.

    PubMed

    Caetano, Bruna Alves; Rocha, Letícia Barboza; Carvalho, Eneas; Piazza, Roxane Maria Fontes; Luz, Daniela

    2017-01-01

    Several pathogenic bacteria are able to induce the attaching and effacing (A/E) lesion. The A/E lesion is caused by effector proteins, such as Escherichia coli-secreted protein B (EspB), responsible together with Escherichia coli-secreted protein D for forming a pore structure on the host cell, which allows the translocation of effector proteins. Different variants of this protein can be found in E. coli strains, and during natural infection or when this protein is injected, this leads to variant-specific production of antibodies, which may not be able to recognize other variants of this bacterial protein. Herein, we describe the production of a hybrid recombinant EspB toxin that comprises all known variants of this protein. This recombinant protein could be useful as an antigen for the production of antibodies with broad-range detection of EspB-bearing bacteria, or as an antigen that could be used in vaccine formulation to generate antibodies against different EspB variants, thereby increasing immunization potential. In addition, the recombinant protein allowed us to analyze its secondary structure, to propose the immunogenic regions of EspB variants, and also to characterize anti-EspB antibodies. Our results suggest that this hybrid protein or a protein composed of the conserved immunogenic regions could be used for a variety of clinical applications.

  19. 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

  20. 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

  1. 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.

  2. 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.

  3. Extracellular production and affinity purification of recombinant proteins with Escherichia coli using the versatility of the maltose binding protein.

    PubMed

    Sommer, Benjamin; Friehs, Karl; Flaschel, Erwin; Reck, Michael; Stahl, Frank; Scheper, Thomas

    2009-03-25

    Recombinant proteins are essential products of today's industrial biotechnology. In this study we address two crucial factors in recombinant protein production: (i) product accessibility and (ii) product recovery. Escherichia coli, one of the most frequently used hosts for recombinant protein expression, does not inherently secrete proteins into the extracellular environment. The major drawback of this expression system is, therefore, to be found in the intracellular protein accumulation and hampered product accessibility. We have constructed a set of expression vectors in order to facilitate extracellular protein production and purification. The maltose binding protein from E. coli is used as fusion partner for several proteins of interest allowing an export to the bacteria's periplasm via both the Sec and the Tat pathway. Upon coexpression of a modified Cloacin DF13 bacteriocin release protein, the hybrid proteins are released into the culture medium. This essentially applies to a distinguished reporter molecule, the green fluorescent protein, for which an extracellular production was not reported so far. The sequestered proteins can be purified to approximate homogeneity by a simple, rapid and cheap procedure which utilizes the affinity of the maltose binding protein to alpha-1,4-glucans.

  4. 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.

  5. 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.

  6. Interaction of Recombinant Myocilin with the Matricellular Protein SPARC: Functional Implications

    PubMed Central

    Aroca-Aguilar, José-Daniel; Sánchez-Sánchez, Francisco; Ghosh, Sikha; Fernández-Navarro, Ana; Coca-Prados, Miguel; Escribano, Julio

    2011-01-01

    Purpose. Myocilin is an extracellular glycoprotein with unknown function that is associated with glaucoma. Calpain II cleaves recombinant myocilin within the linker region of the protein, releasing the C-terminal olfactomedin domain from the N-terminal domain. The authors previously reported that myocilin interacts with the C-terminal region of hevin, a secretory glycoprotein belonging to the SPARC family of matricellular proteins. This study aims to investigate the interaction of myocilin with SPARC. Methods. Protein-protein interactions were evaluated by the yeast two-hybrid system. The positive interactions were confirmed by solid-phase binding assays using Ni-chelating HPLC purified recombinant proteins and coexpression of recombinant proteins in HEK-293T cells. Coexpression of myocilin, SPARC, and hevin in ocular tissues was identified by immunoflorescence microscopy, Western blot, and array-based gene profiling. Results. Yeast two-hybrid analyses showed that myocilin interacted with the highly conserved C-terminal extracellular calcium binding (EC) domain within SPARC and hevin. Solid-phase binding assays confirmed these interactions and showed that both myocilin and its C-terminal olfactomedin fragment interacted noncovalently with SPARC and a peptide containing the EC domain of SPARC. Full-length myocilin interacted with higher affinity with SPARC and its EC domain than the myocilin C-terminal fragment. Coexpression of the two recombinant proteins in HEK-293T cells also indicated their intracellular interaction. Conclusions. Recombinant myocilin and SPARC interact through their C-terminal domains. The data suggest that the proteolytic processing of myocilin modulates this interaction as well as the interactions of myocilin with other extracellular matrix and matricellular proteins, further supporting a functional role for this proteolytic cleavage. PMID:20926826

  7. 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

  8. [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.

  9. 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

  10. Intragenic recombination events and evidence for hybrid speciation in Nicotiana (Solanaceae).

    PubMed

    Kelly, Laura J; Leitch, Andrew R; Clarkson, James J; Hunter, Robin B; Knapp, Sandra; Chase, Mark W

    2010-04-01

    Reticulate evolution may function both at the species level, through homoploid and polyploid hybridization, and below the species level, through inter and intragenic recombination. These processes represent challenges for the reconstruction of evolutionary relationships between species, because they cannot be represented adequately with bifurcating trees. We use data from low-copy nuclear genes to evaluate long-standing hypotheses of homoploid (interspecific) hybrid speciation in Nicotiana (Solanaceae) and reconstruct a complex series of reticulation events that have been important in the evolutionary history of this genus. Hybrid origins for three diploid species (Nicotiana glauca, N. linearis, and N. spegazzinii) are inferred on the basis of gene tree incongruence, evidence for interallelic recombination between likely parental alleles, and support for incompatible splits in Lento plots. Phylogenetic analysis of recombinant gene sequences illustrates that recombinants may be resolved with one of their progenitor lineages with a high posterior probability under Bayesian inference, and thus there is no indication of the conflict between phylogenetic signals that results from reticulation. Our results illustrate the importance of hybridization in shaping evolution in Nicotiana and also show that intragenic recombination may be relatively common. This finding demonstrates that it is important to investigate the possibility of recombination when aiming to detect hybrids from DNA-sequence data and reconstruct patterns of reticulate evolution between species.

  11. Recombination between defective tombusvirus RNAs generates functional hybrid genomes

    SciTech Connect

    White, K.A.; Morris, T.J.

    1994-04-26

    The tombusviruses represent a group of small icosahedral plant viruses that contain monopartite positive-sense RNA genomes. Tombusviruses are able to generate small replicating deletion mutants of their genomes (i.e., defective interfering RNAs) during infections via RNA recombination and/or rearrangement. To further study the process of RNA recombination and to determine whether tombusviruses were capable of trans-recombination, protoplasts were coinoculated with in vitro-generated transcripts of a nonreplicating 3{prime}-truncated genomic RNA of cucumber necrosis tombusvirus and either replicative or replication-defective DI RNAs of tomato bushy stunt tombusvirus. After 48-hr incubation, two dominant replicative chimeric recombinant viral RNA populations were detected that contained various large contiguous 5{prime} segments of the cucumber necrosis tombusvirus genomic RNA fused to 3{prime}-terminal regions of the tomato bushy stunt tombusvirus defective interfering RNA. Some of the larger chimeric recombinants formed in protoplasts were able to systemically infect plants and induce wild-type symptoms. In addition, a functional chimeric genome was generated in planta after direct coinoculation of whole plants with the defective RNA components. These results indicate that (i) RNA recombination can occur relatively efficiently in single-cell infections, (ii) trans-recombination can occur with nonreplicating viral RNA components, and (iii) functional chimeric genomes can be generated via recombination. Possible mechanisms for the formation of the recombinants are proposed, and evolutionary implications are discussed.

  12. 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 ...

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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

  4. 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...

  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. 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.

  8. 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

  9. 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.

  10. 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.

  11. 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

  12. Hybrid system of semiconductor and photosynthetic protein

    NASA Astrophysics Data System (ADS)

    Kim, Younghye; Shin, Seon Ae; Lee, Jaehun; Yang, Ki Dong; Nam, Ki Tae

    2014-08-01

    Photosynthetic protein has the potential to be a new attractive material for solar energy absorption and conversion. The development of semiconductor/photosynthetic protein hybrids is an example of recent progress toward efficient, clean and nanostructured photoelectric systems. In the review, two biohybrid systems interacting through different communicating methods are addressed: (1) a photosynthetic protein immobilized semiconductor electrode operating via electron transfer and (2) a hybrid of semiconductor quantum dots and photosynthetic protein operating via energy transfer. The proper selection of materials and functional and structural modification of the components and optimal conjugation between them are the main issues discussed in the review. In conclusion, we propose the direction of future biohybrid systems for solar energy conversion systems, optical biosensors and photoelectric devices.

  13. Hybrid system of semiconductor and photosynthetic protein.

    PubMed

    Kim, Younghye; Shin, Seon Ae; Lee, Jaehun; Yang, Ki Dong; Nam, Ki Tae

    2014-08-29

    Photosynthetic protein has the potential to be a new attractive material for solar energy absorption and conversion. The development of semiconductor/photosynthetic protein hybrids is an example of recent progress toward efficient, clean and nanostructured photoelectric systems. In the review, two biohybrid systems interacting through different communicating methods are addressed: (1) a photosynthetic protein immobilized semiconductor electrode operating via electron transfer and (2) a hybrid of semiconductor quantum dots and photosynthetic protein operating via energy transfer. The proper selection of materials and functional and structural modification of the components and optimal conjugation between them are the main issues discussed in the review. In conclusion, we propose the direction of future biohybrid systems for solar energy conversion systems, optical biosensors and photoelectric devices.

  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. 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

  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. 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.

  18. Protein fragment swapping: a method for asymmetric, selective site-directed recombination.

    PubMed

    Zheng, Wei; Griswold, Karl E; Bailey-Kellogg, Chris

    2010-03-01

    This article presents a new approach to site-directed recombination, swapping combinations of selected discontiguous fragments from a source protein in place of corresponding fragments of a target protein. By being both asymmetric (differentiating source and target) and selective (swapping discontiguous fragments), our method focuses experimental effort on a more restricted portion of sequence space, constructing hybrids that are more likely to have the properties that are the objective of the experiment. Furthermore, since the source and target need to be structurally homologous only locally (rather than overall), our method supports swapping fragments from functionally important regions of a source into a target "scaffold" (for example, to humanize an exogenous therapeutic protein). A protein fragment swapping plan is defined by the residue position boundaries of the fragments to be swapped; it is assessed by an average potential score over the resulting hybrid library, with singleton and pairwise terms evaluating the importance and fit of the swapped residues. While we prove that it is NP-hard to choose an optimal set of fragments under such a potential score, we develop an integer programming approach, which we call Swagmer, that works very well in practice. We demonstrate the effectiveness of our method in three swapping problems: selective recombination between beta-lactamases, activity swapping between glutathione transferases, and activity swapping between carboxylases and mutases in the purE family. We show that the selective recombination approach generates better plan (in terms of resulting potential score) than traditional site-directed recombination approaches. We also show that in all cases the optimized experiments are significantly better than ones that would result from stochastic methods.

  19. Pairing and recombination at meiosis of Brassica rapa (AA) x Brassica napus (AACC) hybrids.

    PubMed

    Leflon, M; Eber, F; Letanneur, J C; Chelysheva, L; Coriton, O; Huteau, V; Ryder, C D; Barker, G; Jenczewski, E; Chèvre, A M

    2006-11-01

    Interspecific crosses contribute significantly to plant evolution enabling gene exchanges between species. The efficiency of interspecific crosses depends on the similarity between the implicated genomes as high levels of genome similarity are required to ensure appropriate chromosome pairing and genetic recombination. Brassica napus (AACC) is an allopolyploid, resulting from natural hybridization between Brassica rapa (AA) and Brassica oleracea (CC), both being diploid species derived from a common ancestor. To study the relationships between genomes of these Brassica species, we have determined simultaneously the pairing and recombination pattern of A and C chromosomes during meiosis of AAC triploid hybrids, which result from the interspecific cross between natural B. napus and B. rapa. Different AAC triploid hybrids and their progenies have been analysed using cytogenetic, BAC-FISH, and molecular techniques. In 71% of the pollen mother cells, homologous A chromosomes paired regularly, and usually one chromosome of each pair was transmitted to the progeny. C chromosomes remained mainly univalent, but were involved in homoeologous pairing in 21.5% of the cells, and 13% of the transmitted C chromosomes were either recombined or broken. The rate of transmission of C chromosomes depended on the identity of the particular chromosome and on the way the hybrid was crossed, as the male or as the female parent, to B. napus or to B. rapa. Gene transfers in triploid hybrids are favoured between A genomes of B. rapa and B. napus, but also occur between A and C genomes though at lower rates.

  20. Application of a hybrid collisional radiative model to recombining argon plasmas

    NASA Astrophysics Data System (ADS)

    Benoy, D. A.; van der Mullen, J. A. M.; van de Sanden, M. C. M.; van der Sijde, B.; Schram, D. C.

    1993-02-01

    A collisional radiative model, in which a hybrid cut-off technique is used, is applied to recombining plasmas to study the atomic state distribution (ASDF) and the recombination coefficient. Computations of the ASDF using semi-empirical rate coefficients of Vriens and Smeets (V-S) and Drawin (D) are compared with experimental values measured at various positions in a free expanding argon arc jet. Apart from the shock position, where the calculated results are too low, the model calculations are higher than the experimental results. The volumetric recombination coefficient has a Te exp -4 and a Te exp -4.8 dependence when semiempirical rate coefficients of, respectively, V-S and D are used. The differences between the models based on the rate coefficients of V-S and D indicate that the recombination flow is sensitive to the low temperature behavior of the rate coefficients.

  1. 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.

  2. [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.

  3. 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.

  4. 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.

  5. 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.

  6. Mechanism of charge recombination in organic-inorganic hybrid perovskite solar cells

    NASA Astrophysics Data System (ADS)

    Yang, Wenchao; Yao, Yao; Wu, Chang-Qin; organic Group Team

    2015-03-01

    In the recent popular organic-inorganic hybrid perovskite solar cells, the slowness of the charge recombination processes is found to be a key factor for contributing to their high efficiencies and open circuit voltages, but the underlying mechanism remains unclear. In this work we study the recombination mechanism in perovskite solar cells and its roles on determining the device performance. Based on macroscopic device model simulations, the recombination resistances (Rrec) under different applied voltages are calculated to characterize the recombination mechanism, and the current density-voltage (J - V) curves are simulated to describe the device performance under at the same time. Through comparison with the impedance spectroscopy (IS) extracted Rrec data, it is found that bimolecular recombination (BR) is the dominant recombination process in the whole applied voltage regime and can determine the open circuit voltage, while the trap-assisted SRH monomolecular recombination (MR) is only important if the trap density is high or the BR rate is significantly reduced. The different electron injection barriers at the contact can induce different patterns for the Rrec- V characteristics. Under the cases of increased band gap or decreased BR rate, the Rrec's are enhanced which leads to high open circuit voltages. We are grateful to the support from the state key laboratory of surface physics, Fudan University.

  7. Increased expression in vivo and in vitro of foreign genes directed by A-type inclusion body hybrid promoters in recombinant vaccinia viruses.

    PubMed Central

    Funahashi, S; Itamura, S; Iinuma, H; Nerome, K; Sugimoto, M; Shida, H

    1991-01-01

    We constructed A-type inclusion body (ATI) hybrid promoters, that is, late ATI promoters followed by tandemly repeated early regions of the promoter for the 7.5-kDa protein (the 7.5-kDa promoter). The repetition of the whole early promoter sequence of the 7.5-kDa gene, including the upstream consensus sequence and initiation region, efficiently increased the early expression of the bacterial chloramphenicol acetyltransferase gene in recombinant vaccinia virus. Recombinant vaccinia virus could express influenza virus hemagglutinin via the hybrid promoter more efficiently, induced higher levels of neutralizing antibody and cytotoxic T lymphocytes, and consequently protected mice more efficiently against challenge with influenza virus than did recombinant vaccinia virus containing the widely used 7.5-kDa promoter. Images PMID:1654453

  8. 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.

  9. Tracing the recombination and colonization history of hybrid species in space and time.

    PubMed

    Lexer, C; Stölting, K N

    2011-09-01

    Hybrid speciation has long fascinated evolutionary biologists and laymen alike, presumably because it challenges our classical view of evolution as a 'one-way street' leading to strictly tree-like patterns of ancestry and descent. Homoploid hybrid speciation (HHS) has been a particularly interesting puzzle, as it appears to occur extremely rapidly, perhaps within less than 50 generations (McCarthy et al. 1995; Buerkle et al. 2000). Nevertheless, HHS may sometimes involve extended or repeated periods of recombination and gene exchange between populations subject to strong divergent natural selection (Buerkle & Rieseberg 2008). Thus, HHS provides a highly interesting setting for understanding the drivers and tempo of adaptive divergence and speciation in the face of gene flow (Arnold 2006; Rieseberg & Willis 2007; Nolte & Tautz 2009). In the present issue of Molecular Ecology, Wang et al. (2011) explore a particularly challenging issue connected to HHS: they attempt to trace the colonization and recombination history of an ancient (several MYA) hybrid species, from admixture and recombination in the ancestral hybrid zone to subsequent range shifts triggered by tectonic events (uplift of the Tibetan plateau) and climatic shifts (Pleistocene ice ages). This work is important because it addresses key issues related to the origin of the standing genetic variation available for adaptive responses (e.g. to climate change) and speciation in temperate species, which are topics of great current interest (Rieseberg et al. 2003; Barrett & Schluter 2008; de Carvalho et al. 2010).

  10. 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.

  11. 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.

  12. Hybrid Perovskites for Photovoltaics: Charge-Carrier Recombination, Diffusion, and Radiative Efficiencies.

    PubMed

    Johnston, Michael B; Herz, Laura M

    2016-01-19

    Photovoltaic (PV) devices that harvest the energy provided by the sun have great potential as renewable energy sources, yet uptake has been hampered by the increased cost of solar electricity compared with fossil fuels. Hybrid metal halide perovskites have recently emerged as low-cost active materials in PV cells with power conversion efficiencies now exceeding 20%. Rapid progress has been achieved over only a few years through improvements in materials processing and device design. In addition, hybrid perovskites appear to be good light emitters under certain conditions, raising the prospect of applications in low-cost light-emitting diodes and lasers. Further optimization of such hybrid perovskite devices now needs to be supported by a better understanding of how light is converted into electrical currents and vice versa. This Account provides an overview of charge-carrier recombination and mobility mechanisms encountered in such materials. Optical-pump-terahertz-probe (OPTP) photoconductivity spectroscopy is an ideal tool here, because it allows the dynamics of mobile charge carriers inside the perovskite to be monitored following excitation with a short laser pulse whose photon energy falls into the range of the solar spectrum. We first review our insights gained from transient OPTP and photoluminescence spectroscopy on the mechanisms dominating charge-carrier recombination in these materials. We discuss that mono-molecular charge-recombination predominantly originates from trapping of charges, with trap depths being relatively shallow (tens of millielectronvolts) for hybrid lead iodide perovskites. Bimolecular recombination arises from direct band-to-band electron-hole recombination and is found to be in significant violation of the simple Langevin model. Auger recombination exhibits links with electronic band structure, in accordance with its requirement for energy and momentum conservation for all charges involved. We further discuss charge-carrier mobility

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. The Rashba effect and indirect electron-hole recombination in hybrid organic-inorganic perovskites.

    PubMed

    Yu, Zhi-Gang

    2017-06-14

    Slow electron-hole recombination, characterized by the bimolecular coefficient k2 in hybrid organic-inorganic perovskites (HOIPs), is a key to their outstanding photovoltaic performance. The measured k2 in HOIPs strongly deviates from k2 ∝ T(-3/2) (T is the temperature) in typical direct-gap semiconductors. Here we show that the observed temperature dependence can be quantitatively accounted for by phonon-assisted recombination of electrons and holes located at the band extrema, which become indirect due to the Rashba effect. Polar optical phonons are most effective in facilitating this indirect recombination. The variation in k2 in HOIPs among different studies in the literature can be attributed to different Rashba strengths in their samples. Our results indicate that the confluence of the Rashba effect and polar coupling transform HOIPs into a unique indirect semiconductor that can accommodate both strong optical absorption and slow carrier dynamics.

  3. 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

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. [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.

  9. 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

  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. Evolutionary experimentation through hybridization under laboratory condition in Drosophila: evidence for recombinational speciation.

    PubMed

    Harini, Ballagere P; Ramachandra, Nallur B

    2003-10-01

    Drosophila nasuta nasuta (2n = 8) and Drosophila nasuta albomicans (2n = 6) are a pair of sibling allopatric chromosomal cross-fertile races of the nasuta subgroup of immigrans species group of Drosophila. Interracial hybridization between these two races has given rise to new karyotypic strains called Cytorace 1 and Cytorace 2 (first phase). Further hybridization between Thailand strain of D. n. albomicans and D. n. nasuta of Coorg strain has resulted in the evolution of two more Cytoraces, namely Cytorace 3 and Cytorace 4 (second phase). The third phase Cytoraces (Cytorace 5 to Cytorace 16) have evolved through interracial hybridization among first, second phase Cytoraces along with parental races. Each of these Cytoraces is composed of recombined genomes of the parental races. Here, we have made an attempt to systematically assess the impact of hybridization on karyotypes, morphometric and life history traits in all 16 Cytoraces. The results reveal that in most cases, the newly evolved Cytoraces, with different chromosome constitutions, exhibit decreased body size, better fitness and live longer than their parents. Particularly, Cytorace 5, 6 and 8 have evolved with very much higher range values of quantitative traits than the parents and other Cytoraces, which suggests the role of transgressive segregation in the evolution of these Cytoraces. Thus, the rapid divergence recorded in the chromosomes, karyotypes, body size and fitness traits of Cytoraces exhibit the early event of recombinational raciation / speciation in the evolution of the Cytoraces under laboratory conditions.

  12. Evolutionary experimentation through hybridization under laboratory condition in Drosophila: Evidence for Recombinational Speciation

    PubMed Central

    Harini, Ballagere P; Ramachandra, Nallur B

    2003-01-01

    Background Drosophila nasuta nasuta (2n = 8) and Drosophila nasuta albomicans (2n = 6) are a pair of sibling allopatric chromosomal cross-fertile races of the nasuta subgroup of immigrans species group of Drosophila. Interracial hybridization between these two races has given rise to new karyotypic strains called Cytorace 1 and Cytorace 2 (first phase). Further hybridization between Thailand strain of D. n. albomicans and D. n. nasuta of Coorg strain has resulted in the evolution of two more Cytoraces, namely Cytorace 3 and Cytorace 4 (second phase). The third phase Cytoraces (Cytorace 5 to Cytorace 16) have evolved through interracial hybridization among first, second phase Cytoraces along with parental races. Each of these Cytoraces is composed of recombined genomes of the parental races. Here, we have made an attempt to systematically assess the impact of hybridization on karyotypes, morphometric and life history traits in all 16 Cytoraces. Results The results reveal that in most cases, the newly evolved Cytoraces, with different chromosome constitutions, exhibit decreased body size, better fitness and live longer than their parents. Particularly, Cytorace 5, 6 and 8 have evolved with very much higher range values of quantitative traits than the parents and other Cytoraces, which suggests the role of transgressive segregation in the evolution of these Cytoraces. Conclusion Thus, the rapid divergence recorded in the chromosomes, karyotypes, body size and fitness traits of Cytoraces exhibit the early event of recombinational raciation / speciation in the evolution of the Cytoraces under laboratory conditions. PMID:14519211

  13. 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.

  14. Mitochondrial Genome Recombination in the Zone of Contact Between Two Hybridizing Conifers

    PubMed Central

    Jaramillo-Correa, Juan P.; Bousquet, Jean

    2005-01-01

    Variation in mitochondrial DNA was surveyed at four gene loci in and around the zone of contact between two naturally hybridizing conifers, black spruce (Picea mariana) and red spruce (P. rubens) in northeastern North America. Most of the mtDNA diversity of these species was found in populations next to or into the zone of contact, where some individuals bore rare mitotypes intermediate between the common mitotypes observed in the allopatric areas of each species. Sequence analysis and tests for mtDNA recombination point to this phenomenon, rather than to recurrent mutation, as the most tenable hypothesis for the origin of these rare mitotypes. From the 10 mitotypes observed, at least 4 would be the product of recombination between 4 of the 5 putative ancestral mitotypes. Tests for cytonuclear disequilibrium and geographical structure of the putative recombinant mitotypes suggest that mtDNA recombination is not frequent and relatively recent on the geological time scale. mtDNA recombination would have been promoted by transient heteroplasmy due to leakage of paternal mtDNA since the Holocene secondary contact between the two species. PMID:16118197

  15. 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.

  16. 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.

  17. Nanoscale hybrid protein/polymer functionalized materials

    NASA Astrophysics Data System (ADS)

    Ho, Dean; Chu, Ben; Lee, Hyeseung; Montemagno, Carlo D.

    2004-07-01

    Block copolymer-based membrane technology represents a versatile class of nanoscale materials in which biomolecules, such as membrane proteins, can be reconstituted. Our work has demonstrated the fabrication of large-area, protein- enhanced membranes that possess significant performance improvements in protein functionality. Among its many advantages over conventional lipid-based membrane systems, block copolymers can mimic natural cell biomembrane environments in a single chain, enabling large-area membrane fabrication using methods like Langmuir-Blodgett (LB) deposition, or spontaneous protein-functionalized nano-vesicle formation. The membrane protein, Bacteriorhodopsin (BR), found in Halobacterium Halobium, is a light-actuated proton pump that develops gradients towards the demonstration of coupled functionality with other membrane proteins to effect ATP production, or production of electricity through Bacteriorhodopsin activity-dependent reversal of Cytochrome C Oxidase (COX), found in Rhodobacter Sphaeroides. Using quantum dot-labeled, engineered protein constructs, we have demonstrated large-scale insertion of proteins into block copolymer Langmuir-Blodgett (LB) films as well as measurable pH changes based upon light-actuated proton pumping. Light actuated-activity across the protein-functionalized membrane when fully enclosed in a sol-gel matrix has also been observed using impedance spectroscopy. Initial data has suggested a significant pH change of up to 1.75 in a volume of 100 mL and surface area of 0.317cm2, a level that is capable of powering a number of proton-gradient dependent proteins towards the buildup of a robust, hybrid protein/polymer device. Recent atomic force microscopy studies of the protein-embedded polymer film samples have revealed the formation of protein aggregate-based pattern generation with very uniform torus-shaped rings. Current work focused towards characterizing the effects that various pattern formations can have on the

  18. 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

  19. Reduced recombination patterns in Robertsonian hybrids between chromosomal races of the house mouse: chiasma analyses.

    PubMed

    Dumas, D; Catalan, J; Britton-Davidian, J

    2015-01-01

    The recombination suppression models of chromosomal speciation posit that chromosomal rearrangements act as partial barriers to gene flow allowing these regions to accumulate genetic incompatibilities, thus contributing to the divergence of populations. Empirical and theoretical studies exploring the requirements of these models have mostly focused on the role of inversions. Here, the recombination landscape of heterozygosity for Robertsonian (Rb) fusions is investigated in the house mouse. Laboratory-bred F1 males and females between highly differentiated races from Tunisia (Rb: 2n=22, Standard, St: 2n=40) were produced in which all Rb fusions are present as trivalents in meiosis. Recombination patterns were determined by the analysis of chiasmata and compared with previous data on the Tunisian parental mice. A comparative analysis was performed on wild-caught male mice spanning the hybrid zone between two Italian races (2n=40, 2n=22). The results showed that the chiasma characteristics of both male and female Tunisian F1 and Italian hybrids clearly differed from those of Rb and St mice. Not only was the mean chiasma number (CN) intermediate between those of the parental mice in both geographic samples, but the distribution of chiasmata along the chromosomal arms of the F1 showed a distinct mosaic pattern. In short, the proximal region in the F1 exhibited a reduced CN similar to that observed in homozygous Rb, whereas distal regions more closely matched those in St mice. These results suggest that Rb rearrangements (homozygous or heterozygous) reduce recombination in the proximal regions of the chromosomes supporting their potential role in recombination-mediated speciation models.

  20. 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.

  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. [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.

  3. 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.

  4. Recombinant Human Peptidoglycan Recognition Proteins Reveal Antichlamydial Activity.

    PubMed

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

    2016-07-01

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

  5. 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

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

    PubMed

    Wiesler, Simone C; Weinzierl, Robert O J

    2012-08-26

    X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity. In order to attribute the different phenotypes to the nature of the mutation--rather than to fluctuating experimental conditions--it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates. Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex. We exploited the high-throughput purification method to

  7. Hybrid tandem photovoltaic devices with a transparent conductive interconnecting recombination layer

    SciTech Connect

    Kim, Taehee; Choi, Jin Young; Jeon, Jun Hong; Kim, Youn-Su; Kim, Bong-Soo; Lee, Doh-Kwon; Kim, Honggon; Han, Seunghee; Kim, Kyungkon

    2012-10-15

    Highlights: ► This work enhanced power conversion efficiency of the hybrid tandem solar cell from 1.0% to 2.6%. ► The interfacial series resistance of the tandem solar cell was eliminated by inserting ITO layer. ► This work shows the feasibility of the highly efficient hybrid tandem solar cells. -- Abstract: We demonstrate hybrid tandem photovoltaic devices with a transparent conductive interconnecting recombination layer. The series-connected hybrid tandem photovoltaic devices were developed by combining hydrogenated amorphous silicon (a-Si:H) and polymer-based organic photovoltaics (OPVs). In order to enhance the interfacial connection between the subcells, we employed highly transparent and conductive indium tin oxide (ITO) thin layer. By using the ITO interconnecting layer, the power conversion efficiency of the hybrid tandem solar cell was enhanced from 1.0% (V{sub OC} = 1.041 V, J{sub SC} = 2.97 mA/cm{sup 2}, FF = 32.3%) to 2.6% (V{sub OC} = 1.336 V, J{sub SC} = 4.65 mA/cm{sup 2}, FF = 41.98%) due to the eliminated interfacial series resistance.

  8. 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

  9. Research Update: Relativistic origin of slow electron-hole recombination in hybrid halide perovskite solar cells

    NASA Astrophysics Data System (ADS)

    Azarhoosh, Pooya; McKechnie, Scott; Frost, Jarvist M.; Walsh, Aron; van Schilfgaarde, Mark

    2016-09-01

    The hybrid perovskite CH3NH3PbI3 (MAPI) exhibits long minority-carrier lifetimes and diffusion lengths. We show that slow recombination originates from a spin-split indirect-gap. Large internal electric fields act on spin-orbit-coupled band extrema, shifting band-edges to inequivalent wavevectors, making the fundamental gap indirect. From a description of photoluminescence within the quasiparticle self-consistent GW approximation for MAPI, CdTe, and GaAs, we predict carrier lifetime as a function of light intensity and temperature. At operating conditions we find radiative recombination in MAPI is reduced by a factor of more than 350 compared to direct gap behavior. The indirect gap is retained with dynamic disorder.

  10. 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.

  11. 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.

  12. 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

  13. 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.

  14. 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

  15. 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 ...

  16. 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

  17. 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

  18. Recombinant azurin-CdSe/ZnS hybrid structures for nanoscale resistive random access memory device.

    PubMed

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

    2017-04-15

    In the present study, we developed a biohybrid material composed of recombinant azurin and CdSe-ZnS quantum dot to perform as a resistive random access memory (ReRAM) device. Site specific amino acid sequences were introduced in azurin to bind with the surface of CdSe-ZnS nanoparticle allowing the formation of a hybrid and voltage-driven switching enabled to develop a resistive random access memory (ReRAM) device. The analytical measurements confirmed that the azurin and CdSe-ZnS nanoparticles were well conjugated and formed into a single hybrid. Further, reversible, bistable switching along with repeatable writing-reading-erasing processes on individual azurin/CdSe-ZnS hybrid at nanoscale was achieved on the hybrid device. The device was programmed tested for 50 cycles with an ON/OFF ratio and measured to be of three orders of magnitude. The developed device shown good stability and repeatability and operates at low voltages thus makes it promising candidate for future memory device applications.

  19. Hybrid metabolic flux analysis: combining stoichiometric and statistical constraints to model the formation of complex recombinant products.

    PubMed

    Carinhas, Nuno; Bernal, Vicente; Teixeira, Ana P; Carrondo, Manuel Jt; Alves, Paula M; Oliveira, Rui

    2011-02-25

    Stoichiometric models constitute the basic framework for fluxome quantification in the realm of metabolic engineering. A recurrent bottleneck, however, is the establishment of consistent stoichiometric models for the synthesis of recombinant proteins or viruses. Although optimization algorithms for in silico metabolic redesign have been developed in the context of genome-scale stoichiometric models for small molecule production, still rudimentary knowledge of how different cellular levels are regulated and phenotypically expressed prevents their full applicability for complex product optimization. A hybrid framework is presented combining classical metabolic flux analysis with projection to latent structures to further link estimated metabolic fluxes with measured productivities. We first explore the functional metabolic decomposition of a baculovirus-producing insect cell line from experimental data, highlighting the TCA cycle and mitochondrial respiration as pathways strongly associated with viral replication. To reduce uncertainty in metabolic target identification, a Monte Carlo sampling method was used to select meaningful associations with the target, from which 66% of the estimated fluxome had to be screened out due to weak correlations and/or high estimation errors. The proposed hybrid model was then validated using a subset of preliminary experiments to pinpoint the same determinant pathways, while predicting the productivity of independent cultures. Overall, the results indicate our hybrid metabolic flux analysis framework is an advantageous tool for metabolic identification and quantification in incomplete or ill-defined metabolic networks. As experimental and computational solutions for constructing comprehensive global cellular models are in development, the contribution of hybrid metabolic flux analysis should constitute a valuable complement to current computational platforms in bridging the metabolic state with improved cell culture performance.

  20. 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.

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

    PubMed

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

    2016-01-01

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

  2. 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

  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. 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.

  5. Enrichment of apoplastic fluid with therapeutic recombinant protein for efficient biofarming.

    PubMed

    Chatterjee, Aparajita; Das, Narayan C; Raha, Sumita; Maiti, Indu B; Shrestha, Ankita; Khan, Ahamed; Acharya, Sefali; Dey, Nrisingha

    2017-05-01

    For efficient biofarming we attempted to enrich plant interstitial fluid (IF)/apoplastic fluid with targeted recombinant therapeutic protein. We employed a synthetic human Glucocerebrosidase (GCB), a model biopharmaceutical protein gene in this study. Twenty one Nicotiana varieties, species and hybrids were initially screened for individual IF recovery and based on the findings, we selected Nicotiana tabacum NN (S-9-6), Nicotiana tabacum nn (S-9-7) and Nicotiana benthamiana (S-6-6) as model plants for raising transgenic expressing GCB via Agrobacterium mediated transformation under the control of M24 promoter; GCB specific activity in each transgenic lines were analyzed and we observed higher concentration of recombinant GCB in IF of these transgenic lines (S-9-6, S-9-7, and S-6-6) in comparison to their concentration in crude leaf extracts. Recovery of valuable therapeutics in plant IF as shown in the present study holds great promise for promoting plant based biofarming. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:726-736, 2017. © 2017 American Institute of Chemical Engineers.

  6. 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.

  7. 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.

  8. 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.

  9. Advances in recombinant DNA technology: corifollitropin alfa, a hybrid molecule with sustained follicle-stimulating activity and reduced injection frequency.

    PubMed

    Fauser, B C J M; Mannaerts, B M J L; Devroey, P; Leader, A; Boime, I; Baird, D T

    2009-01-01

    Recombinant DNA technologies have been used to develop longer-acting therapeutic proteins. One approach is to introduce sequences containing additional glycosylation sites. Using this technique, a new chimeric gene has been developed containing the coding sequences of the FSH beta-subunit and the C-terminal peptide of the hCG beta-subunit, which bears four O-linked oligosaccharide binding sites. Co-expression of the alpha-subunit and the chimeric FSH beta-subunit produces a new recombinant molecule, named corifollitropin alfa, with a prolonged elimination half-life and enhanced in vivo bioactivity compared with wild-type FSH. Medline searches by subject and additional searching by hand. Initial studies in pituitary suppressed female volunteers confirmed the extended half-life of the compound. Phase II studies have shown that corifollitropin alfa is able to induce and sustain multi-follicular growth for an entire week in women undergoing ovarian stimulation using GnRH antagonist co-treatment for IVF. Corifollitropin alfa regimens have been developed with dosages of 100 and 150 microg, for patients with body weight 60 kg, respectively. Corifollitropin alfa is the first long-acting hybrid molecule with sustained follicle-stimulating activity developed for the induction of multi-follicular growth along with GnRH antagonist co-treatment for IVF. This new treatment option may be simpler and more convenient for patients compared with conventional long protocols of daily FSH injections in combination with GnRH agonist co-treatment. The safety and efficacy of such regimens is currently being evaluated in large comparative phase III clinical trials. The development of corifollitropin alfa is the first step towards a new generation of recombinant gonadotrophins.

  10. [Identification of proteins interacting with the circadian clock protein PER1 in tumors using bacterial two-hybrid system technique].

    PubMed

    Zhang, Yu; Yao, Youlin; Jiang, Siyuan; Lu, Yilu; Liu, Yunqiang; Tao, Dachang; Zhang, Sizhong; Ma, Yongxin

    2015-04-01

    To identify protein-protein interaction partners of PER1 (period circadian protein homolog 1), key component of the molecular oscillation system of the circadian rhythm in tumors using bacterial two-hybrid system technique. Human cervical carcinoma cell Hela library was adopted. Recombinant bait plasmid pBT-PER1 and pTRG cDNA plasmid library were cotransformed into the two-hybrid system reporter strain cultured in a special selective medium. Target clones were screened. After isolating the positive clones, the target clones were sequenced and analyzed. Fourteen protein coding genes were identified, 4 of which were found to contain whole coding regions of genes, which included optic atrophy 3 protein (OPA3) associated with mitochondrial dynamics and homo sapiens cutA divalent cation tolerance homolog of E. coli (CUTA) associated with copper metabolism. There were also cellular events related proteins and proteins which are involved in biochemical reaction and signal transduction-related proteins. Identification of potential interacting proteins with PER1 in tumors may provide us new insights into the functions of the circadian clock protein PER1 during tumorigenesis.

  11. PRDM9 interactions with other proteins provide a link between recombination hotspots and the chromosomal axis in meiosis.

    PubMed

    Parvanov, Emil D; Tian, Hui; Billings, Timothy; Saxl, Ruth L; Spruce, Catrina; Aithal, Rakesh; Krejci, Lumir; Paigen, Kenneth; Petkov, Petko M

    2017-02-01

    In mammals, meiotic recombination occurs at 1- to 2-kb genomic regions termed hotspots, whose positions and activities are determined by PRDM9, a DNA-binding histone methyltransferase. We show that the KRAB domain of PRDM9 forms complexes with additional proteins to allow hotspots to proceed into the next phase of recombination. By a combination of yeast-two hybrid assay, in vitro binding, and coimmunoprecipitation from mouse spermatocytes, we identified four proteins that directly interact with PRDM9's KRAB domain, namely CXXC1, EWSR1, EHMT2, and CDYL. These proteins are coexpressed in spermatocytes at the early stages of meiotic prophase I, the limited period when PRDM9 is expressed. We also detected association of PRDM9-bound complexes with the meiotic cohesin REC8 and the synaptonemal complex proteins SYCP3 and SYCP1. Our results suggest a model in which PRDM9-bound hotspot DNA is brought to the chromosomal axis by the action of these proteins, ensuring the proper chromatin and spatial environment for subsequent recombination events.

  12. 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.

  13. 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.

  14. Protein-directed assembly of cobalt phosphate hybrid nanoflowers.

    PubMed

    Kim, Kyung Hoon; Jeong, Jae-Min; Lee, Seok Jae; Choi, Bong Gill; Lee, Kyoung G

    2016-12-15

    The understanding and controlling of biomimetic hybrid materials are a key objective in bio-nanotechnology, materials chemistry, and colloid science fields. Biomaterials, such as, enzyme, DNA, RNA, and proteins have become important templates for the construction of inorganic-organic hybrid nanoflowers. From this perspective, we present a simple approach to synthesize protein and metal hybrid flower-like structure using bovine serum albumin (BSA) and cobalt phosphate, and the results of our study on the formation mechanism involved. The time dependent growing stage and formation mechanism were analyzed by electron microscopes and spectroscopic techniques. The protein-directed assembly method for preparation of hybrid nanoflowers described in this work could be used to fabricate other bio-metal hybrid materials with possible applications in biosensors, bioanalytical devices, and industrial biocatalyst fields.

  15. 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.

  16. 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.

  17. 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

  18. Chromosome rearrangements, recombination suppression, and limited segregation distortion in hybrids between Yellowstone cutthroat trout (Oncorhynchus clarkii bouvieri) and rainbow trout (O. mykiss)

    USGS Publications Warehouse

    Ostberg, Carl O.; Hauser, Lorenz; Pritchard, Victoria L.; Garza, John C.; Naish, Kerry A.

    2013-01-01

    Chromosome rearrangements suppressed recombination in the hybrids. This result supports several previous findings demonstrating that recombination suppression restricts gene flow between chromosomes that differ by arrangement. Conservation of synteny and map order between the hybrid and rainbow trout maps and minimal segregation distortion in the hybrids suggest rainbow and Yellowstone cutthroat trout genomes freely introgress across chromosomes with similar arrangement. Taken together, these results suggest that rearrangements impede introgression. Recombination suppression across rearrangements could enable large portions of non-recombined chromosomes to persist within admixed populations.

  19. 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.

  20. 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.

  1. 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.)

  2. 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

  3. 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.

  4. 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.

  5. Green Fluorescent Protein as a Visual Marker in Somatic Hybridization

    PubMed Central

    OLIVARES‐FUSTER, O.; PEÑA, L.; DURAN‐VILA, N.; NAVARRO, L.

    2002-01-01

    Using a transgenic citrus plant expressing Green Fluorescent Protein (GFP) as a parent in somatic fusion experiments, we investigated the suitability of GFP as an in vivo marker to follow the processes of protoplast fusion, regeneration and selection of hybrid plants. A high level of GFP expression was detected in transgenic citrus protoplasts, hybrid callus, embryos and plants. It is demonstrated that GFP can be used for the continuous monitoring of the fusion process, localization of hybrid colonies and callus, and selection of somatic hybrid embryos and plants. PMID:12096810

  6. 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.

  7. Unique Characteristics of Recombinant Hybrid Manganese Superoxide Dismutase from Staphylococcus equorum and S. saprophyticus.

    PubMed

    Retnoningrum, Debbie S; Rahayu, Anis Puji; Mulyanti, Dina; Dita, Astrid; Valerius, Oliver; Ismaya, Wangsa T

    2016-04-01

    A recombinant hybrid of manganese dependent-superoxide dismutase of Staphylococcus equorum and S. saprophyticus has successfully been overexpressed in Escherichia coli BL21(DE3), purified, and characterized. The recombinant enzyme suffered from degradation and aggregation upon storage at -20 °C, but not at room temperature nor in cold. Chromatographic analysis in a size exclusion column suggested the occurrence of dimeric form, which has been reported to contribute in maintaining the stability of the enzyme. Effect of monovalent (Na(+), K(+)), divalent (Ca(2+), Mg(2+)), multivalent (Mn(2+/4+), Zn(2+/4+)) cations and anions (Cl(-), SO4 (2-)) to the enzyme stability or dimeric state depended on type of cation or anion, its concentration, and pH. However, tremendous effect was observed with 50 mM ZnSO4, in which thermostability of both the dimer and monomer was increased. Similar situation was not observed with MnSO4, and its presence was detrimental at 200 mM. Finally, chelating agent appeared to destabilize the dimer around neutral pH and dissociate it at basic pH. The monomer remained stable upon addition of ethylene diamine tetraacetic acid. Here we reported unique characteristics and stability of manganese dependent-superoxide dismutase from S. equorum/saprophyticus.

  8. Application of hybrid LRR technique to protein crystallization.

    PubMed

    Jin, Mi Sun; Lee, Jie-Oh

    2008-05-31

    LRR family proteins play important roles in a variety of physiological processes. To facilitate their production and crystallization, we have invented a novel method termed "Hybrid LRR Technique". Using this technique, the first crystal structures of three TLR family proteins could be determined. In this review, design principles and application of the technique to protein crystallization will be summarized. For crystallization of TLRs, hagfish VLR receptors were chosen as the fusion partners and the TLR and the VLR fragments were fused at the conserved LxxLxLxxN motif to minimize local structural incompatibility. TLR-VLR hybridization did not disturb structures and functions of the target TLR proteins. The Hybrid LRR Technique is a general technique that can be applied to structural studies of other LRR proteins. It may also have broader application in biochemical and medical application of LRR proteins by modifying them without compromising their structural integrity.

  9. Chimeric Protein Complexes in Hybrid Species Generate Novel Phenotypes

    PubMed Central

    Piatkowska, Elzbieta M.; Naseeb, Samina; Knight, David; Delneri, Daniela

    2013-01-01

    Hybridization between species is an important mechanism for the origin of novel lineages and adaptation to new environments. Increased allelic variation and modification of the transcriptional network are the two recognized forces currently deemed to be responsible for the phenotypic properties seen in hybrids. However, since the majority of the biological functions in a cell are carried out by protein complexes, inter-specific protein assemblies therefore represent another important source of natural variation upon which evolutionary forces can act. Here we studied the composition of six protein complexes in two different Saccharomyces “sensu stricto” hybrids, to understand whether chimeric interactions can be freely formed in the cell in spite of species-specific co-evolutionary forces, and whether the different types of complexes cause a change in hybrid fitness. The protein assemblies were isolated from the hybrids via affinity chromatography and identified via mass spectrometry. We found evidence of spontaneous chimericity for four of the six protein assemblies tested and we showed that different types of complexes can cause a variety of phenotypes in selected environments. In the case of TRP2/TRP3 complex, the effect of such chimeric formation resulted in the fitness advantage of the hybrid in an environment lacking tryptophan, while only one type of parental combination of the MBF complex allowed the hybrid to grow under respiratory conditions. These phenotypes were dependent on both genetic and environmental backgrounds. This study provides empirical evidence that chimeric protein complexes can freely assemble in cells and reveals a new mechanism to generate phenotypic novelty and plasticity in hybrids to complement the genomic innovation resulting from gene duplication. The ability to exchange orthologous members has also important implications for the adaptation and subsequent genome evolution of the hybrids in terms of pattern of gene loss. PMID

  10. 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.

  11. 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.

  12. 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

  13. Adipose tissue insulin receptor knockdown via a new primate-derived hybrid recombinant AAV serotype

    PubMed Central

    Liu, Xianglan; Magee, Daniel; Wang, Chuansong; McMurphy, Travis; Slater, Andrew; During, Matthew; Cao, Lei

    2014-01-01

    Adipose tissue plays an essential role in metabolic homeostasis and holds promise as an alternative depot organ in gene therapy. However, efficient methods of gene transfer into adipose tissue in vivo have yet to be established. Here, we assessed the transduction efficiency to fat depots by a family of novel engineered hybrid capsid serotypes (Rec1~4) recombinant adeno-associated viral (AAV) vectors in comparison with natural serotypes AAV1, AAV8, and AAV9. Rec2 serotype led to widespread transduction in both brown fat and white fat with the highest efficiency among the seven serotypes tested. As a proof-of-efficacy, Rec2 serotype was used to deliver Cre recombinase to adipose tissues of insulin receptor floxed animals. Insulin receptor knockdown led to decreased fat pad mass and morphological and molecular changes in the targeted depot. These novel hybrid AAV vectors can serve as powerful tools to genetically manipulate adipose tissue and provide valuable vehicles to gene therapy targeting adipose tissue. PMID:25383359

  14. Climate-Driven Reshuffling of Species and Genes: Potential Conservation Roles for Species Translocations and Recombinant Hybrid Genotypes

    PubMed Central

    Scriber, Jon Mark

    2013-01-01

    Comprising 50%–75% of the world’s fauna, insects are a prominent part of biodiversity in communities and ecosystems globally. Biodiversity across all levels of biological classifications is fundamentally based on genetic diversity. However, the integration of genomics and phylogenetics into conservation management may not be as rapid as climate change. The genetics of hybrid introgression as a source of novel variation for ecological divergence and evolutionary speciation (and resilience) may generate adaptive potential and diversity fast enough to respond to locally-altered environmental conditions. Major plant and herbivore hybrid zones with associated communities deserve conservation consideration. This review addresses functional genetics across multi-trophic-level interactions including “invasive species” in various ecosystems as they may become disrupted in different ways by rapid climate change. “Invasive genes” (into new species and populations) need to be recognized for their positive creative potential and addressed in conservation programs. “Genetic rescue” via hybrid translocations may provide needed adaptive flexibility for rapid adaptation to environmental change. While concerns persist for some conservationists, this review emphasizes the positive aspects of hybrids and hybridization. Specific implications of natural genetic introgression are addressed with a few examples from butterflies, including transgressive phenotypes and climate-driven homoploid recombinant hybrid speciation. Some specific examples illustrate these points using the swallowtail butterflies (Papilionidae) with their long-term historical data base (phylogeographical diversity changes) and recent (3-decade) climate-driven temporal and genetic divergence in recombinant homoploid hybrids and relatively recent hybrid speciation of Papilio appalachiensis in North America. Climate-induced “reshuffling” (recombinations) of species composition, genotypes, and genomes

  15. Bacteriorhodopsin protein hybrids for chemical and biological sensing

    NASA Astrophysics Data System (ADS)

    Winder, Eric Michael

    Bacteriorhodopsin (bR), an optoelectric protein found in Halobacterium salinarum, has the potential for use in protein hybrid sensing systems. Bacteriorhodopsin has no intrinsic sensing properties, however molecular and chemical tools permit production of bR protein hybrids with transducing and sensing properties. As a proof of concept, a maltose binding protein-bacteriorhodopsin ([MBP]-bR) hybrid was developed. It was proposed that the energy associated with target molecule binding, maltose, to the hybrid sensor protein would provide a means to directly modulate the electrical output from the MBP-bR bio-nanosensor platform. The bR protein hybrid is produced by linkage between bR (principal component of purified purple membrane [PM]) and MBP, which was produced by use of a plasmid expression vector system in Escherichia coli and purified utilizing an amylose affinity column. These proteins were chemically linked using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), which facilitates formation of an amide bond between a primary carboxylic acid and a primary amine. The presence of novel protein hybrids after chemical linkage was analyzed by SDS-PAGE. Soluble proteins (MBP-only derivatives and unlinked MBP) were separated from insoluble proteins (PM derivatives and unlinked PM) using size exclusion chromatography. The putatively identified MBP-bR protein hybrid, in addition to unlinked bR, was collected. This sample was normalized for bR concentration to native PM and both were deposited onto indium tin oxide (ITO) coated glass slides by electrophoretic sedimentation. The photoresponse of both samples, activated using 100 Watt tungsten lamp at 10 cm distance, were equal at 175 mV. Testing of deposited PM with 1 mM sucrose or 1 mM maltose showed no change in the photoresponse of the material, however addition of 1 mM maltose to the deposited MBP-bR linked hybrid material elicited a 57% decrease in photoresponse

  16. Two-hybrid system for characterization of protein-protein interactions in E. coli.

    PubMed

    Hays, L B; Chen, Y S; Hu, J C

    2000-08-01

    The yeast two-hybrid system has been used to characterize many protein-protein interactions. A two-hybrid system for E. coli was constructed in which one hybrid protein bound to a specific DNA site recruits another to an adjacent DNA binding site. The first hybrid comprises a test protein, the bait, fused to a chimeric protein containing the 434 repressor DNA binding domain. In the second hybrid, a second test protein, the prey, is fused downstream of a chimeric protein with the DNA binding specificity of the lambda repressor. Reporters were designed to express cat and lacZ under the control of a low-affinity lambda operator. At low expression levels, lambda repressor hybrids weakly repress the reporter genes. A high-affinity operator recognized by 434 repressor was placed nearby, in a position that does not yield repression by 434 repressor alone. If the test proteins interact, the 434 hybrid bound to the 434 operator stabilizes the binding of the lambda repressor hybrid to the lambda operator, causing increased repression of the reporter genes. Reconstruction experiments with the fos and jun leucine zippers detected protein-protein interactions between either homodimeric or heterodimeric leucine zippers.

  17. 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.

  18. Using Protein Dimers to Maximize the Protein Hybridization Efficiency with Multisite DNA Origami Scaffolds

    PubMed Central

    Verma, Vikash; Mallik, Leena; Hariadi, Rizal F.; Sivaramakrishnan, Sivaraj; Skiniotis, Georgios; Joglekar, Ajit P.

    2015-01-01

    DNA origami provides a versatile platform for conducting ‘architecture-function’ analysis to determine how the nanoscale organization of multiple copies of a protein component within a multi-protein machine affects its overall function. Such analysis requires that the copy number of protein molecules bound to the origami scaffold exactly matches the desired number, and that it is uniform over an entire scaffold population. This requirement is challenging to satisfy for origami scaffolds with many protein hybridization sites, because it requires the successful completion of multiple, independent hybridization reactions. Here, we show that a cleavable dimerization domain on the hybridizing protein can be used to multiplex hybridization reactions on an origami scaffold. This strategy yields nearly 100% hybridization efficiency on a 6-site scaffold even when using low protein concentration and short incubation time. It can also be developed further to enable reliable patterning of a large number of molecules on DNA origami for architecture-function analysis. PMID:26348722

  19. Using Protein Dimers to Maximize the Protein Hybridization Efficiency with Multisite DNA Origami Scaffolds.

    PubMed

    Verma, Vikash; Mallik, Leena; Hariadi, Rizal F; Sivaramakrishnan, Sivaraj; Skiniotis, Georgios; Joglekar, Ajit P

    2015-01-01

    DNA origami provides a versatile platform for conducting 'architecture-function' analysis to determine how the nanoscale organization of multiple copies of a protein component within a multi-protein machine affects its overall function. Such analysis requires that the copy number of protein molecules bound to the origami scaffold exactly matches the desired number, and that it is uniform over an entire scaffold population. This requirement is challenging to satisfy for origami scaffolds with many protein hybridization sites, because it requires the successful completion of multiple, independent hybridization reactions. Here, we show that a cleavable dimerization domain on the hybridizing protein can be used to multiplex hybridization reactions on an origami scaffold. This strategy yields nearly 100% hybridization efficiency on a 6-site scaffold even when using low protein concentration and short incubation time. It can also be developed further to enable reliable patterning of a large number of molecules on DNA origami for architecture-function analysis.

  20. Chemical and Biological Sensing Utilizing Fused Bacteriorhodopsin Protein Hybrids

    DTIC Science & Technology

    2008-12-01

    Utilizing this purified DNA and a plasmid expression vector system, a fused protein hybrid consisting of maltose binding protein and bacterio-opsin has...prior to transcription, or post-expression. Therefore, for the development of the current proof of concept biosensor, maltose binding protein has...been chosen for attachment to the N-terminus of bR by genetic fusion and subsequent expression in E. coli. The maltose binding protein is a

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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...

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. Protection of mice against Staphylococcus aureus infection by a recombinant protein ClfA-IsdB-Hlg as a vaccine candidate.

    PubMed

    Delfani, Somayeh; Mohabati Mobarez, Ashraf; Imani Fooladi, Abbas Ali; Amani, Jafar; Emaneini, Mohammad

    2016-02-01

    Staphylococcus aureus is one of the most important causes of nosocomial infections. An effective vaccine to prevent S. aureus infections is urgently required due to the dramatic increase in the number of antibiotic-resistant strains. In this report, we evaluated a newly recombinant protein composed of selected antigenic regions of clumping factor A (ClfA), iron surface determinant B (IsdB) and gamma hemolysin B (HlgB) of S. aureus and sequence coding for hydrophobic linkers between three domains. The recombinant gene was constructed in pET-28a (+) and expressed in Escherichia coli BL21. In addition, sequence coding for a His(6)-tag was added followed by a hybrid procedure of nickel chelate protein purification. Immunization of BALB/c mice with the recombinant protein ClfA-IsdB-Hlg evoked antigen-specific antibodies that could opsonize S. aureus cells, enhancing in vitro phagocytosis by macrophages. Vaccination with the recombinant protein also reduced the bacterial load recovered from mice spleen samples and increased survival following the intraperitoneal challenge with pathogenic S. aureus compared to the control mice. Our results showed that the recombinant protein ClfA-IsdB-Hlg is a promising vaccine candidate for the prevention of S. aureus bacteremia infections.

  17. Extracellular self-assembly of virus-like particles from secreted recombinant polyoma virus major coat protein.

    PubMed

    Ng, J; Koechlin, O; Ramalho, M; Raman, D; Krauzewicz, N

    2007-12-01

    Mouse polyoma virus major coat protein (VP1) expressed from a recombinant baculovirus is efficiently transported to infected cell nuclei and assembles into protein nanospheres morphologically similar to natural capsids. The nanospheres readily combine with plasmid DNA to form a hybrid gene therapy agent known as virus-like particles (VLPs). To facilitate large-scale production of VLPs free from cellular contaminants, the use of stable Drosophila cell lines expressing either wild-type protein, or VP1 tagged with a secretion signal for targeting to the extracellular medium, was investigated. Both wild-type and tagged VP1 expressed at 2-4 mg VP1/litre of culture. As expected, the wild-type protein self-assembled into VLPs. The tagged VP1 was efficiently secreted to the extracellular medium but was also glycosylated, unlike wild-type VP1. Despite this fact, a small fraction of the recombinant secreted protein assembled into VLP-like structures that had altered disulphide bonding, but were still biologically active. These results demonstrate the considerable tolerance in the nanosphere assembly to structural (i.e. aberrant glycosylation) and environmental (i.e. extracellular medium vs. nuclear milieu) changes. Thus, with modifications to improve nanosphere assembly, the secretion method could be adapted to large-scale preparation of VLPs, providing significant advantages over current methods of production of the vector.

  18. 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

  19. Hybrid Sterility in Rice (Oryza sativa L.) Involves the Tetratricopeptide Repeat Domain Containing Protein

    PubMed Central

    Yu, Yang; Zhao, Zhigang; Shi, Yanrong; Tian, Hua; Liu, Linglong; Bian, Xiaofeng; Xu, Yang; Zheng, Xiaoming; Gan, Lu; Shen, Yumin; Wang, Chaolong; Yu, Xiaowen; Wang, Chunming; Zhang, Xin; Guo, Xiuping; Wang, Jiulin; Ikehashi, Hiroshi; Jiang, Ling; Wan, Jianmin

    2016-01-01

    Intersubspecific hybrid sterility is a common form of reproductive isolation in rice (Oryza sativa L.), which significantly hampers the utilization of heterosis between indica and japonica varieties. Here, we elucidated the mechanism of S7, which specially causes Aus-japonica/indica hybrid female sterility, through cytological and genetic analysis, map-based cloning, and transformation experiments. Abnormal positioning of polar nuclei and smaller embryo sac were observed in F1 compared with male and female parents. Female gametes carrying S7cp and S7i were aborted in S7ai/S7cp and S7ai/S7i, respectively, whereas they were normal in both N22 and Dular possessing a neutral allele, S7n. S7 was fine mapped to a 139-kb region in the centromere region on chromosome 7, where the recombination was remarkably suppressed due to aggregation of retrotransposons. Among 16 putative open reading frames (ORFs) localized in the mapping region, ORF3 encoding a tetratricopeptide repeat domain containing protein was highly expressed in the pistil. Transformation experiments demonstrated that ORF3 is the candidate gene: downregulated expression of ORF3 restored spikelet fertility and eliminated absolutely preferential transmission of S7ai in heterozygote S7ai/S7cp; sterility occurred in the transformants Cpslo17-S7ai. Our results may provide implications for overcoming hybrid embryo sac sterility in intersubspecific hybrid rice and utilization of hybrid heterosis for cultivated rice improvement. PMID:27182946

  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. 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

  2. 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.

  3. 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.

  4. 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

  5. The devil and holy water: protein and carbon nanotube hybrids.

    PubMed

    Calvaresi, Matteo; Zerbetto, Francesco

    2013-11-19

    Integrating carbon nanotubes (CNTs) with biological systems to form hybrid functional assemblies is an innovative research area with great promise for medical, nanotechnology, and materials science applications. The specifics of molecular recognition and catalytic activity of proteins combined with the mechanical and electronic properties of CNTs provides opportunities for physicists, chemists, biologists, and materials scientists to understand and develop new nanomachines, sensors, or any of a number of other molecular assemblies. Researchers know relatively little about the structure, function, and spatial orientation of proteins noncovalently adsorbed on CNTs, yet because the interaction of CNTs with proteins depends strongly on the tridimensional structure of the proteins, many of these questions can be answered in simple terms. In this Account, we describe recent research investigating the properties of CNT/protein hybrids. Proteins act to solvate CNTs and may sort them according to diameter or chirality. In turn, CNTs can support and immobilize enzymes, creating functional materials. Additional applications include proteins that assemble ordered hierarchical objects containing CNTs, and CNTs that act as protein carriers for vaccines, for example. Protein/CNT hybrids can form bioscaffolds and can serve as therapeutic and imaging materials. Proteins can detect CNTs or coat them to make them biocompatible. One of the more challenging applications for protein/CNT hybrids is to make CNT substrates for cell growth and neural interfacing applications. The challenge arises from the structures' interactions with living cells, which poses questions surrounding the (nano)toxicology of CNTs and whether and how CNTs can detect biological processes or sense them as they occur. The surface chemistry of CNTs and proteins, including interactions such as π-π stacking interactions, hydrophobic interactions, surfactant-like interactions, and charge-π interactions, governs the

  6. 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

  7. 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.

  8. 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

  9. Comparison of Recombinant Trypanosoma cruzi Peptide Mixtures versus Multiepitope Chimeric Proteins as Sensitizing Antigens for Immunodiagnosis▿

    PubMed Central

    Camussone, Cecilia; Gonzalez, Verónica; Belluzo, María S.; Pujato, Nazarena; Ribone, María E.; Lagier, Claudia M.; Marcipar, Iván S.

    2009-01-01

    The aim of this work was to determine the best strategy to display antigens (Ags) on immunochemical devices to improve test selectivity and sensitivity. We comparatively evaluated five Trypanosoma cruzi antigenic recombinant peptides, chose the three more sensitive ones, built up chimeras bearing these selected Ags, and systematically compared by enzyme-linked immunosorbent assay the performance of the assortments of those peptides with that of the multiepitope constructions bearing all those peptides lineally fused. The better-performing Ags that were compared included peptides homologous to the previously described T. cruzi flagellar repetitive Ag (here named RP1), shed acute-phase Ag (RP2), B13 (RP5), and the chimeric recombinant proteins CP1 and CP2, bearing repetitions of RP1-RP2 and RP1-RP2-RP5, respectively. The diagnostic performances of these Ags were assessed for discrimination efficiency by the formula +OD/cutoff value (where +OD is the mean optical density value of the positive serum samples tested), in comparison with each other either alone, in mixtures, or as peptide-fused chimeras and with total parasite homogenate (TPH). The discrimination efficiency values obtained for CP1 and CP2 were 25% and 52% higher, respectively, than those of their individual-Ag mixtures. CP2 was the only Ag that showed enhanced discrimination efficiency between Chagas' disease-positive and -negative samples, compared with TPH. This study highlights the convenience of performing immunochemical assays using hybrid, single-molecule, chimeric Ags instead of peptide mixtures. CP2 preliminary tests rendered 98.6% sensitivity when evaluated with a 141-Chagas' disease-positive serum sample panel and 99.4% specificity when assessed with a 164-Chagas' disease-negative serum sample panel containing 15 samples from individuals infected with Leishmania spp. PMID:19339486

  10. Comparison of recombinant Trypanosoma cruzi peptide mixtures versus multiepitope chimeric proteins as sensitizing antigens for immunodiagnosis.

    PubMed

    Camussone, Cecilia; Gonzalez, Verónica; Belluzo, María S; Pujato, Nazarena; Ribone, María E; Lagier, Claudia M; Marcipar, Iván S

    2009-06-01

    The aim of this work was to determine the best strategy to display antigens (Ags) on immunochemical devices to improve test selectivity and sensitivity. We comparatively evaluated five Trypanosoma cruzi antigenic recombinant peptides, chose the three more sensitive ones, built up chimeras bearing these selected Ags, and systematically compared by enzyme-linked immunosorbent assay the performance of the assortments of those peptides with that of the multiepitope constructions bearing all those peptides lineally fused. The better-performing Ags that were compared included peptides homologous to the previously described T. cruzi flagellar repetitive Ag (here named RP1), shed acute-phase Ag (RP2), B13 (RP5), and the chimeric recombinant proteins CP1 and CP2, bearing repetitions of RP1-RP2 and RP1-RP2-RP5, respectively. The diagnostic performances of these Ags were assessed for discrimination efficiency by the formula +OD/cutoff value (where +OD is the mean optical density value of the positive serum samples tested), in comparison with each other either alone, in mixtures, or as peptide-fused chimeras and with total parasite homogenate (TPH). The discrimination efficiency values obtained for CP1 and CP2 were 25% and 52% higher, respectively, than those of their individual-Ag mixtures. CP2 was the only Ag that showed enhanced discrimination efficiency between Chagas' disease-positive and -negative samples, compared with TPH. This study highlights the convenience of performing immunochemical assays using hybrid, single-molecule, chimeric Ags instead of peptide mixtures. CP2 preliminary tests rendered 98.6% sensitivity when evaluated with a 141-Chagas' disease-positive serum sample panel and 99.4% specificity when assessed with a 164-Chagas' disease-negative serum sample panel containing 15 samples from individuals infected with Leishmania spp.

  11. 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.

  12. 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.

  13. 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.

  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 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

  16. 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.

  17. 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.

  18. Protein structure prediction using hybrid AI methods

    SciTech Connect

    Guan, X.; Mural, R.J.; Uberbacher, E.C.

    1993-11-01

    This paper describes a new approach for predicting protein structures based on Artificial Intelligence methods and genetic algorithms. We combine nearest neighbor searching algorithms, neural networks, heuristic rules and genetic algorithms to form an integrated system to predict protein structures from their primary amino acid sequences. First we describe our methods and how they are integrated, and then apply our methods to several protein sequences. The results are very close to the real structures obtained by crystallography. Parallel genetic algorithms are also implemented.

  19. PREDICTION OF PROTEIN CRYSTALLIZATION OUTCOME USING A HYBRID METHOD

    PubMed Central

    Zucker, Frank H.; Stewart, Christine; Rosa, Jaclyn dela; Kim, Jessica; Zhang, Li; Xiao, Liren; Ross, Jenni; Napuli, Alberto J.; Mueller, Natascha; Castaneda, Lisa J.; Nakazawa Hewitt, Stephen R.; Arakaki, Tracy L.; Larson, Eric T.; Subramanian, Easwara; Verlinde, Christophe L.M.J.; Fan, Erkang; Buckner, Frederick S.; Van Voorhis, Wesley C.; Merritt, Ethan A.; Hol, Wim G. J.

    2010-01-01

    The great power of protein crystallography to reveal biological structure is often limited by the tremendous effort required to produce suitable crystals. A hybrid crystal growth predictive model is presented that combines both experimental and sequence-derived data from target proteins, including novel variables derived from physico-chemical characterization such as R30, the ratio between a protein‘s DSF intensity at 30 °C and at Tm. This hybrid model is shown to be more powerful than sequence-based prediction alone – and more likely to be useful for prioritizing and directing the efforts of structural genomics and individual structural biology laboratories. PMID:20347992

  20. 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.

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

    PubMed

    Becherel, Olivier J; Yeo, Abrey J; Stellati, Alissa; Heng, Evelyn Y H; Luff, John; Suraweera, Amila M; Woods, Rick; Fleming, Jean; Carrie, Dianne; McKinney, Kristine; Xu, Xiaoling; Deng, Chuxia; Lavin, Martin F

    2013-04-01

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

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. [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.

  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. 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.

  14. 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.

  15. 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

  16. 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.

  17. Evolution of a quadripartite hybrid virus by interspecific exchange and recombination between replicase components of two related tripartite RNA viruses.

    PubMed

    Masuta, C; Ueda, S; Suzuki, M; Uyeda, I

    1998-09-01

    Cucumber mosaic virus (CMV) and tomato aspermy virus (TAV) belong to the Cucumovirus genus. They have a tripartite genome consisting of single-stranded RNAs, designated 1, 2, and 3. Previous studies have shown that viable pseudorecombinants could be created in vitro by reciprocal exchanges between CMV and TAV RNA 3, but exchanges of RNAs 1 and 2 were replication deficient. When we coinoculated CMV RNAs 2 and 3 along with TAV RNAs 1 and 2 onto Nicotiana benthamiana, a hybrid quadripartite virus appeared that consisted of TAV RNA 1, CMV RNAs 2 and 3, and a distinctive chimeric RNA originating from a recombination between CMV RNA 2 and the 3'-terminal 320 nucleotides of TAV RNA 2. This hybrid arose by means of segment reassortment and RNA recombination to produce an interspecific hybrid with the TAV helicase subunit and the CMV polymerase subunit. To our knowledge, this is the first report demonstrating the evolution of a new plant or animal virus strain containing an interspecific hybrid replicase complex.

  18. Yeast two-hybrid analysis of jasmonate signaling proteins.

    PubMed

    Cuéllar, Amparo Pérez; Pauwels, Laurens; De Clercq, Rebecca; Goossens, Alain

    2013-01-01

    Protein-protein interaction studies are crucial to unravel how jasmonate (JA) signals are transduced. Among the different techniques available, yeast two-hybrid (Y2H) is commonly used within the JA research community to identify proteins belonging to the core JA signaling module. The technique is based on the reconstitution of a transcriptional activator that drives the reporter gene expression upon protein-protein interactions. The method is sensitive and straightforward and can be adapted for different approaches. In this chapter, we provide a detailed protocol to perform targeted Y2H assays to test known proteins and/or protein domains for direct interaction in a pairwise manner and present the possibility to study ternary protein complexes through Y3H.

  19. Rad51/Dmc1 paralogs and mediators oppose DNA helicases to limit hybrid DNA formation and promote crossovers during meiotic recombination

    PubMed Central

    Lorenz, Alexander; Mehats, Alizée; Osman, Fekret; Whitby, Matthew C.

    2014-01-01

    During meiosis programmed DNA double-strand breaks (DSBs) are repaired by homologous recombination using the sister chromatid or the homologous chromosome (homolog) as a template. This repair results in crossover (CO) and non-crossover (NCO) recombinants. Only CO formation between homologs provides the physical linkages guiding correct chromosome segregation, which are essential to produce healthy gametes. The factors that determine the CO/NCO decision are still poorly understood. Using Schizosaccharomyces pombe as a model we show that the Rad51/Dmc1-paralog complexes Rad55-Rad57 and Rdl1-Rlp1-Sws1 together with Swi5-Sfr1 play a major role in antagonizing both the FANCM-family DNA helicase/translocase Fml1 and the RecQ-type DNA helicase Rqh1 to limit hybrid DNA formation and promote Mus81-Eme1-dependent COs. A common attribute of these protein complexes is an ability to stabilize the Rad51/Dmc1 nucleoprotein filament, and we propose that it is this property that imposes constraints on which enzymes gain access to the recombination intermediate, thereby controlling the manner in which it is processed and resolved. PMID:25414342

  20. Rad51/Dmc1 paralogs and mediators oppose DNA helicases to limit hybrid DNA formation and promote crossovers during meiotic recombination.

    PubMed

    Lorenz, Alexander; Mehats, Alizée; Osman, Fekret; Whitby, Matthew C

    2014-12-16

    During meiosis programmed DNA double-strand breaks (DSBs) are repaired by homologous recombination using the sister chromatid or the homologous chromosome (homolog) as a template. This repair results in crossover (CO) and non-crossover (NCO) recombinants. Only CO formation between homologs provides the physical linkages guiding correct chromosome segregation, which are essential to produce healthy gametes. The factors that determine the CO/NCO decision are still poorly understood. Using Schizosaccharomyces pombe as a model we show that the Rad51/Dmc1-paralog complexes Rad55-Rad57 and Rdl1-Rlp1-Sws1 together with Swi5-Sfr1 play a major role in antagonizing both the FANCM-family DNA helicase/translocase Fml1 and the RecQ-type DNA helicase Rqh1 to limit hybrid DNA formation and promote Mus81-Eme1-dependent COs. A common attribute of these protein complexes is an ability to stabilize the Rad51/Dmc1 nucleoprotein filament, and we propose that it is this property that imposes constraints on which enzymes gain access to the recombination intermediate, thereby controlling the manner in which it is processed and resolved.

  1. 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 recombinants between homeologous repeats yielded evidence for heteroduplex formation and preferential migration of the Holliday junctions to the boundaries of sequence homology. These findings broaden the target and off-target mutagenic potential of AID and establish a novel system to study induced homologous recombination in vertebrate cells.DOI: http://dx.doi.org/10.7554/eLife.03110.001.

  2. A novel bi-directional promoter system allows tunable recombinant protein production in Pichia pastoris.

    PubMed

    Rajamanickam, Vignesh; Metzger, Karl; Schmid, Christian; Spadiut, Oliver

    2017-09-13

    The methylotrophic yeast Pichia pastoris is a well-studied host organism for recombinant protein production, which is usually regulated either by a constitutive promoter (e.g. promoter of glyceraldehyde-3-phosphate dehydrogenase; PGAP) or an inducible promoter (e.g. promoter of alcohol oxidase 1; PAOX1). Both promoter systems have several advantages and disadvantages; with one of the main disadvantages being their lack of tunability. Various novel promoter systems, which are either inducible or de-repressed, allowing higher degrees of freedom, have been reported. Recently, bi-directional promoter systems in P. pastoris with two promoter systems regulating recombinant expression of one or more genes were developed. In this study, we introduce a novel bi-directional promoter system combining a modified catalase promoter system (PDC; derepressible and inducible) and the traditional PAOX1, allowing tunable recombinant protein production. We characterized a recombinant P. pastoris strain, carrying the novel bi-directional promoter system, during growth and production in three dynamic bioreactor cultivations. We cloned the model enzyme cellobiohydralase downstream of either promoter and applied different feeding strategies to determine the physiological boundaries of the strain. We succeeded in demonstrating tunability of recombinant protein production solely in response to the different feeding strategies and identified a mixed feed regime allowing highest productivity. In this feasibility study, we present the first controlled bioreactor experiments with a recombinant P. pastoris strain carrying a novel bi-directional promotor combination of a catalase promoter variant (PDC) and the traditional PAOX1. We demonstrated that this bi-directional promoter system allows tunable recombinant protein expression only in response to the available C-sources. This bi-directional promoter system offers a high degree of freedom for bioprocess design and development, making bi

  3. A simplified method for purification of recombinant soluble DnaA proteins.

    PubMed

    Zawilak-Pawlik, Anna M; Kois, Agnieszka; Zakrzewska-Czerwinska, Jolanta

    2006-07-01

    An improved, simplified method for the purification of recombinant, tagged DnaA proteins is described. The presented protocol allowed us to purify soluble DnaA proteins from two different bacterial species: Helicobacter pylori and Streptomyces coelicolor, but it can most likely also be used for the isolation of DnaA proteins from other bacteria, as it was adapted for Mycobacterium tuberculosis DnaA. The isolation procedure consists of protein precipitation with ammonium sulphate followed by affinity chromatography. The composition of the buffers used at each purification step is crucial for the successful isolation of the recombinant DnaA proteins. The universality of the method in terms of its application to differently tagged proteins (His-tagged or GST-tagged) as well as different properties of purified proteins (e.g., highly aggregating truncated forms) makes the protocol highly useful for all studies requiring purified and active DnaA proteins.

  4. Mycobacterium avium Subspecies paratuberculosis Recombinant Proteins Modulate Antimycobacterial Functions of Bovine Macrophages.

    PubMed

    Bannantine, John P; Stabel, Judith R; Laws, Elizabeth; D Cardieri, Maria Clara; Souza, Cleverson D

    2015-01-01

    It has been shown that Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) activates the Mitogen Activated Protein Kinase (MAPK) p38 pathway, yet it is unclear which components of M. paratuberculosis are involved in the process. Therefore, a set of 42 M. paratuberculosis recombinant proteins expressed from coding sequences annotated as lipoproteins were screened for their ability to induce IL-10 expression, an indicator of MAPKp38 activation, in bovine monocyte-derived macrophages. A recombinant lipoprotein, designated as MAP3837c, was among a group of 6 proteins that strongly induced IL-10 gene transcription in bovine macrophages, averaging a 3.1-fold increase compared to non-stimulated macrophages. However, a parallel increase in expression of IL-12 and TNF-α was only observed in macrophages exposed to a subset of these 6 proteins. Selected recombinant proteins were further analyzed for their ability to enhance survival of M. avium within bovine macrophages as measured by recovered viable bacteria and nitrite production. All 6 IL-10 inducing MAP recombinant proteins along with M. paratuberculosis cells significantly enhanced phosphorylation of MAPK-p38 in bovine macrophages. Although these proteins are likely not post translationally lipidated in E. coli and thus is a limitation in this study, these results form the foundation of how the protein component of the lipoprotein interacts with the immune system. Collectively, these data reveal M. paratuberculosis proteins that might play a role in MAPK-p38 pathway activation and hence in survival of this organism within bovine macrophages.

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

    PubMed

    Rasala, Beth A; Mayfield, Stephen P

    2015-03-01

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

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

    PubMed

    Shirvan, Ali Nazari; Aitken, Robert

    2016-01-01

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

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

    PubMed

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

    2013-09-01

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

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

    PubMed

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

    2017-02-01

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

  9. 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.

  10. Mechanism of charge recombination in meso-structured organic-inorganic hybrid perovskite solar cells: A macroscopic perspective

    SciTech Connect

    Yang, Wenchao; Yao, Yao Wu, Chang-Qin

    2015-04-21

    In the currently popular organic-inorganic hybrid perovskite solar cells, the slowness of the charge recombination processes is found to be a key factor for contributing to their high efficiencies and high open circuit voltages, but the underlying recombination mechanism remains unclear. In this work, we investigate the bimolecular recombination (BR) and the trap-assisted monomolecular recombination (MR) in meso-structured perovskite solar cells under steady state working condition, and try to reveal their roles on determining the device performance. Some interfacial effects such as the injection barriers at the selective contacts are examined as well. Based on the macroscopic device modeling, the recombination resistance-voltage (R{sub rec}−V) and the current density-voltage (J–V) curves are calculated to characterize the recombination mechanism and describe the device performance, respectively. Through comparison with the impedance spectroscopy extracted R{sub rec} data, it is found that under the typical BR reduction factor and deep trap densities observed in experiments, the MR dominates the charge recombination in the low voltage regime, while the BR dominates in the high voltage regime. The short circuit current and the fill factor could be reduced by the significant MR but the open circuit voltage is generally determined by the BR. The different electron injection barriers at the contact can change the BR rate and induce different patterns for the R{sub rec}–V characteristics. For the perovskites of increased band gaps, the R{sub rec}'s are significantly enhanced, corresponding to the high open circuit voltages. Finally, it is revealed that the reduced effective charge mobility due to the transport in electron and hole transporting material makes the R{sub rec} decrease slowly with the increasing voltage, which leads to increased open circuit voltage.

  11. Mechanism of charge recombination in meso-structured organic-inorganic hybrid perovskite solar cells: A macroscopic perspective

    NASA Astrophysics Data System (ADS)

    Yang, Wenchao; Yao, Yao; Wu, Chang-Qin

    2015-04-01

    In the currently popular organic-inorganic hybrid perovskite solar cells, the slowness of the charge recombination processes is found to be a key factor for contributing to their high efficiencies and high open circuit voltages, but the underlying recombination mechanism remains unclear. In this work, we investigate the bimolecular recombination (BR) and the trap-assisted monomolecular recombination (MR) in meso-structured perovskite solar cells under steady state working condition, and try to reveal their roles on determining the device performance. Some interfacial effects such as the injection barriers at the selective contacts are examined as well. Based on the macroscopic device modeling, the recombination resistance-voltage (Rrec-V) and the current density-voltage (J-V) curves are calculated to characterize the recombination mechanism and describe the device performance, respectively. Through comparison with the impedance spectroscopy extracted Rrec data, it is found that under the typical BR reduction factor and deep trap densities observed in experiments, the MR dominates the charge recombination in the low voltage regime, while the BR dominates in the high voltage regime. The short circuit current and the fill factor could be reduced by the significant MR but the open circuit voltage is generally determined by the BR. The different electron injection barriers at the contact can change the BR rate and induce different patterns for the Rrec-V characteristics. For the perovskites of increased band gaps, the Rrec's are significantly enhanced, corresponding to the high open circuit voltages. Finally, it is revealed that the reduced effective charge mobility due to the transport in electron and hole transporting material makes the Rrec decrease slowly with the increasing voltage, which leads to increased open circuit voltage.

  12. Monitoring of recombinant protein production using bioluminescence in a semiautomated fermentation process.

    PubMed

    Trezzani, I; Nadri, M; Dorel, C; Lejeune, P; Bellalou, J; Lieto, J; Hammouri, H; Longin, R; Dhurjati, P

    2003-01-01

    On-line optimization of fermentation processes can be greatly aided by the availability of information on the physiological state of the cell. The goal of our "BioLux" research project was to design a recombinant cell capable of intracellular monitoring of product synthesis and to use it as part of an automated fermentation system. A recombinant plasmid was constructed containing an inducible promoter that controls the gene coding for a model protein and the genes necessary for bioluminescence. The cells were cultured in microfermenters equipped with an on-line turbidity sensor and a specially designed on-line light sensor capable of continuous measurement of bioluminescence. Initial studies were done under simple culture conditions, and a linear correlation between luminescence and protein production was obtained. Such specially designed recombinant bioluminescent cells can potentially be applied for model-based inference of intracellular product formation, as well as for optimization and control of recombinant fermentation processes.

  13. [Prokaryotic expression and activity identification of gene recombinant protein of brain-derivedneurotrophic factor precursor].

    PubMed

    Chen, Jia; Liang, Xiaomin; Xu, Zhiqiang

    2014-08-19

    To generate the gene recombinant protein of brain-derived neurotrophic factor precursor (proBDNF) in prokaryotic cells and investigate its biological activity. Rat-derived cDNA of proBDNF with point mutation was amplified by polymerase chain reaction (PCR) and cloned into plasmid pET-28a for expression in E. coli BL21. Western blot was used to identify the product and DAPI performed to test its effect on apoptosis of PC12 cells. PCR product of recombinant gene was successfully expressed in E. coli. And the product agreed with the target protein in molecular weight and showed reactivity with its specific antibody. Apoptosis of PC12 cells was induced by a certain concentration of recombinant proBDNF. The prokaryotic expression vector has been successfully constructed for recombinant gene of proBDNF. And the product has biological toxicity and it may induce the apoptosis of PC12 cells.

  14. Targeted In Vivo Inhibition of Specific Protein–Protein Interactions Using Recombinant Antibodies

    PubMed Central

    Zábrady, Matej; Hrdinová, Vendula; Müller, Bruno; Conrad, Udo; Hejátko, Jan; Janda, Lubomír

    2014-01-01

    With the growing availability of genomic sequence information, there is an increasing need for gene function analysis. Antibody-mediated “silencing” represents an intriguing alternative for the precise inhibition of a particular function of biomolecules. Here, we describe a method for selecting recombinant antibodies with a specific purpose in mind, which is to inhibit intrinsic protein–protein interactions in the cytosol of plant cells. Experimental procedures were designed for conveniently evaluating desired properties of recombinant antibodies in consecutive steps. Our selection method was successfully used to develop a recombinant antibody inhibiting the interaction of ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 3 with such of its upstream interaction partners as the receiver domain of CYTOKININ INDEPENDENT HISTIDINE KINASE 1. The specific down-regulation of the cytokinin signaling pathway in vivo demonstrates the validity of our approach. This selection method can serve as a prototype for developing unique recombinant antibodies able to interfere with virtually any biomolecule in the living cell. PMID:25299686

  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. 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.

  17. Extraction and purification methods in downstream processing of plant-based recombinant proteins.

    PubMed

    Łojewska, Ewelina; Kowalczyk, Tomasz; Olejniczak, Szymon; Sakowicz, Tomasz

    2016-04-01

    During the last two decades, the production of recombinant proteins in plant systems has been receiving increased attention. Currently, proteins are considered as the most important biopharmaceuticals. However, high costs and problems with scaling up the purification and isolation processes make the production of plant-based recombinant proteins a challenging task. This paper presents a summary of the information regarding the downstream processing in plant systems and provides a comprehensible overview of its key steps, such as extraction and purification. To highlight the recent progress, mainly new developments in the downstream technology have been chosen. Furthermore, besides most popular techniques, alternative methods have been described.

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

    DTIC Science & Technology

    2016-08-01

    Frazier, A.; Crotty, S.; Zajonc, D.M.; Peters, B.; Xiang, Y. Potent Neutralization of Vaccinia Virus by Divergent Murine Antibodies Targeting a Common ...AVAILABILITY STATEMENT Approved for public release; distribution is unlimited. 13. SUPPLEMENTARY NOTES 14. ABSTRACT: The Vaccinia virus is...immunoassay development. 15. SUBJECT TERMS Vaccinia virus (VACV) Smallpox Recombinant Protein expression Vaccinia surface protein, L1R Purification

  19. Immunological characterization of a recombinant 27-kilodalton antigenic protein from Paracoccidioides brasiliensis.

    PubMed

    Ortiz, B L; Garcia, A M; Restrepo, A; McEwen, J G

    1996-03-01

    We report the expression in Escherichia coli of a 27-kDa antigenic protein from Paracoccidioides brasiliensis. When analyzed by immunoblotting, this recombinant antigenic protein was recognized by antibodies present in the sera of 40 of the 44 paracoccidioidomycosis patients studied. No cross-reactions were observed with sera from patients with other mycoses (histoplasmosis, aspergillosis, cryptococcosis, sporotrichosis, and chromoblastomycosis) or with tuberculosis.

  20. 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...

  1. Impact of Profiling Technologies in the Understanding of Recombinant Protein Production

    NASA Astrophysics Data System (ADS)

    Vijayendran, Chandran; Flaschel, Erwin

    Since expression profiling methods have been available in a high throughput fashion, the implication of these technologies in the field of biotechnology has increased dramatically. Microarray technology is one such unique and efficient methodology for simultaneous exploration of expression levels of numerous genes. Likewise, two-dimensional gel electrophoresis or multidimensional liquid chromatography coupled with mass spectrometry are extensively utilised for studying expression levels of numerous proteins. In the field of biotechnology these highly parallel analytical methods have paved the way to study and understand various biological phenomena depending on expression patterns. The next phenomenological level is represented by the metabolome and the (metabolic) fluxome. However, this chapter reviews gene and protein profiling and their impact on understanding recombinant protein production. We focus on the computational methods utilised for the analyses of data obtained from these profiling technologies as well as prominent results focusing on recombinant protein expression with Escherichia coli. Owing to the knowledge accumulated with respect to cellular signals triggered during recombinant protein production, this field is on the way to design strategies for developing improved processes. Both gene and protein profiling have exhibited a handful of functional categories to concentrate on in order to identify target genes and proteins, respectively, involved in the signalling network with major impact on recombinant protein production.

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

    USDA-ARS?s Scientific Manuscript database

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

  3. Production of a recombinant hybrid hemoflavoprotein: engineering a functional NADH:cytochrome c reductase.

    PubMed

    Barber, M J; Quinn, G B

    2001-11-01

    A gene has been constructed coding for a unique fusion protein, NADH:cytochrome c reductase, that comprises the soluble heme-containing domain of rat hepatic cytochrome b(5) as the amino-terminal portion of the protein and the soluble flavin-containing domain of rat hepatic cytochrome b(5) reductase as the carboxyl terminus. The gene has been expressed in Escherichia coli resulting in the highly efficient production of a functional hybrid hemoflavoprotein which has been purified to homogeneity by a combination of ammonium sulfate precipitation, affinity chromatography on 5'-ADP agarose, and size-exclusion chromatography. The purified protein exhibited a molecular mass of approximately 46 kDa by polyacrylamide gel electrophoresis and 40,875 Da, for the apoprotein, using mass spectrometry which also confirmed the presence of both heme and FAD prosthetic groups. The fusion protein showed immunological cross-reactivity with both anti-rat cytochrome b(5) and anti-rat cytochrome b(5) reductase antibodies indicating the conservation of antigenic determinants from both native domains. Spectroscopic analysis indicated the fusion protein contained both a b-type cytochrome and flavin chromophors with properties identical to those of the native proteins. Amino-terminal and internal amino acid sequencing confirmed the identity of peptides derived from both the heme- and flavin-binding domains with sequences identical to the deduced amino acid sequence. The isolated fusion protein retained NADH:ferricyanide reductase activity (k(cat) = 8.00 x 10(2) s(-1), K(NADH)(m) = 4 microM, K(FeCN(6))(m) = 11 microM) comparable to that of that of native NADH:cytochrome b(5) reductase and also exhibited both NADH:cytochrome c reductase activity (k(cat) = 2.17 x 10(2) s(-1), K(NADH)(m) = 2 microM, K(FeCN(6))(m) = 11 microM, K(Cyt.c)(m) = 1 microM) and NADH:methemoglobin reductase activity (k(cat) = 4.40 x 10(-1) s(-1), K(NADH)(m) = 3 microM, K(mHb)(m) = 47 microM), the latter two activities

  4. 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.

  5. Recombinant sheep pox virus proteins elicit neutralizing antibodies

    USDA-ARS?s Scientific Manuscript database

    The aim of this study was to evaluate the immunogenicity and neutralizing activity of bacterially-expressed sheep pox virus (SPPV) structural proteins as candidate subunit vaccines to control sheep pox disease. SPPV structural proteins were identified by sequence homology with proteins from vaccinia...

  6. Transforming the treatment for hemophilia B patients: update on the clinical development of recombinant fusion protein linking recombinant coagulation factor IX with recombinant albumin (rIX-FP).

    PubMed

    Santagostino, Elena

    2016-05-01

    Recombinant fusion protein linking recombinant coagulation factor IX with recombinant albumin (rIX-FP; Idelvion®(†)) is an innovative new treatment designed to extend the half-life of factor IX (FIX) and ease the burden of care for hemophilia B patients. The rIX-FP clinical development program - PROLONG-9FP - is in its advanced phases, with pivotal studies in previously treated adults, adolescents, and pediatrics now completed. Across all age groups studied, rIX-FP has demonstrated a markedly improved pharmacokinetic profile compared with plasma-derived and recombinant FIX treatments, with a 30-40% higher incremental recovery, an approximately 5-fold longer half-life, a lower clearance, and a greater area under the curve. rIX-FP has been very well tolerated with an excellent safety profile. In the pivotal studies, there have been no reports of FIX inhibitors or antidrug antibodies, and few treatment-related adverse events have been observed. Prophylactic regimens of rIX-FP administered once weekly to once every 14 days have been highly effective. When used for surgical prophylaxis, a single infusion of rIX-FP has been sufficient to maintain hemostasis, even during major orthopedic surgery. An ongoing study is now enrolling previously untreated patients and evaluating the possibility of extending the dosing interval to every 21 days. There is little doubt that rIX-FP will transform the treatment of hemophilia B. © 2016 Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

  7. Recombinant neural protein PrP can bind with both recombinant and native apolipoprotein E in vitro.

    PubMed

    Gao, Chen; Lei, Yan-Jun; Han, Jun; Shi, Qi; Chen, Lan; Guo, Yan; Gao, Yong-Jun; Chen, Jian-Ming; Jiang, Hui-Ying; Zhou, Wei; Dong, Xiao-Ping

    2006-09-01

    The most essential and crucial step during the pathogenesis of transmissible spongiform encephalopathy is the conformational change of cellular prion protein (PrP(C)) to pathologic isoform (PrP(Sc)). A lot of data revealed that caveolae-like domains (CLDs) in the cell surface were the probable place where the conversion of PrP proteins happened. Apolipoprotein E (ApoE) is an apolipoprotein which is considered to play an important role in the development of Alzheimer's disease and other neurodegenerative diseases by forming protein complex through binding to the receptor located in the clathrin-coated pits of the cell surface. In this study, a 914-bp cDNA sequence encoding human ApoE3 was amplified from neuroblastoma cell line SH-SY5Y. Three human ApoE isomers were expressed and purified from Escherichia coli. ApoE-specific antiserum was prepared by immunizing rabbits with the purified ApoE3. GST/His pull-down assay, immunoprecipitation and ELISA revealed that three full-length ApoE isomers interact with the recombinant full-length PrP protein in vitro. The regions corresponding to protein binding were mapped in the N-terminal segment of ApoE (amino acid 1-194) and the N-terminal of PrP (amino acid 23-90). Moreover, the recombinant PrP showed the ability to form a complex with the native ApoE from liver tissues. Our data provided direct evidence of molecular interaction between ApoE and PrP. It also supplied scientific clues for assessing the significance of CLDs on the surface of cellular membrane in the process of conformational conversion from PrP(C) to PrP(Sc) and probing into the pathogenesis of transmissible spongiform encephalopathy.

  8. Organizing protein-DNA hybrids as nanostructures with programmed functionalities.

    PubMed

    Teller, Carsten; Willner, Itamar

    2010-12-01

    The structural and functional information encoded in the base sequence of nucleic acids provides a means to organize hybrid protein-DNA nanostructures with pre-designed, programmed functionality. This review discusses the activation of enzyme cascades in supramolecular DNA-protein hybrid structures, the bioelectrocatalytic activation of redox enzymes on DNA scaffolds, and the programmed positioning of enzymes on 1D, 2D and 3D DNA nanostructures. These systems provide starting points towards the design of interconnected enzyme networks. Substantial progress in the tailoring of functional protein-DNA nanostructures has been accomplished in recent years, and advances in this field warrant a comprehensive discussion. The application of these systems for the control of biocatalytic transformations, for amplified biosensing, and for the synthesis of metallic nanostructures are addressed, and future prospects for these systems are highlighted.

  9. Recombinant human bone morphogenetic protein 2 (rhBMP-2) immobilized on laser-fabricated 3D scaffolds enhance osteogenesis.

    PubMed

    Chatzinikolaidou, Maria; Pontikoglou, Charalampos; Terzaki, Konstantina; Kaliva, Maria; Kalyva, Athanasia; Papadaki, Eleni; Vamvakaki, Maria; Farsari, Maria

    2017-01-01

    The regeneration of bone via a tissue engineering approach involves components from the macroscopic to the nanoscopic level, including appropriate 3D scaffolds, cells and growth factors. In this study, hexagonal scaffolds of different diagonals were fabricated by Direct Laser Writing using a photopolymerizable hybrid material. The proliferation of bone marrow (BM) mesenchymal stem cells (MSCs) cultured on structures with various diagonals, 50, 100, 150 and 200μm increased significantly after 10days in culture, however without significant differences among them. Next, recombinant human bone morphogenetic protein 2 (rhBMP-2) was immobilized onto the hybrid material both via covalent binding and physical adsorption. Both immobilization types exhibited similar high releaseate bioactivity profiles and a sustained delivery of rhBMP-2. The collagen and calcium levels produced in the extracellular matrix (ECM) were significantly elevated for the samples functionalized with BMP-2 compared to those in the osteogenic medium. Furthermore, significant upregulation of gene expression in both types of BMP-2 immobilized scaffolds was observed for alkaline phosphatase (ALPL) and osteocalcin (BGLAP) at days 7, 14, and 21, for RUNX2 at day 21, and for osteonectin (SPARC) at days 7 and 14. The results suggest that the release of bioactive rhBMP-2 from the hybrid scaffolds enhance the control over the osteogenic differentiation during cell culture.

  10. A Comparison of Two-Hybrid Approaches for Detecting Protein-Protein Interactions.

    PubMed

    Mehla, J; Caufield, J H; Sakhawalkar, N; Uetz, P

    2017-01-01

    Two-hybrid systems are one of the most popular, preferred, cost effective, and scalable in vivo genetic approaches for screening protein-protein interactions. A number of variants of yeast and bacterial two-hybrid systems exist, rendering them ideal for modern, flexible proteomics-driven studies. For mapping protein interactions at genome scales (that is, constructing an interactome), the yeast two-hybrid system has been extensively tested and is preferred over bacterial two-hybrid systems, given that users have created more resources such as a variety of vectors and other modifications. Each system has its own advantages and limitations and thus needs to be compared directly. For instance, the bacterial two-hybrid method seems a better fit than the yeast two-hybrid system to screen membrane-associated proteins. In this chapter, we provide detailed protocols for yeast and bacterial two-hybrid systems as well as a comparison of outcomes for each approach using our own and published data. © 2017 Elsevier Inc. All rights reserved.

  11. Footprint Analysis of the RAG Protein Recombination Signal Sequence Complex for V(D)J Type Recombination

    PubMed Central

    Nagawa, Fumikiyo; Ishiguro, Kei-ichiro; Tsuboi, Akio; Yoshida, Tomoyuki; Ishikawa, Akiko; Takemori, Toshitada; Otsuka, Anthony J.; Sakano, Hitoshi

    1998-01-01

    We have studied the interaction between recombination signal sequences (RSSs) and protein products of the truncated forms of recombination-activating genes (RAG) by gel mobility shift, DNase I footprinting, and methylation interference assays. Methylation interference with dimethyl sulfate demonstrated that binding was blocked by methylation in the nonamer at the second-position G residue in the bottom strand and at the sixth- and seventh-position A residues in the top strand. DNase I footprinting experiments demonstrated that RAG1 alone, or even a RAG1 homeodomain peptide, gave footprint patterns very similar to those obtained with the RAG1-RAG2 complex. In the heptamer, partial methylation interference was observed at the sixth-position A residue in the bottom strand. In DNase I footprinting, the heptamer region was weakly protected in the bottom strand by RAG1. The effects of RSS mutations on RAG binding were evaluated by DNA footprinting. Comparison of the RAG-RSS footprint data with the published Hin model confirmed the notion that sequence-specific RSS-RAG interaction takes place primarily between the Hin domain of the RAG1 protein and adjacent major and minor grooves of the nonamer DNA. PMID:9418911

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

    PubMed

    Mamipour, Mina; Yousefi, Mohammadreza; Hasanzadeh, Mohammad

    2017-09-01

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

  13. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2011-02-01

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

  15. 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.

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

    PubMed Central

    Farahmand, Mahin; Nahrevanian, Hossein

    2016-01-01

    Visceral leishmaniasis (VL) is a zoonotic disease caused by leishmania species. Dogs are considered to be the main reservoir of VL. A number of methods and antigen-based assays are used for the diagnosis of leishmaniasis. However, currently available methods are mainly based on direct examination of tissues for the presence of parasites, which is highly invasive. A variety of serological tests are commonly applied for VL diagnosis, including indirect fluorescence antibody test, enzyme-linked immunosorbent assay (ELISA), dot-ELISA, direct agglutination test, Western-blotting, and immunochromatographic test. However, when soluble antigens are used, serological tests are less specific due to cross-reactivity with other parasitic diseases. Several studies have attempted to replace soluble antigens with recombinant proteins to improve the sensitivity and the specificity of the immunodiagnostic tests. Major technological advances in recombinant antigens as reagents for the serological diagnosis of VL have led to high sensitivity and specificity of these serological tests. A great number of recombinant proteins have been shown to be effective for the diagnosis of leishmania infection in dogs, the major reservoir of L. infantum. Although few recombinant proteins with high efficacy provide reasonable results for the diagnosis of human and canine VL, more optimization is still needed for the appropriate antigens to provide high-throughput performance. This review aims to explore the application of different recombinant proteins for the serodiagnosis of VL in humans and dogs. PMID:26883952

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

    PubMed

    Farahmand, Mahin; Nahrevanian, Hossein

    2016-07-01

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

  18. RecO Protein Initiates DNA Recombination and Strand Annealing through Two Alternative DNA Binding Mechanisms*

    PubMed Central

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

    2014-01-01

    Recombination mediator proteins (RMPs) are important for genome stability in all organisms. Several RMPs support two alternative reactions: initiation of homologous recombination and DNA annealing. We examined mechanisms of RMPs in both reactions with Mycobacterium smegmatis RecO (MsRecO) and demonstrated that MsRecO interacts with ssDNA by two distinct mechanisms. Zinc stimulates MsRecO binding to ssDNA during annealing, whereas the recombination function is zinc-independent and is regulated by interaction with MsRecR. Thus, different structural motifs or conformations of MsRecO are responsible for interaction with ssDNA during annealing and recombination. Neither annealing nor recombinase loading depends on MsRecO interaction with the conserved C-terminal tail of single-stranded (ss) DNA-binding protein (SSB), which is known to bind Escherichia coli RecO. However, similarly to E. coli proteins, MsRecO and MsRecOR do not dismiss SSB from ssDNA, suggesting that RMPs form a complex with SSB-ssDNA even in the absence of binding to the major protein interaction motif. We propose that alternative conformations of such complexes define the mechanism by which RMPs initiate the repair of stalled replication and support two different functions during recombinational repair of DNA breaks. PMID:25170075

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

    PubMed

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

    2006-04-01

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

  20. Optimisation of contained Nicotiana tabacum cultivation for the production of recombinant protein pharmaceuticals.

    PubMed

    Colgan, Richard; Atkinson, Christopher J; Paul, Matthew; Hassan, Sally; Drake, Pascal M W; Sexton, Amy L; Santa-Cruz, Simon; James, David; Hamp, Keith; Gutteridge, Colin; Ma, Julian K-C

    2010-04-01

    Nicotiana tabacum is emerging as a crop of choice for production of recombinant protein pharmaceuticals. Although there is significant commercial expertise in tobacco farming, different cultivation practices are likely to be needed when the objective is to optimise protein expression, yield and extraction, rather than the traditional focus on biomass and alkaloid production. Moreover, pharmaceutical transgenic tobacco plants are likely to be grown initially within a controlled environment, the parameters for which have yet to be established. Here, the growth characteristics and functional recombinant protein yields for two separate transgenic tobacco plant lines were investigated. The impacts of temperature, day-length, compost nitrogen content, radiation and plant density were examined. Temperature was the only environmental variable to affect IgG concentration in the plants, with higher yields observed in plants grown at lower temperature. In contrast, temperature, supplementary radiation and plant density all affected the total soluble protein yield in the same plants. Transgenic plants expressing a second recombinant protein (cyanovirin-N) responded differently to IgG transgenic plants to elevated temperature, with an increase in cyanovirin-N concentration, although the effect of the environmental variables on total soluble protein yields was the same as the IgG plants. Planting density and radiation levels were important factors affecting variability of the two recombinant protein yields in transgenic plants. Phenotypic differences were observed between the two transgenic plant lines and non-transformed N. tabacum, but the effect of different growing conditions was consistent between the three lines. Temperature, day length, radiation intensity and planting density all had a significant impact on biomass production. Taken together, the data suggest that recombinant protein yield is not affected substantially by environmental factors other than growth

  1. Predicting the solubility of recombinant proteins in Escherichia coli.

    PubMed

    Harrison, Roger G; Bagajewicz, Miguel J

    2015-01-01

    We describe a statistical model that uses binomial logistic regression for predicting the solubility of heterologous proteins expressed in E. coli. The model is based on a set of proteins reported to have been expressed in E. coli in either soluble or insoluble form. The 22 parameters used in the final model based on proteins' amino acid composition are discussed. The overall accuracy of the model developed is 94%. The way to use this model on the website http://www.ou.edu/ for the prediction of protein solubility is explained.

  2. Chloroplast-targeted expression of recombinant crystal-protein gene in cotton: an unconventional combat with resistant pests.

    PubMed

    Kiani, Sarfraz; Mohamed, Bahaeldeen Babiker; Shehzad, Kamran; Jamal, Adil; Shahid, Muhammad Naveed; Shahid, Ahmad Ali; Husnain, Tayyab

    2013-07-10

    Plants transformed with single Bt gene are liable to develop insect resistance and this has already been reported in a number of studies carried out around the world where Bt cotton was cultivated on commercial scale. Later, it was envisaged to transform plants with more than one Bt genes in order to combat with resistant larvae. This approach seems valid as various Bt genes possess different binding domains which could delay the likely hazards of insect resistance against a particular Bt toxin. But it is difficult under field conditions to develop homozygous plants expressing all Bt genes equally after many generations without undergoing recombination effects. A number of researches claiming to transform plants from three to seven transgenes in a single plant were reported during the last decade but none has yet applied for patent of homozygous transgenic lines. A better strategy might be to use hybrid-Bt gene(s) modified for improved lectin-binding domains to boost Bt receptor sites in insect midgut. These recombinant-Bt gene(s) would express different lectin domains in a single polypeptide and it is relatively easy to develop homozygous transgenic lines under field conditions. Enhanced chloroplast-localized expression of hybrid-Bt gene would leave no room for insects to develop resistance. We devised and successfully applied this strategy in cotton (Gossypium hirsutum) and data up to T3 generation showed that our transgenic cotton plants were displaying enhanced chloroplast-targeted Cry1Ac-RB expression. Laboratory and field bioassays gave promising results against American bollworm (Heliothis armigera), pink bollworm (Pictinophora scutigera) and fall armyworm (Spodoptera frugiperda) that otherwise, were reported to have evolved resistance against Cry1Ac toxin. Elevated levels of hybrid-Bt toxin were confirmed by ELISA of chloroplast-enriched protein samples extracted from leaves of transgenic cotton lines. While, localization of recombinant Cry1Ac-RB protein in

  3. Overproduction, purification, crystallization and preliminary X-ray analysis of the peroxiredoxin domain of a larger natural hybrid protein from Thermotoga maritima

    SciTech Connect

    Barbey, Carole; Rouhier, Nicolas; Haouz, Ahmed; Navaza, Alda; Jacquot, Jean-Pierre

    2008-01-01

    Crystals of the peroxiredoxin domain of a larger natural hybrid protein from T. maritima were obtained which diffracted to 2.9 Å resolution on a synchrotron source. Thermotoga maritima contains a natural hybrid protein constituted of two moieties: a peroxiredoxin domain at the N-terminus and a nitroreductase domain at the C-terminus. The peroxiredoxin (Prx) domain has been overproduced and purified from Escherichia coli cells. The recombinant Prx domain, which is homologous to bacterial Prx BCP and plant Prx Q, folds properly into a stable protein that possesses biological activity. The recombinant protein was crystallized and synchrotron data were collected to 2.9 Å resolution. The crystals belonged to the tetragonal space group I422, with unit-cell parameters a = b = 176.67, c = 141.20 Å.

  4. 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

  5. [Purification of recombinant Bacillus cereus ResD-ResE proteins expressed in Escherichia coli strains].

    PubMed

    Shapyrina, E V; Shadrin, A M; Solonin, A S

    2013-01-01

    Recombinant E. coli strains expressing the Bacillus cereus ATCC 14579T resD and resEgenes fused with the ubiquitin gene were constructed, and purification of the ResD and ResE proteins was performed. The approach used in the study allowed us to increase the protein yield of the electrophoretic homogeneous ResD andResE proteins without denaturation steps up to 150 mg per gram of wet cell weight.

  6. 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

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

    PubMed

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

    2015-12-29

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

  8. Hybridization-based antibody cDNA recovery for the production of recombinant antibodies identified by repertoire sequencing.

    PubMed

    Valdés-Alemán, Javier; Téllez-Sosa, Juan; Ovilla-Muñoz, Marbella; Godoy-Lozano, Elizabeth; Velázquez-Ramírez, Daniel; Valdovinos-Torres, Humberto; Gómez-Barreto, Rosa E; Martinez-Barnetche, Jesús

    2014-01-01

    High-throughput sequencing of the antibody repertoire is enabling a thorough analysis of B cell diversity and clonal selection, which may improve the novel antibody discovery process. Theoretically, an adequate bioinformatic analysis could allow identification of candidate antigen-specific antibodies, requiring their recombinant production for experimental validation of their specificity. Gene synthesis is commonly used for the generation of recombinant antibodies identified in silico. Novel strategies that bypass gene synthesis could offer more accessible antibody identification and validation alternatives. We developed a hybridization-based recovery strategy that targets the complementarity-determining region 3 (CDRH3) for the enrichment of cDNA of candidate antigen-specific antibody sequences. Ten clonal groups of interest were identified through bioinformatic analysis of the heavy chain antibody repertoire of mice immunized with hen egg white lysozyme (HEL). cDNA from eight of the targeted clonal groups was recovered efficiently, leading to the generation of recombinant antibodies. One representative heavy chain sequence from each clonal group recovered was paired with previously reported anti-HEL light chains to generate full antibodies, later tested for HEL-binding capacity. The recovery process proposed represents a simple and scalable molecular strategy that could enhance antibody identification and specificity assessment, enabling a more cost-efficient generation of recombinant antibodies.

  9. 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

  10. High Level Expression and Purification of Recombinant Proteins from Escherichia coli with AK-TAG

    PubMed Central

    Luo, Dan; Wen, Caixia; Zhao, Rongchuan; Liu, Xinyu; Liu, Xinxin; Cui, Jingjing; Liang, Joshua G.; Liang, Peng

    2016-01-01

    Adenylate kinase (AK) from Escherichia coli was used as both solubility and affinity tag for recombinant protein production. When fused to the N-terminus of a target protein, an AK fusion protein could be expressed in soluble form and purified to near homogeneity in a single step from Blue-Sepherose via affinity elution with micromolar concentration of P1, P5- di (adenosine—5’) pentaphosphate (Ap5A), a transition-state substrate analog of AK. Unlike any other affinity tags, the level of a recombinant protein expression in soluble form and its yield of recovery during each purification step could be readily assessed by AK enzyme activity in near real time. Coupled to a His-Tag installed at the N-terminus and a thrombin cleavage site at the C terminus of AK, the streamlined method, here we dubbed AK-TAG, could also allow convenient expression and retrieval of a cleaved recombinant protein in high yield and purity via dual affinity purification steps. Thus AK-TAG is a new addition to the arsenal of existing affinity tags for recombinant protein expression and purification, and is particularly useful where soluble expression and high degree of purification are at stake. PMID:27214237

  11. [Prokaryotic expression, purification and biological activity analysis of recombinant β-Lactamase protein].

    PubMed

    Zhou, Xiao-liang; Shi, Pei-ji; Wang, Hao

    2011-01-01

    To prepare RGD4CβL fusion protein using prokaryotic expression system and evaluate the biological activity of the RGD4CβL. RGD4CβL gene was cloned into pColdII to contruct β-Lactamase prokaryotic expression vector. After transformation, the recombinant vector was induced to express recombinant protein RGD4CβL by IPTG in E.coli BL(DE3). The recombinant protein was purified by Ni-NTA resin under denaturing condition and then dialyzed to renature. The tumor cell targeting ability of the recombinant protein was analyzed by flow cytometric analysis. After cleavage and purification, β-Lactamase moiety showed the expected size of 42 000 on Tricine-SDS-PAGE, and was further confirmed by Western blotting. Based on flow cytometric analysis, the purified protein specially targeted breast cancer cell line MCF-7. This research successfully estiblished a method for prokaryotic expression and purification of β-lactamase. These results suggest the potential use of the protein as an agent for ADEPT.

  12. 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

  13. 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.

  14. Development of a novel recombinant LHRH fusion protein for therapy of androgen and estrogen dependent cancers.

    PubMed

    Gupta, Jagdish C; Hada, Rohit S; Sahai, P; Talwar, G P

    2017-06-01

    LHRH based vaccines are promising candidates for therapy of androgen and estrogen dependent cancers. We report in this communication development of a novel recombinant protein vaccine candidate against LHRH. A synthetic gene was designed in which the codon sequence in the LHRH decapeptide was modified by substituting the codon for 6-glycine with that of l-leucine. Further the LHRH(6leu) gene was linked to heat-labile enterotoxin of E. coli (LTB) as carrier. This LHRH(6leu)-LTB gene was cloned into a prokaryotic expression vector under the control of inducible and strong bacteriophage T7 promoter to over-express LHRH(leu) fused to LTB as recombinant protein in E. coli. Recombinant LHRH(leu)-LTB protein of ∼14 kDa size, was purified from inclusion bodies using in-situ refolding on the column and Ni-NTA based immobilized affinity chromatography. Western blot confirmed the immunoreactivity of purified LHRH(leu)-LTB fusion protein with anti-LHRH monoclonal antibody. The vaccine protein was further characterized by mass spectroscopy, circular dichroism and fluorescence spectroscopy. This communication reports a recombinant LHRH fusion protein with potential for blocking of sex hormones production for eventual therapy of sex hormones dependent neoplasms. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. 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.

  16. A Comparative Analysis of Recombinant Protein Expression in Different Biofactories: Bacteria, Insect Cells and Plant Systems

    PubMed Central

    Brozzetti, Annalisa; Falorni, Alberto; Pezzotti, Mario; Avesani, Linda

    2015-01-01

    Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the flexible, low-cost production of high-quality, bioactive products. In this study, we compared the expression of a target human recombinant protein in traditional fermenter-based cell cultures (bacterial and insect) with plant-based expression systems, both transient and stable. For each platform, we described the set-up, optimization and length of the production process, the final product quality and the yields and we evaluated provisional production costs, specific for the selected target recombinant protein. Overall, our results indicate that bacteria are unsuitable for the production of the target protein due to its accumulation within insoluble inclusion bodies. On the other hand, plant-based systems are versatile platforms that allow the production of the selected protein at lower-costs than Baculovirus/insect cell system. In particular, stable transgenic lines displayed the highest-yield of the final product and transient expressing plants the fastest process development. However, not all recombinant proteins may benefit from plant-based systems but the best production platform should be determined empirically with a case-by-case approach, as described here. PMID:25867956

  17. Recombinant Sheep Pox Virus Proteins Elicit Neutralizing Antibodies

    PubMed Central

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

    2016-01-01

    The aim of this work was to evaluate the immunogenicity and neutralizing activity of sheep pox virus (SPPV; genus Capripoxvirus, family Poxviridae) structural proteins as candidate subunit vaccines to control sheep pox disease. SPPV structural proteins were identified by sequence homology with proteins of vaccinia virus (VACV) strain Copenhagen. Four SPPV proteins (SPPV-ORF 060, SPPV-ORF 095, SPPV-ORF 117, and SPPV-ORF 122), orthologs of immunodominant L1, A4, A27, and A33 VACV proteins, respectively, were produced in Escherichia coli. Western blot analysis revealed the antigenic and immunogenic properties of SPPV-060, SPPV-095, SPPV-117 and SPPV-122 proteins when injected with adjuvant into experimental rabbits. Virus-neutralizing activity against SPPV in lamb kidney cell culture was detected for polyclonal antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins. To our knowledge, this is the first report demonstrating the virus-neutralizing activities of antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins. PMID:27338444

  18. 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. Choreography of recombination proteins during the DNA damage response.

    PubMed

    Lisby, Michael; Rothstein, Rodney

    2009-09-02

    Genome integrity is frequently challenged by DNA lesions from both endogenous and exogenous sources. A single DNA double-strand break (DSB) is lethal if unrepaired and may lead to loss of heterozygosity, mutations, deletions, genomic rearrangements and chromosome loss if repaired improperly. Such genetic alterations are the main causes of cancer and other genetic diseases. Consequently, DNA double-strand break repair (DSBR) is an important process in all living organisms. DSBR is also the driving mechanism in most strategies of gene targeting, which has applications in both genetic and clinical research. Here we review the cell biological response to DSBs in mitotically growing cells with an emphasis on homologous recombination pathways in yeast Saccharomyces cerevisiae and in mammalian cells.

  20. 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.

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

    PubMed

    Viitanen, Mikko I; Vasala, Antti; Neubauer, Peter; Alatossava, Tapani

    2003-04-09

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

  2. 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.

  3. 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.

  4. 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

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

    PubMed

    Ramos-Martinez, E M; Fimognari, L; Sakuragi, Y

    2017-02-16

    Microalga-based biomanufacturing of recombinant proteins is attracting growing attention due to its advantages in safety, metabolic diversity, scalability, and sustainability. Secretion of recombinant proteins can accelerate the use of microalgal platforms by allowing post-translational modifications and easy recovery of products from the culture media. However, currently, the yields of secreted recombinant proteins are low, which hampers the commercial application of this strategy. This study aimed at expanding the genetic tools for enhancing secretion of recombinant proteins in Chlamydomonas reinhardtii, a widely used green microalga as a model organism and a potential industrial biotechnology platform. We demonstrated that the putative signal sequence from C. reinhardtii gametolysin can assist the secretion of the yellow fluorescent protein Venus into the culture media. In order to increase the secretion yields, Venus was C-terminally fused with synthetic glycomodules comprised of tandem serine (Ser) and proline (Pro) repeats of 10 and 20 units [hereafter (SP)n, wherein n=10 or 20]. The yields of the (SP)n-fused Venus were higher than Venus without the glycomodule by up to 12 folds, with the maximum yield of 15 mg L(-1) . Moreover, the presence of the glycomodules confererred an enhanced proteolytic protein stability. The Venus-(SP)n proteins were shown to be glycosylated, and a treatment of the cells with Brefeldin A led to a suggestion that glycosylation of the (SP)n glycomodules starts in the endoplasmic reticulum (ER). Taken together, the results demonstrate the utility of the gametolysin signal sequence and (SP)n glycomodule to promote a more efficient biomanufacturing of microalgae-based recombinant proteins. This article is protected by copyright. All rights reserved.

  6. Expression and affinity purification of recombinant proteins from plants

    NASA Technical Reports Server (NTRS)

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

    2002-01-01

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

  7. Recombinant methods in protein and whole-cell biosensing

    NASA Astrophysics Data System (ADS)

    Shetty, R. S.; Salins, Lyndon L.; Ramanathan, S.; Daunert, Sylvia

    1999-12-01

    In this paper, we investigate the use of fluorescently- labeled binding proteins and genetically engineered bacterial cells for sensing of phosphate, glucose, and L- arabinose. To optimize the performance of the labeled binding proteins for biosensing purposes, a few key considerations were taken into account. A site-selective labeling protocol of the fluorescent reporter to the protein was used to ensure that the probe reported from a specific domain of the protein. The labeling sites chosen were hypothesized to undergo a physicochemical change when the biorecognition element binds the analyte. Cysteine mutations were introduced into the binding proteins by site-directed mutagenesis using the polymerase chain reaction. The residues selected were all in close proximity to the binding cleft, a region that is affected the most by the conformational change that accompanies ligand binding. The cysteine residues were then labeled with environment- sensitive fluorophores and changes in the fluorescence properties of the conjugates were monitored and related to the amount of ligand present. The application of microorganisms in sensing systems represent new advances in the development of novel analytical techniques for the detection of a target analyte. In these systems, a genetically engineered organism generates an analytically useful signal when it encounters a specific target substance due to selective recognition and binding properties towards that particular compound. This concept has been demonstrated using an optical bacteria-based sensing system capable of detecting the monosaccharide L-arabinose that employed the green fluorescent protein as a reporter protein.

  8. Expression and affinity purification of recombinant proteins from plants

    NASA Technical Reports Server (NTRS)

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

    2002-01-01

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

  9. Hybrid Sterility in Rice (Oryza sativa L.) Involves the Tetratricopeptide Repeat Domain Containing Protein.

    PubMed

    Yu, Yang; Zhao, Zhigang; Shi, Yanrong; Tian, Hua; Liu, Linglong; Bian, Xiaofeng; Xu, Yang; Zheng, Xiaoming; Gan, Lu; Shen, Yumin; Wang, Chaolong; Yu, Xiaowen; Wang, Chunming; Zhang, Xin; Guo, Xiuping; Wang, Jiulin; Ikehashi, Hiroshi; Jiang, Ling; Wan, Jianmin

    2016-07-01

    Intersubspecific hybrid sterility is a common form of reproductive isolation in rice (Oryza sativa L.), which significantly hampers the utilization of heterosis between indica and japonica varieties. Here, we elucidated the mechanism of S7, which specially causes Aus-japonica/indica hybrid female sterility, through cytological and genetic analysis, map-based cloning, and transformation experiments. Abnormal positioning of polar nuclei and smaller embryo sac were observed in F1 compared with male and female parents. Female gametes carrying S7(cp) and S7(i) were aborted in S7(ai)/S7(cp) and S7(ai)/S7(i), respectively, whereas they were normal in both N22 and Dular possessing a neutral allele, S7(n) S7 was fine mapped to a 139-kb region in the centromere region on chromosome 7, where the recombination was remarkably suppressed due to aggregation of retrotransposons. Among 16 putative open reading frames (ORFs) localized in the mapping region, ORF3 encoding a tetratricopeptide repeat domain containing protein was highly expressed in the pistil. Transformation experiments demonstrated that ORF3 is the candidate gene: downregulated expression of ORF3 restored spikelet fertility and eliminated absolutely preferential transmission of S7(ai) in heterozygote S7(ai)/S7(cp); sterility occurred in the transformants Cpslo17-S7(ai) Our results may provide implications for overcoming hybrid embryo sac sterility in intersubspecific hybrid rice and utilization of hybrid heterosis for cultivated rice improvement. Copyright © 2016 by the Genetics Society of America.

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

    SciTech Connect

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

    2012-02-03

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

  11. Backbone fractal dimension and fractal hybrid orbital of protein structure

    NASA Astrophysics Data System (ADS)

    Peng, Xin; Qi, Wei; Wang, Mengfan; Su, Rongxin; He, Zhimin

    2013-12-01

    Fractal geometry analysis provides a useful and desirable tool to characterize the configuration and structure of proteins. In this paper we examined the fractal properties of 750 folded proteins from four different structural classes, namely (1) the α-class (dominated by α-helices), (2) the β-class (dominated by β-pleated sheets), (3) the (α/β)-class (α-helices and β-sheets alternately mixed) and (4) the (α + β)-class (α-helices and β-sheets largely segregated) by using two fractal dimension methods, i.e. "the local fractal dimension" and "the backbone fractal dimension" (a new and useful quantitative parameter). The results showed that the protein molecules exhibit a fractal behavior in the range of 1 ⩽ N ⩽ 15 (N is the number of the interval between two adjacent amino acid residues), and the value of backbone fractal dimension is distinctly greater than that of local fractal dimension for the same protein. The average value of two fractal dimensions decreased in order of α > α/β > α + β > β. Moreover, the mathematical formula for the hybrid orbital model of protein based on the concept of backbone fractal dimension is in good coincidence with that of the similarity dimension. So it is a very accurate and simple method to analyze the hybrid orbital model of protein by using the backbone fractal dimension.

  12. Several recombinant capsid proteins of equine rhinitis a virus show potential as diagnostic antigens.

    PubMed

    Li, Fan; Stevenson, Rachel A; Crabb, Brendan S; Studdert, Michael J; Hartley, Carol A

    2005-06-01

    Equine rhinitis A virus (ERAV) is a significant pathogen of horses and is also closely related to Foot-and-mouth disease virus (FMDV). Despite these facts, knowledge of the prevalence and importance of ERAV infections remains limited, largely due to the absence of a simple, robust diagnostic assay. In this study, we compared the antigenicities of recombinant full-length and fragmented ERAV capsid proteins expressed in Escherichia coli by using sera from experimentally infected and naturally exposed horses. We found that, from the range of antigens tested, recombinant proteins encompassing the C-terminal region of VP1, full-length VP2, and the N-terminal region of VP2 reacted specifically with antibodies present in sera from each of the five experimentally infected horses examined. Antibodies to epitopes on VP2 (both native and recombinant forms) persisted longer postinfection (>105 days) than antibodies specific for epitopes on other fragments. Our data also suggest that B-cell epitopes within the C terminus of VP1 and N terminus of VP2 contribute to a large proportion of the total reactivity of recombinant VP1 and VP2, respectively. Importantly, the reactivity of these VP1 and VP2 recombinant proteins in enzyme-linked immunosorbent assays (ELISAs) correlated well with the results from a range of native antigen-based serological assays using sera from 12 field horses. This study provides promising candidates for development of a diagnostic ERAV ELISA.

  13. Functions of the Snf2/Swi2 family Rad54 motor protein in homologous recombination

    PubMed Central

    Ceballos, Shannon J.; Heyer, Wolf-Dietrich

    2011-01-01

    Homologous recombination is a central pathway to maintain genomic stability and is involved in the repair of DNA damage and replication fork support, as well as accurate chromosome segregation during meiosis. Rad54 is a dsDNA-dependent ATPase of the Snf2/Swi2 family of SF2 helicases, although Rad54 lacks classical helicase activity and cannot carry out the strand displacement reactions typical for DNA helicases. Rad54 is a potent and processive motor protein that translocates on dsDNA, potentially executing several functions in recombinational DNA repair. Rad54 acts in concert with Rad51, the central protein of recombination that performs the key reactions of homology search and DNA strand invasion. Here, we will review the role of the Rad54 protein in homologous recombination with an emphasis on mechanistic studies with the yeast and human enzymes. We will discuss how these results relate to in vivo functions of Rad54 during homologous recombination in somatic cells and during meiosis. PMID:21704205

  14. 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.

  15. Synthesis of recombinant human parainfluenza virus 1 and 3 nucleocapsid proteins in yeast Saccharomyces cerevisiae.

    PubMed

    Juozapaitis, Mindaugas; Zvirbliene, Aurelija; Kucinskaite, Indre; Sezaite, Indre; Slibinskas, Rimantas; Coiras, Mayte; de Ory Manchon, Fernando; López-Huertas, María Rosa; Pérez-Breña, Pilar; Staniulis, Juozas; Narkeviciute, Irena; Sasnauskas, Kestutis

    2008-05-01

    Human parainfluenza virus types 1 and 3 (HPIV1 and HPIV3, respectively), members of the virus family Paramyxoviridae, are common causes of lower respiratory tract infections in infants, young children, the immunocompromised, the chronically ill, and the elderly. In order to synthesize recombinant HPIV1 and HPIV3 nucleocapsid proteins, the coding sequences were cloned into the yeast Saccharomyces cerevisiae expression vector pFGG3 under control of GAL7 promoter. A high level of recombinant virus nucleocapsid proteins expression (20-24 mg l(-1) of yeast culture) was obtained. Electron microscopy demonstrated the assembly of typical herring-bone structures of purified recombinant nucleocapsid proteins, characteristic for other paramyxoviruses. These structures contained host RNA, which was resistant to RNase treatment. The nucleocapsid proteins were stable in yeast and were easily purified by caesium chloride gradient ultracentrifugation. Therefore, this system proved to be simple, efficient and cost-effective, suitable for high-level production of parainfluenza virus nucleocapsids as nucleocapsid-like particles. When used as coating antigens in an indirect ELISA, the recombinant N proteins reacted with sera of patients infected with HPIV1 or 3. Serological assays to detect HPIV-specific antibodies could be designed on this basis.

  16. Recombinant Flagellin-Porcine Circovirus Type 2 Cap Fusion Protein Promotes Protective Immune Responses in Mice

    PubMed Central

    Zhang, Chunyan; Zhu, Shanshan; Wei, Li; Yan, Xu; Wang, Jing; Quan, Rong; She, Ruiping; Hu, Fengjiao; Liu, Jue

    2015-01-01

    The Cap protein of porcine circovirus type 2 (PCV2) that serves as a major host-protective immunogen was used to develop recombinant vaccines for control of PCV2-associated diseases. Growing research data have demonstrated the high effectiveness of flagellin as an adjuvant for humoral and cellular immune responses. Here, a recombinant protein was designed by fusing a modified version of bacterial flagellin to PCV2 Cap protein and expressed in a baculovirus system. When administered without adjuvant to BALB/c mice, the flagellin-Cap fusion protein elicited stronger PCV2-specific IgG antibody response, higher neutralizing antibody levels, milder histopathological changes and lower viremia, as well as higher secretion of cytokines such as TNF-α and IFN-γ that conferred better protection against virus challenge than those in the recombinant Cap alone-inoculated mice. These results suggest that the recombinant Cap protein when fused to flagellin could elicit better humoral and cellular immune responses against PCV2 infection in a mouse model, thereby acting as an attractive candidate vaccine for control of the PCV2-associated diseases. PMID:26070075

  17. Cloning, expression and immunoreactivity of recombinant Toxoplasma gondii GRA5 protein

    PubMed Central

    Arab-Mazar, Zahra; Fallahi, Shirzad; Koochaki, Ameneh; Mirahmadi, Hadi; Tabaei, Seyyed Javad Seyyed

    2016-01-01

    Background and Objectives: Toxoplasma gondii is an obligatory intracellular parasite which causes severe diseases in the fetus of pregnant women and immunocopmromised patients. Serological tests based on recombinant protein are one of the main diagnosis methods for the detection of specific antibodies in serum samples. Dense granule antigenic proteins derived from T. gondii (TgGRAs) are potential antigens for the development of diagnostic tools. Materials and Methods: DNA was extracted from T. gondii (RH-strain) tachyzoites and PCR reaction was done using corresponding primers for GRA5 antigen. The PCR product was purified and ligated into pTG19-t vector and then subcloned into XhoI and BamHI digested pGEX6p-1 expression vector. Recombinant plasmid was transformed into E. coli (BL21 DE3) and induced by 1mM IPTG and analyzed by 15% SDS-PAGE. Expressed protein was confirmed by western blot analysis. Results: There was no difference among the sequences of T. gondii GRA5 gene from different isolates. The recombinant plasmid pGEX-6p-1/GRA5 induced by IPTG was expressed in E. coli. It was a GST fusion protein and could react with human positive sera analyzed by western blot. Conclusion: The GRA5 gene of T. gondii isolates is highly conservative. This antigen as a recombinant protein was successfully expressed in E. coli, which showed high immunoreactivity. PMID:28149494

  18. 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.

  19. 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.

  20. Structure and pH-induced alterations of recombinant and natural spider silk proteins in solution.

    PubMed

    Leclerc, Jérémie; Lefèvre, Thierry; Pottier, Fabien; Morency, Louis-Philippe; Lapointe-Verreault, Camille; Gagné, Stéphane M; Auger, Michèle

    2012-06-01

    The spinning process of spiders can modulate the mechanical properties of their silk fibers. It is therefore of primary importance to understand what are the key elements of the spider spinning process to develop efficient industrial spinning processes. We have exhaustively investigated the native conformation of major ampullate silk (MaS) proteins by comparing the content of the major ampullate gland of Nephila clavipes, solubilized MaS (SolMaS) fibers and the recombinant proteins rMaSpI and rMaSpII using (1) H solution NMR spectroscopy. The results indicate that the protein secondary structure is basically identical for the recombinant protein rMaSpI, SolMaS proteins, and the proteins in the dope, and corresponds to a disordered protein rich in 3(1) -helices. The data also show that glycine proton chemical shifts of rMaSpI and SolMaS are affected by pH, but that this change is not due to a modification of the secondary structure. Using a combination of NMR and dynamic light scattering, we have found that the spectral alteration of glycine is concomitant to a modification of the hydrodynamical diameter of recombinant and solubilized MaS. This led us to suggest new potential roles for the pH acidification in the spinning process of MaS proteins. Copyright © 2011 Wiley Periodicals, Inc.

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

    PubMed

    Li, Zhaopeng; Carstensen, Bettina; Rinas, Ursula

    2014-11-05

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

  2. Characterization and reactivity of broiler chicken sera to selected recombinant Campylobacter jejuni chemotactic proteins.

    PubMed

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

    2014-05-01

    Campylobacter jejuni, a Gram-negative rod bacterium, is the leading causative agent of human acute bacterial gastroenteritis worldwide. Consumption and handling of raw or undercooked poultry are regarded as a major source for human infection. Because bacterial chemotaxis guides microorganisms to colonization and invasion in the host cells, proteins involved in chemotactic processes can be novel targets for vaccine development. In this communication, we report amplification, cloning and expression of the C. jejuni chemotactic proteins in an Escherichia coli expression system. A total of 15 chemotactic protein genes were successfully expressed. These recombinant proteins were confirmed by nucleotide sequencing, SDS-PAGE analysis and immunoblot analysis of six-His and hemagglutinin tags. Twelve recombinant chemotactic proteins were further tested whether they were antigenic using sera from broiler chickens older than 4 weeks. The immunoblot results show that each chicken serum reacted to a variety of the recombinant proteins, but all sera reacted to the Cjj0473 gene product (annotated as a methyl-accepting chemotaxis protein), suggesting that anti-Campylobacter antibodies may be prevalent in the poultry population. These antibody screening results provide a rationale for further evaluation of the Cjj0473 protein as a potential vaccine for broilers to improve human food safety.

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

    PubMed

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

    2016-04-01

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

  4. Effect of metal catalyzed oxidation in recombinant viral protein assemblies

    PubMed Central

    2014-01-01

    Background Protein assemblies, such as virus-like particles, have increasing importance as vaccines, delivery vehicles and nanomaterials. However, their use requires stable assemblies. An important cause of loss of stability in proteins is oxidation, which can occur during their production, purification and storage. Despite its importance, very few studies have investigated the effect of oxidation in protein assemblies and their structural units. In this work, we investigated the role of in vitro oxidation in the assembly and stability of rotavirus VP6, a polymorphic protein. Results The susceptibility to oxidation of VP6 assembled into nanotubes (VP6NT) and unassembled VP6 (VP6U) was determined and compared to bovine serum albumin (BSA) as control. VP6 was more resistant to oxidation than BSA, as determined by measuring protein degradation and carbonyl content. It was found that assembly protected VP6 from in vitro metal-catalyzed oxidation. Oxidation provoked protein aggregation and VP6NT fragmentation, as evidenced by dynamic light scattering and transmission electron microscopy. Oxidative damage of VP6 correlated with a decrease of its center of fluorescence spectral mass. The in vitro assembly efficiency of VP6U into VP6NT decreased as the oxidant concentration increased. Conclusions Oxidation caused carbonylation, quenching, and destruction of aromatic amino acids and aggregation of VP6 in its assembled and unassembled forms. Such modifications affected protein functionality, including its ability to assemble. That assembly protected VP6 from oxidation shows that exposure of susceptible amino acids to the solvent increases their damage, and therefore the protein surface area that is exposed to the solvent is determinant of its susceptibility to oxidation. The inability of oxidized VP6 to assemble into nanotubes highlights the importance of avoiding this modification during the production of proteins that self-assemble. This is the first time that the role of

  5. Optical sensing systems based on biomolecular recognition of recombinant proteins

    NASA Astrophysics Data System (ADS)

    Salins, Lyndon L.; Schauer-Vukasinovic, Vesna; Daunert, Sylvia

    1998-05-01

    SIte-directed mutagenesis and the associated site-specific fluorescent labeling of proteins can be used to rationally design reagentless fluorescent molecular senors. The phosphate binding protein (PBP) and calmodulin (CaM) bind to phosphate and calcium in a highly specific manner. These ions induce a hinge motion in the proteins, and the resultant conformational change constitutes the basis of the sensor development. By labeling each protein at a specific site with environment-sensitive fluorescent probes, these conformational changes can be monitored and related to the amount of analyte ion present. In this study, the polymerase chain reaction was used to construct mutants of PBP and CaM that have a single cysteine at positions 197 and 109, respectively. Each protein was site-specifically labeled through the sulfhydryl group of the introduced cysteine residue at a single location with an environment-sensitive fluorescent probe. Characterization of the steady-state fluorescence indicated an enhancement of signal intensity upon binding of the analyte ion. Highly sensitive and selective and selective sensing systems for phosphate and calcium were obtained by using this approach.

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

    PubMed

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

    2015-12-22

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

  7. Recent advances in recombinant protein-based malaria vaccines

    PubMed Central

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

    2015-01-01

    Plasmodium parasites are the causative agent of human malaria, and the development of a highly effective vaccine against infection, disease and transmission remains a key priority. It is widely established that multiple stages of the parasite's complex lifecycle within the human host and mosquito vector are susceptible to vaccine-induced antibodies. The mainstay approach to antibody induction by subunit vaccination has been the delivery of protein antigen formulated in adjuvant. Extensive efforts have been made in this endeavor with respect to malaria vaccine development, especially with regard to target antigen discovery, protein expression platforms, adjuvant testing, and development of soluble and virus-like particle (VLP) delivery platforms. The breadth of approaches to protein-based vaccines is continuing to expand as innovative new concepts in next-generation subunit design are explored, with the prospects for the development of a highly effective multi-component/multi-stage/multi-antigen formulation seeming ever more likely. This review will focus on recent progress in protein vaccine design, development and/or clinical testing for a number of leading malaria antigens from the sporozoite-, merozoite- and sexual-stages of the parasite's lifecycle–including PfCelTOS, PfMSP1, PfAMA1, PfRH5, PfSERA5, PfGLURP, PfMSP3, Pfs48/45 and Pfs25. Future prospects and challenges for the development, production, human delivery and assessment of protein-based malaria vaccines are discussed. PMID:26458807

  8. Baculovirus expression of the glycoprotein gene of Lassa virus and characterization of the recombinant protein.

    PubMed

    Hummel, K B; Martin, M L; Auperin, D D

    1992-09-01

    A recombinant baculovirus was constructed that expresses the glycoprotein gene of Lassa virus (Josiah strain) under the transcriptional control of the polyhedrin promoter. The expressed protein (B-LSGPC) comigrated with the authentic viral glycoprotein as observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), was reactive with monoclonal antibodies (MAbs) in Western blots, and was glycosylated. Although the recombinant protein was not processed into the mature glycoproteins, G1 and G2, it demonstrated reactivity with all known epitopes as measured by indirect immunofluorescence (IFA), and it was immunogenic, eliciting antisera in rabbits that recognized whole virus in IFAs. Regarding future applications to diagnostic assays, the recombinant glycoprotein proved to be an effective substitute for Lassa virus-infected mammalian cells in IFAs and it was able to distinguish sera from several human cases of Lassa fever, against a panel of known negative sera of African origin, in an enzyme immunoassay (EIA).

  9. The RAG proteins in V(D)J recombination: more than just a nuclease

    PubMed Central

    Sadofsky, Moshe J.

    2001-01-01

    V(D)J recombination is the process that generates the diversity among T cell receptors and is one of three mechanisms that contribute to the diversity of antibodies in the vertebrate immune system. The mechanism requires precise cutting of the DNA at segment boundaries followed by rejoining of particular pairs of the resulting termini. The imprecision of aspects of the joining reaction contributes significantly to increasing the variability of the resulting functional genes. Signal sequences target DNA recombination and must participate in a highly ordered protein–DNA complex in order to limit recombination to appropriate partners. Two proteins, RAG1 and RAG2, together form the nuclease that cleaves the DNA at the border of the signal sequences. Additional roles of these proteins in organizing the reaction complex for subsequent steps are explored. PMID:11266539

  10. Cell culture process operations for recombinant protein production.

    PubMed

    Abu-Absi, Susan; Xu, Sen; Graham, Hugh; Dalal, Nimish; Boyer, Marcus; Dave, Kedar

    2014-01-01

    The market for protein therapeutics has grown significantly over the past two decades and the pace of development continues to increase. It is a challenge to the industry to maintain the desired quality attributes while accelerating delivery to patients, reducing the cost of goods, and providing production flexibility. Efficient manufacturing scale production of protein therapeutics is required to continue to meet the needs of the patients and stockholders. This chapter describes batch, fed-batch, and perfusion processes and their utilization in the production of monoclonal antibodies and other therapeutic proteins. In addition, we have provided detailed discussions of the ongoing challenges of lactate metabolism and the future prospects of process monitoring and control.

  11. Engineering functional artificial hybrid proteins between poplar peroxiredoxin II and glutaredoxin or thioredoxin

    SciTech Connect

    Rouhier, Nicolas . E-mail: nrouhier@scbiol.uhp-nancy.fr; Gama, Filipe; Wingsle, Gunnar; Gelhaye, Eric; Gans, Pierre; Jacquot, Jean-Pierre

    2006-03-24

    The existence of natural peroxiredoxin-glutaredoxin hybrid enzymes in several bacteria is in line with previous findings indicating that poplar peroxiredoxin II can use glutaredoxin as an electron donor. This peroxiredoxin remains however unique since it also uses thioredoxin with a quite good efficiency. Based on the existing fusions, we have created artificial enzymes containing a poplar peroxiredoxin module linked to glutaredoxin or thioredoxin modules. The recombinant fusion enzymes folded properly into non-covalently bound homodimers or homotetramers. Two of the three protein constructs exhibit peroxidase activity, a reaction where the two modules need to function together, but they also display enzymatic activities specific of each module. In addition, mass spectrometry analyses indicate that the Prx module can be both glutathiolated or overoxidized in vitro. This is discussed in the light of the Prx reactivity.

  12. Improved hybrid optimization algorithm for 3D protein structure prediction.

    PubMed

    Zhou, Changjun; Hou, Caixia; Wei, Xiaopeng; Zhang, Qiang

    2014-07-01

    A new improved hybrid optimization algorithm - PGATS algorithm, which is based on toy off-lattice model, is presented for dealing with three-dimensional protein structure prediction problems. The algorithm combines the particle swarm optimization (PSO), genetic algorithm (GA), and tabu search (TS) algorithms. Otherwise, we also take some different improved strategies. The factor of stochastic disturbance is joined in the particle swarm optimization to improve the search ability; the operations of crossover and mutation that are in the genetic algorithm are changed to a kind of random liner method; at last tabu search algorithm is improved by appending a mutation operator. Through the combination of a variety of strategies and algorithms, the protein structure prediction (PSP) in a 3D off-lattice model is achieved. The PSP problem is an NP-hard problem, but the problem can be attributed to a global optimization problem of multi-extremum and multi-parameters. This is the theoretical principle of the hybrid optimization algorithm that is proposed in this paper. The algorithm combines local search and global search, which overcomes the shortcoming of a single algorithm, giving full play to the advantage of each algorithm. In the current universal standard sequences, Fibonacci sequences and real protein sequences are certified. Experiments show that the proposed new method outperforms single algorithms on the accuracy of calculating the protein sequence energy value, which is proved to be an effective way to predict the structure of proteins.

  13. 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.

  14. Mycobacterium avium Subspecies paratuberculosis Recombinant Proteins Modulate Antimycobacterial Functions of Bovine Macrophages

    PubMed Central

    Bannantine, John P.; Stabel, Judith R.; Laws, Elizabeth; D. Cardieri, Maria Clara; Souza, Cleverson D.

    2015-01-01

    It has been shown that Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) activates the Mitogen Activated Protein Kinase (MAPK) p38 pathway, yet it is unclear which components of M. paratuberculosis are involved in the process. Therefore, a set of 42 M. paratuberculosis recombinant proteins expressed from coding sequences annotated as lipoproteins were screened for their ability to induce IL-10 expression, an indicator of MAPKp38 activation, in bovine monocyte-derived macrophages. A recombinant lipoprotein, designated as MAP3837c, was among a group of 6 proteins that strongly induced IL-10 gene transcription in bovine macrophages, averaging a 3.1-fold increase compared to non-stimulated macrophages. However, a parallel increase in expression of IL-12 and TNF-α was only observed in macrophages exposed to a subset of these 6 proteins. Selected recombinant proteins were further analyzed for their ability to enhance survival of M. avium within bovine macrophages as measured by recovered viable bacteria and nitrite production. All 6 IL-10 inducing MAP recombinant proteins along with M. paratuberculosis cells significantly enhanced phosphorylation of MAPK-p38 in bovine macrophages. Although these proteins are likely not post translationally lipidated in E. coli and thus is a limitation in this study, these results form the foundation of how the protein component of the lipoprotein interacts with the immune system. Collectively, these data reveal M. paratuberculosis proteins that might play a role in MAPK-p38 pathway activation and hence in survival of this organism within bovine macrophages. PMID:26076028

  15. Optical determination of Shockley-Read-Hall and interface recombination currents in hybrid perovskites.

    PubMed

    Sarritzu, Valerio; Sestu, Nicola; Marongiu, Daniela; Chang, Xueqing; Masi, Sofia; Rizzo, Aurora; Colella, Silvia; Quochi, Francesco; Saba, Michele; Mura, Andrea; Bongiovanni, Giovanni

    2017-03-20

    Metal-halide perovskite solar cells rival the best inorganic solar cells in power conversion efficiency, providing the outlook for efficient, cheap devices. In order for the technology to mature and approach the ideal Shockley-Queissier efficiency, experimental tools are needed to diagnose what processes limit performances, beyond simply measuring electrical characteristics often affected by parasitic effects and difficult to interpret. Here we study the microscopic origin of recombination currents causing photoconversion losses with an all-optical technique, measuring the electron-hole free energy as a function of the exciting light intensity. Our method allows assessing the ideality factor and breaks down the electron-hole recombination current into bulk defect and interface contributions, providing an estimate of the limit photoconversion efficiency, without any real charge current flowing through the device. We identify Shockley-Read-Hall recombination as the main decay process in insulated perovskite layers and quantify the additional performance degradation due to interface recombination in heterojunctions.

  16. Optical determination of Shockley-Read-Hall and interface recombination currents in hybrid perovskites

    NASA Astrophysics Data System (ADS)

    Sarritzu, Valerio; Sestu, Nicola; Marongiu, Daniela; Chang, Xueqing; Masi, Sofia; Rizzo, Aurora; Colella, Silvia; Quochi, Francesco; Saba, Michele; Mura, Andrea; Bongiovanni, Giovanni

    2017-03-01

    Metal-halide perovskite solar cells rival the best inorganic solar cells in power conversion efficiency, providing the outlook for efficient, cheap devices. In order for the technology to mature and approach the ideal Shockley-Queissier efficiency, experimental tools are needed to diagnose what processes limit performances, beyond simply measuring electrical characteristics often affected by parasitic effects and difficult to interpret. Here we study the microscopic origin of recombination currents causing photoconversion losses with an all-optical technique, measuring the electron-hole free energy as a function of the exciting light intensity. Our method allows assessing the ideality factor and breaks down the electron-hole recombination current into bulk defect and interface contributions, providing an estimate of the limit photoconversion efficiency, without any real charge current flowing through the device. We identify Shockley-Read-Hall recombination as the main decay process in insulated perovskite layers and quantify the additional performance degradation due to interface recombination in heterojunctions.

  17. 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.

  18. SMC proteins constitute two subunits of the mammalian recombination complex RC-1.

    PubMed Central

    Jessberger, R; Riwar, B; Baechtold, H; Akhmedov, A T

    1996-01-01

    Recombination protein complex RC-1, purified from calf thymus nuclear extracts, catalyzes cell-free DNA strand transfer and repair of gaps and deletions through DNA recombination. DNA polymerase E, DNA ligase III and a DNA structure-specific endonuclease co-purify with the five polypeptide complex. Here we describe the identification of two hitherto unknown subunits of RC-1. N-terminal amino acid sequences of the 160 and 130 kDa polypeptides display up to 100% identity to proteins of the structural maintenance of chromosomes (SMC) subfamilies 1 and 2. SMC proteins are involved in mitotic chromosome segregation and condensation, as well as in certain DNA repair pathways in fission (rad18 gene) and budding (RHC18 gene) yeast. The assignment was substantiated by immuno-cross-reactivity of the RC-1 subunits with polyclonal antibodies specific for Xenopus laevis SMC proteins. These antibodies, and polyclonal antibodies directed against the bovine 160 and 130 kDa polypeptides, named BSMC1 and BSMC2 (bovine SMC), inhibited RC-1-mediated DNA transfer, indicating that the SMC proteins are necessary components of the reaction. Two independent assays revealed DNA reannealing activity of RC-1, which resides in its BSMC subunits, thereby demonstrating a novel function of these proteins. To our knowledge, this is the first evidence for the association of mammalian SMC proteins with a multiprotein complex harboring, among others, DNA recombination, DNA ligase and DNA polymerase activities. Images PMID:8670910

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

    PubMed

    Raran-Kurussi, Sreejith; Waugh, David S

    2016-07-01

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

  20. Optimization of recombinant protein expression in the chloroplasts of green algae.

    PubMed

    Fletcher, Samuel P; Muto, Machiko; Mayfield, Stephen P

    2007-01-01

    Through advances in molecular and genetic techniques, protein expression in the chloroplasts of green algae has been optimized for high-level expression. Recombinant proteins expressed in algae have the potential to provide novel and safe treatment of disease and infection where current, high-cost drugs are the only option, or worse, where therapeutic drugs are not available due to their prohibitively high-cost to manufacture. Optimization of recombinant protein expression in Chlamydomonas reinhardtii chloroplasts has been accomplished by employing chloroplast codon bias and combinatorial examination of promoter and UTR combinations. In addition, as displayed by the expression of an anti-herpes antibody, the C. reinhardtii chloroplast is capable of correctly folding and assembling complex mammalian proteins. These data establish algal chloroplasts as a system for the production of complex human therapeutic proteins in soluble and active form, and at significantly reduced time and cost compared to existing production systems. Production of recombinant proteins in algal chloroplasts may enable further development of safe, efficacious and cost-effective protein therapeutics.

  1. Recombinant Expression, Purification, and Functional Characterisation of Connective Tissue Growth Factor and Nephroblastoma-Overexpressed Protein

    PubMed Central

    Bohr, Wilhelm; Kupper, Michael; Hoffmann, Kurt; Weiskirchen, Ralf

    2010-01-01

    The CCN family of proteins, especially its prominent member, the Connective tissue growth factor (CTGF/CCN2) has been identified as a possible biomarker for the diagnosis of fibrotic diseases. As a downstream mediator of TGF-β1 signalling, it is involved in tissue scarring, stimulates interstitial deposition of extracellular matrix proteins, and promotes proliferation of several cell types. Another member of this family, the Nephroblastoma-Overexpressed protein (NOV/CCN3), has growth-inhibiting properties. First reports further suggest that these two CCN family members act opposite to each other in regulating extracellular matrix protein expression and reciprocally influence their own expression when over-expressed. We have established stable HEK and Flp-In-293 clones as productive sources for recombinant human CCN2/CTGF. In addition, we generated an adenoviral vector for recombinant expression of rat NOV and established protocols to purify large quantities of these CCN proteins. The identity of purified human CCN2/CTGF and rat CCN3/NOV was proven by In-gel digest followed by ESI-TOF/MS mass spectrometry. The biological activity of purified proteins was demonstrated using a Smad3-sensitive reporter gene and BrdU proliferation assay in permanent cell line EA•hy 926 cells. We further demonstrate for the first time that both recombinant CCN proteins are N-glycosylated. PMID:21209863

  2. 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.

  3. 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

  4. A Novel Hybrid Clonal Selection Algorithm with Combinatorial Recombination and Modified Hypermutation Operators for Global Optimization

    PubMed Central

    Lin, Jingjing; Jing, Honglei

    2016-01-01

    Artificial immune system is one of the most recently introduced intelligence methods which was inspired by biological immune system. Most immune system inspired algorithms are based on the clonal selection principle, known as clonal selection algorithms (CSAs). When coping with complex optimization problems with the characteristics of multimodality, high dimension, rotation, and composition, the traditional CSAs often suffer from the premature convergence and unsatisfied accuracy. To address these concerning issues, a recombination operator inspired by the biological combinatorial recombination is proposed at first. The recombination operator could generate the promising candidate solution to enhance search ability of the CSA by fusing the information from random chosen parents. Furthermore, a modified hypermutation operator is introduced to construct more promising and efficient candidate solutions. A set of 16 common used benchmark functions are adopted to test the effectiveness and efficiency of the recombination and hypermutation operators. The comparisons with classic CSA, CSA with recombination operator (RCSA), and CSA with recombination and modified hypermutation operator (RHCSA) demonstrate that the proposed algorithm significantly improves the performance of classic CSA. Moreover, comparison with the state-of-the-art algorithms shows that the proposed algorithm is quite competitive. PMID:27698662

  5. New perspectives for use of native and engineered recombinant food proteins in treatment of food allergy.

    PubMed

    Nowak-Wegrzyn, Anna

    2007-02-01

    Food allergy has emerged as an important target for research on curative treatment and prevention, with most efforts focusing on peanut, cow's milk, and egg allergy. This article reviews the recent developments in the potential treatments for IgE-mediated food allergy using native and engineered recombinant food proteins.

  6. 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.

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

    NASA Astrophysics Data System (ADS)

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

    1999-01-01

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

  8. DNA-dependent protein kinase mediates V(D)J recombination via RAG2 phosphorylation.

    PubMed

    Hah, Young-Sool; Lee, Jung Hwa; Kim, Deok Ryong

    2007-05-31

    V(D)J recombination, a site-specific gene rearrangement process occurring during the lymphocyte development, begins with DNA double strand breaks by two recombination activating gene products (RAG1/2) and finishes with the repair process by several proteins including DNA-dependent protein kinase (DNA-PK). In this report, we found that RAG2 was specifically phosphorylated by DNA-PK at the 365(th) serine residue, and this phosphorylated RAG2 affected the V(D)J recombination activity in cells in the GFP expression-based assay. While the V(D)J recombination activity between wild-type RAG2 and mutant S365A RAG2 in the assay using a signal joint substrate was undistinguishable in DNA-PK deficient cells (M059J), the activity with wild-type RAG2 was largely increased in DNA-PK proficient cells (M059K) in comparison with mutant RAG2, suggesting that RAG2 phosphorylation by DNA-PK plays a crucial role in the signal joint formation during V(D)J recombination.

  9. 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.

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

    PubMed Central

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

    1999-01-01

    Serology remains the method of choice for laboratory diagnosis of Mycoplasma pneumoniae infection. Currently available serological tests employ complex cellular fractions of M. pneumoniae as antigen. To improve the specificity of M. pneumoniae diagnosis, a recombinant protein was assessed as a serodiagnostic reagent. A panel of recombinant proteins were expressed from a cloned M. pneumoniae gene that encodes a 116-kDa surface protein antigen. The recombinant proteins were assessed for reactivity with patient sera and the most antigenic was further assessed for its serodiagnostic potential by indirect enzyme-linked immunosorbent assay (ELISA). The ELISA based on the recombinant protein was equivalent in sensitivity to the commercial test (Serodia Myco II; Fujirebio Inc.) to which it was compared. Southern and Western blotting data suggested that the recombinant protein derived from the 116-kDa protein of M. pneumoniae could provide a species-specific diagnostic tool, although further assessment is required. PMID:10074521

  11. 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.

  12. Immunological characterization of a recombinant 27-kilodalton antigenic protein from Paracoccidioides brasiliensis.

    PubMed Central

    Ortiz, B L; Garcia, A M; Restrepo, A; McEwen, J G

    1996-01-01

    We report the expression in Escherichia coli of a 27-kDa antigenic protein from Paracoccidioides brasiliensis. When analyzed by immunoblotting, this recombinant antigenic protein was recognized by antibodies present in the sera of 40 of the 44 paracoccidioidomycosis patients studied. No cross-reactions were observed with sera from patients with other mycoses (histoplasmosis, aspergillosis, cryptococcosis, sporotrichosis, and chromoblastomycosis) or with tuberculosis. PMID:8991645

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

    PubMed Central

    Saraswat, Mayank; Ravidá, Alessandra; Holthofer, Harry

    2013-01-01

    Advances in fermentation technologies have resulted in the production of increased yields of proteins of economic, biopharmaceutical, and medicinal importance. Consequently, there is an absolute requirement for the development of rapid, cost-effective methodologies which facilitate the purification of such products in the absence of contaminants, such as superfluous proteins and endotoxins. Here, we provide a comprehensive overview of a selection of key purification methodologies currently being applied in both academic and industrial settings and discuss how innovative and effective protocols such as aqueous two-phase partitioning, membrane chromatography, and high-performance tangential flow filtration may be applied independently of or in conjunction with more traditional protocols for downstream processing applications. PMID:24455685

  14. Oocyte shuttle, a recombinant protein transporting donor DNA into the Xenopus oocyte in situ.

    PubMed

    Rungger, Duri; Muster, Lisbeth; Georgiev, Oleg; Rungger-Brändle, Elisabeth

    2017-02-15

    The newly developed oocyte shuttle protein contains a streptavidin moiety that tightly binds biotinylated DNA. Injected intravenously into adult Xenopus females, the protein-DNA complex is rapidly transported through the bloodstream and, within the ovary, the vitellogenin ligand present in the protein binds to the receptors at the surface of the oocytes. The bound complex is internalized and translocates into the oocyte nucleus thanks to an SV40 nuclear localization signal, enhanced by an adjacent casein kinase phosphorylation site. Functioning of the shuttle protein is documented by transporting DNA molecules that, upon intramolecular homologous recombination within the oocyte nucleus, express easily traceable markers such as green fluorescence or tetracycline resistance.

  15. IMAC capture of recombinant protein from unclarified mammalian cell feed streams

    PubMed Central

    Kinna, Alexander; Tolner, Berend; Rota, Enrique Miranda; Titchener‐Hooker, Nigel; Nesbeth, Darren

    2015-01-01

    ABSTRACT Fusion‐tag affinity chromatography is a key technique in recombinant protein purification. Current methods for protein recovery from mammalian cells are hampered by the need for feed stream clarification. We have developed a method for direct capture using immobilized metal affinity chromatography (IMAC) of hexahistidine (His6) tagged proteins from unclarified mammalian cell feed streams. The process employs radial flow chromatography with 300–500 μm diameter agarose resin beads that allow free passage of cells but capture His‐tagged proteins from the feed stream; circumventing expensive and cumbersome centrifugation and/or filtration steps. The method is exemplified by Chinese Hamster Ovary (CHO) cell expression and subsequent recovery of recombinant His‐tagged carcinoembryonic antigen (CEA); a heavily glycosylated and clinically relevant protein. Despite operating at a high NaCl concentration necessary for IMAC binding, cells remained over 96% viable after passage through the column with host cell proteases and DNA detected at ∼8 U/mL and 2 ng/μL in column flow‐through, respectively. Recovery of His‐tagged CEA from unclarified feed yielded 71% product recovery. This work provides a basis for direct primary capture of fully glycosylated recombinant proteins from unclarified mammalian cell feed streams. Biotechnol. Bioeng. 2016;113: 130–140. © 2015 Wiley Periodicals, Inc. PMID:26174988

  16. 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

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

    PubMed Central

    Zhang, Litai; Huang, Xiaofeng; Xue, Bai; Peng, Quanhui; Wang, Zhisheng; Yan, Tianhai; Wang, Lizhi

    2015-01-01

    Vaccination through recombinant proteins against rumen methanogenesis provides a mitigation approach to reduce enteric methane (CH4) emissions in ruminants. The objective of present study was to evaluate the in vivo efficacy of a new vaccine candidate protein (EhaF) on methanogenesis and microbial population in the rumen of goats. We amplified the gene mru 1407 encoding protein EhaF using fresh rumen fluid samples of mature goats and successfully expressed recombinant protein (EhaF) in Escherichia coli Rosetta. This product was evaluated using 12 mature goats with half for control and other half injected with 400ug/goat the purified recombinant protein in day 1 and two subsequent booster immunizations in day 35 and 49. All measurements were undertaken from 63 to 68 days after the initial vaccination, with CH4 emissions determined using respiration calorimeter chambers. The results showed that the vaccination caused intensive immune responses in serum and saliva, although it had no significant effect on total enteric CH4 emissions and methanogen population in the rumen, when compared with the control goats. However, the vaccination altered the composition of rumen bacteria, especially the abundance of main phylum Firmicutes and genus Prevotella. The results indicate that protein EhaF might not be an effective vaccine to reduce enteric CH4 emissions but our vaccine have potential to influence the rumen ecosystem of goats. PMID:26445479

  18. 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.

  19. Transient gene expression: recombinant protein production with suspension-adapted HEK293-EBNA cells.

    PubMed

    Meissner, P; Pick, H; Kulangara, A; Chatellard, P; Friedrich, K; Wurm, F M

    2001-10-20

    Transient gene expression (TGE) in mammalian cells at the reactor scale is becoming increasingly important for the rapid production of recombinant proteins. We improved a process for transient calcium phosphate-based transfection of HEK293-EBNA cells in a 1-3 L bioreactor volume. Cells were adapted to suspension culture using a commercially available medium (BioWhittaker, Walkersville, MD). Process parameters were optimized using a plasmid reporter vector encoding the enhanced green fluorescent protein (EGFP/CLONTECH, Palo Alto, CA, USA). Using GFP as a marker-protein, we observed by microscopic examination transfection efficiencies between 70-100%. Three different recombinant proteins were synthesized within a timeframe of 7 days from time of transfection to harvest. The first, a human recombinant IgG(1)-type antibody, was secreted into the supernatant of the cell culture and achieved a final concentration of >20 mg/L. An E. coli-derived DNA-binding protein remained intracellular, as expected, but accumulated to such a concentration that the lysate of cells, taken up into the entire culture volume, gave a concentration of 18 mg/L. The third protein, a transmembrane receptor, was expressed at 3-6 x 10(6) molecules/cell.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-12-15

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

  2. 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.

  3. 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.

  4. Antigenic validation of recombinant hemagglutinin-neuraminidase protein of Newcastle disease virus expressed in Saccharomyces cerevisiae.

    PubMed

    Khulape, S A; Maity, H K; Pathak, D C; Mohan, C Madhan; Dey, S

    2015-09-01

    The outer membrane glycoprotein, hemagglutinin-neuraminidase (HN) of Newcastle disease virus (NDV) is important for virus infection and subsequent immune response by host, and offers target for development of recombinant antigen-based immunoassays and subunit vaccines. In this study, the expression of HN protein of NDV is attempted in yeast expression system. Yeast offers eukaryotic environment for protein processing and posttranslational modifications like glycosylation, in addition to higher growth rate and easy genetic manipulation. Saccharomyces cerevisiae was found to be better expression system for HN protein than Pichia pastoris as determined by codon usage analysis. The complete coding  sequence of HN gene was amplified with the histidine tag, cloned in pESC-URA under GAL10 promotor and transformed in Saccharomyces cerevisiae. The recombinant HN (rHN) protein was characterized by western blot, showing glycosylation heterogeneity as observed with other eukaryotic expression systems. The recombinant protein was purified by affinity column purification. The protein could be further used as subunit vaccine.

  5. 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.

  6. Rift Valley Fever Virus Structural and Nonstructural Proteins: Recombinant Protein Expression and Immunoreactivity Against Antisera from Sheep

    PubMed Central

    Faburay, Bonto; Wilson, William; McVey, D. Scott; Drolet, Barbara S.; Weingartl, Hana; Madden, Daniel; Young, Alan; Ma, Wenjun

    2013-01-01

    Abstract The Rift Valley fever virus (RVFV) encodes the structural proteins nucleoprotein (N), aminoterminal glycoprotein (Gn), carboxyterminal glycoprotein (Gc), and L protein, 78-kD, and the nonstructural proteins NSm and NSs. Using the baculovirus system, we expressed the full-length coding sequence of N, NSs, NSm, Gc, and the ectodomain of the coding sequence of the Gn glycoprotein derived from the virulent strain of RVFV ZH548. Western blot analysis using anti-His antibodies and monoclonal antibodies against Gn and N confirmed expression of the recombinant proteins, and in vitro biochemical analysis showed that the two glycoproteins, Gn and Gc, were expressed in glycosylated form. Immunoreactivity profiles of the recombinant proteins in western blot and in indirect enzyme-linked immunosorbent assay against a panel of antisera obtained from vaccinated or wild type (RVFV)-challenged sheep confirmed the results obtained with anti-His antibodies and demonstrated the suitability of the baculo-expressed antigens for diagnostic assays. In addition, these recombinant proteins could be valuable for the development of diagnostic methods that differentiate infected from vaccinated animals (DIVA). PMID:23962238

  7. Analysis of Mycobacterium tuberculosis genoma and production of a recombinant protein containing specific B and T cell antigenic determinants--new approaches to second generation antituberculosis vaccines.

    PubMed

    Colizzi, V; Vismara, D; D'Urso, C; Mezzopreti, M F; Lombardi, G; Piccolella, E; Damiani, G; Marelli, P; Campa, M

    1989-01-01

    Tuberculosis is still a major health problem in almost all over the world. Thus, new directions in basic and applied research of tuberculosis are under investigation in several laboratories. In this paper, we provide recent data obtained in our laboratory with the recombinant DNA technology which allow a systematic survey of the microbial genome. Screening of the M. tuberculosis genomic DNA library in the phage lambda gt11 expression vector, using E. coli as surrogate host, has evidenced the possibility of producing recombinant M. tuberculosis proteins recognized by sera from tuberculosis patients and by specific monoclonal antibodies. Using this technology, we have isolated a recombinant protein (molecular weight 130 kilodaltons) which is identified by a murine monoclonal antibody recognizing a mycobacterial cell wall antigenic determinant present on a mycobacterial protein. This protein is only present on the mycobacterial species related to the tuberculosis complex (M. tuberculosis, M. bovis, M. africanum) and does not crossreact with nonpathogenic Mycobacteria. Since the immune response to mycobacterial infections is cell-mediated, the question arises about the use of M. tuberculosis-specific T lymphocytes to screen this gene bank. Thus, the recombinant mycobacterial protein isolated by antibodies has been then used to stimulate the proliferation of T lymphocytes from patients with tubercular pleuritis. This experiment indicates that the recombinant protein contains antigenic determinants recognized by T cells. Moreover, such protein is able to elicit delayed type hypersensitivity skin reaction in mice immunized or infected with M. tuberculosis and M. bovis. Finally, gene mapping and hybridization studies with native M. tuberculosis DNA confirme the mycobacterial nature of the recombinant DNA insert. Thus a good candidate for the prophylaxis and the immunodiagnosis of tuberculosis has been identified. The identification and selection of genes encoding antigenic

  8. Host SAMHD1 Protein Promotes HIV-1 Recombination in Macrophages*

    PubMed Central

    Nguyen, Laura A.; Kim, Dong-Hyun; Daly, Michele B.; Allan, Kevin C.; Kim, Baek

    2014-01-01

    Template switching can occur during the reverse transcription of HIV-1. Deoxynucleotide triphosphate (dNTP) concentrations have been biochemically shown to impact HIV-1 reverse transcriptase (RT)-mediated strand transfer. Lowering the dNTP concentrations promotes RT pausing and RNA template degradation by RNase H activity of the RT, subsequently leading to strand transfer. Terminally differentiated/nondividing macrophages, which serve as a key HIV-1 reservoir, contain extremely low dNTP concentrations (20–50 nm), which results from the cellular dNTP hydrolyzing sterile α motif and histidine aspartic domain containing protein 1 (SAMHD1) protein, when compared with activated CD4+ T cells (2–5 μm). In this study, we first observed that HIV-1 template switching efficiency was nearly doubled in human primary macrophages when compared with activated CD4+ T cells. Second, SAMHD1 degradation by viral protein X (Vpx), which elevates cellular dNTP concentrations, decreased HIV-1 template switching efficiency in macrophages to the levels comparable with CD4+ T cells. Third, differentiated SAMHD1 shRNA THP-1 cells have a 2-fold increase in HIV-1 template switching efficiency. Fourth, SAMHD1 degradation by Vpx did not alter HIV-1 template switching efficiency in activated CD4+ T cells. Finally, the HIV-1 V148I RT mutant that is defective in dNTP binding and has DNA synthesis delay promoted RT stand transfer when compared with wild type RT, particularly at low dNTP concentrations. Here, we report that SAMHD1 regulation of the dNTP concentrations influences HIV-1 template switching efficiency, particularly in macrophages. PMID:24352659

  9. Development of a SARS Coronavirus Vaccine from Recombinant Spike Protein Plus Delta Inulin Adjuvant.

    PubMed

    McPherson, Clifton; Chubet, Richard; Holtz, Kathy; Honda-Okubo, Yoshikazu; Barnard, Dale; Cox, Manon; Petrovsky, Nikolai

    2016-01-01

    Given periodic outbreaks of fatal human infections caused by coronaviruses, development of an optimal coronavirus vaccine platform capable of rapid production is an ongoing priority. This chapter describes the use of an insect cell expression system for rapid production of a recombinant vaccine against severe acute respiratory syndrome coronavirus (SARS). Detailed methods are presented for expression, purification, and release testing of SARS recombinant spike protein antigen, followed by adjuvant formulation and animal testing. The methods herein described for rapid development of a highly protective SARS vaccine are equally suited to rapid development of vaccines against other fatal human coronavirus infections, e.g., the MERS coronavirus.

  10. Attenuated vaccinia virus-circumsporozoite protein recombinants confer protection against rodent malaria.

    PubMed Central

    Lanar, D E; Tine, J A; de Taisne, C; Seguin, M C; Cox, W I; Winslow, J P; Ware, L A; Kauffman, E B; Gordon, D; Ballou, W R; Paoletti, E; Sadoff, J C

    1996-01-01

    NYVAC-based vaccinia virus recombinants expressing the circumsporozoite protein (CSP) were evaluated in the Plasmodium berghei rodent malaria model system. Immunization of mice with a NYVAC-based CSP recombinant elicited a high level of protection (60 to 100%). Protection did not correlate with CS repeat-specific antibody responses and was abrogated by in vivo CD8+ T-cell depletion. Protection was not enhanced by modification of the subcellular localization of CSP. These results suggest the potential of poxvirus-based vectors for the development of vaccine candidates for human malaria. PMID:8613376

  11. 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.

  12. 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.

  13. 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.

  14. Essential bacterial helicases that counteract the toxicity of recombination proteins

    PubMed Central

    Petit, Marie-Agnès; Ehrlich, Dusko

    2002-01-01

    PcrA, Rep and UvrD are three closely related bacterial helicases with a DExx signature. PcrA is encoded by Gram-positive bacteria and is essential for cell growth. Rep and UvrD are encoded by Gram-negative bacteria, and mutants lacking both helicases are also not viable. To understand the non-viability of the helicase mutants, we characterized spontaneous extragenic suppressors of a Bacillus subtilis pcrA null mutation. Here we report that one of these suppressors maps in recF and that previously isolated mutations in B.subtilis recF, recL, recO and recR, which belong to the same complementation group, all suppress the lethality of a pcrA mutation. Similarly, recF, recO or recR mutations suppress the lethality of the Escherichia coli rep uvrD double mutant. We conclude that RecFOR proteins are toxic in cells devoid of PcrA in Gram-positive bacteria, or Rep and UvrD in Gram-negative bacteria, and propose that the RecFOR proteins interfere with an essential cellular process, possibly replication, when DExx helicases PcrA, or Rep and UvrD are absent. PMID:12065426

  15. Surface display of recombinant proteins on Escherichia coli by BclA exosporium of Bacillus anthracis

    PubMed Central

    2013-01-01

    Background The anchoring motif is one of the most important aspects of cell surface display as well as efficient and stable display of target proteins. Thus, there is currently a need for the identification and isolation of novel anchoring motifs. Results A system for the display of recombinant proteins on the surface of Escherichia coli was developed using the Bacillus anthracis exosporal protein (BclA) as a new anchoring motif. For the surface display of recombinant proteins, the BAN display platform was constructed in which a target protein is linked to the C-terminus of N-terminal domain (21 amino acids) of BclA. The potential application of BAN platform for cell surface display was demonstrated with two model proteins of different size, the Bacillus sp. endoxylanase (XynA) and monooxygenase (P450 BM3m2). Through experimental analysis including outer membrane fractionation, confocal microscopy and activity assay, it was clearly confirmed that both model proteins were successfully displayed with high activities on the E. coli cell surface. Conclusions These results of this study suggest that the strategy employing the B. anthracis BclA as an anchoring motif is suitable for the display of heterologous proteins on the surface of E. coli and consequently for various biocatalytic applications as well as protein engineering. PMID:24053632

  16. Versatile method for production and controlled polymer-immobilization of biologically active recombinant proteins.

    PubMed

    Allard, Laure; Cheynet, Valérie; Oriol, Guy; Mandrand, Bernard; Delair, Thierry; Mallet, François

    2002-11-05

    The immobilization of a protein by covalent attachment to a support matrix should involve only functional groups of the protein that are not essential for its biological activity. A general strategy for obtaining recombinant proteins designed for oriented covalent grafting onto copolymers was investigated. The rationale involves the definition of seven p24-derived recombinant proteins as fused to either distant or adjacent tags comprising primary amine rich tag consisting of six contiguous lysines suitable for oriented covalent immobilization and a hexa-histidine tag suitable for metal chelate affinity purification. High-level expression, efficient affinity purification, and coupling yields onto maleic anhydride-alt-methyl vinyl ether copolymers higher than 95% were obtained for all proteins. Afterwards, an investigation of the biological features of the immobilized vs. nonimmobilized protein onto the copolymer allowed us to select one bioconjugate which was used in a diagnostic context, i.e., as a capture antigen in an ELISA format test. Sera from 107 HIV-seropositive individuals at various stages of HIV infection, including two seroconversion panels and 104 healthy HIV-seronegative controls, were tested using either RH24 or RK24H-copolymer coated onto the microtiter plate. These assays showed that the use of such a protein-copolymer bioconjugate allowed detection of lower antibody titers than the RH24 protein, illustrating the potential of applications of such doubly tagged proteins. Thus, a set of expression vectors was designed containing four different combinations of hexa-lysine and hexa-histidine tags and a multiple cloning site, allowing the production of different recombinant fusion proteins suitable for biological reactivity conservation after immobilization. Copyright 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 341-348, 2002.

  17. Monitoring the centrifugal recovery of recombinant protein inclusion bodies.

    PubMed

    Middelberg, A P; O'Neill, B K

    1991-04-01

    The industrial processing of proteins expressed as insoluble inclusion bodies employs a reasonably standard sequence of unit operations. One of these is centrifugation, which serves to concentrate the inclusion bodies after disruption of the host microorganism, and also separates the inclusion bodies from other cellular debris. Monitoring the performance of the centrifuge is essential if excessive product and hence financial loss is to be avoided and a reasonable separation obtained. The analytical disc centrifuge may be used to monitor the centrifugation. This instrument returns the sample size distribution with high resolution and without fouling. By obtaining size distributions of the centrifuge feed, supernatant and concentrate, the fractional collection efficiency of the centrifuge may be determined as a function of the Stokes diameter, and a mass balance constructed.

  18. Manipulating corn germplasm to increase recombinant protein accumulation.

    PubMed

    Hood, Elizabeth E; Devaiah, Shivakumar P; Fake, Gina; Egelkrout, Erin; Teoh, Keat Thomas; Requesens, Deborah Vicuna; Hayden, Celine; Hood, Kendall R; Pappu, Kameshwari M; Carroll, Jennifer; Howard, John A

    2012-01-01

    Using plants as biofactories for industrial enzymes is a developing technology. The application of this technology to plant biomass conversion for biofuels and biobased products has potential for significantly lowering the cost of these products because of lower enzyme production costs. However, the concentration of the enzymes in plant tissue must be high to realize this goal. We describe the enhancement of the accumulation of cellulases in transgenic maize seed as a part of the process to lower the cost of these dominant enzymes for the bioconversion process. We have used breeding to move these genes into elite and high oil germplasm to enhance protein accumulation in grain. We have also explored processing of the grain to isolate the germ, which preferentially contains the enzymes, to further enhance recovery of enzyme on a dry weight basis of raw materials. The enzymes are active on microcrystalline cellulose to release glucose and cellobiose.

  19. Nanopatterning of recombinant proteins and viruses using block copolymer templates

    NASA Astrophysics Data System (ADS)

    Cresce, Arthur Von Wald

    The study of interfaces is important in understanding biological interactions, including cellular signaling and virus infection. This thesis is an original effort to examine the interaction between a block copolymer and both a protein and a virus. Block copolymers intrinsically form nanometer-scale structures over large areas without expensive processing, making them ideal for the synthesis of the nanopatterned surfaces used in this study. The geometry of these nanostructures can be easily tuned for different applications by altering the block ratio and composition of the block copolymer. Block copolymers can be used for controlled uptake of metal ions, where one block selectively binds metal ions while the other does not. 5-norbornene-2,3-dicarboxylic acid is synthesized through ring-opening metathesis polymerization. It formed spherical domains with spheres approximately 30 nm in diameter, and these spheres were then subsequently loaded with nickel ion. This norbornene block copolymer was tested for its ability to bind histidine-tagged green fluorescent protein (hisGFP), and it was found that the nickel-loaded copolymer was able to retain hisGFP through chelation between the histidine tag and the metal-containing portions of the copolymer surface. Poly(styrene-b-4-vinylpyridine) (PS/P4VP) was also loaded with nickel, forming a cylindrical microstructure. The binding of Tobacco mosaic virus and Tobacco necrosis virus was tested through Tween 20 detergent washes. Electron microscopy allowed for observation of both block copolymer nanostructures and virus particles. Results showed that Tween washes could not remove bound Tobacco mosaic virus from the surface of PS/P4VP. It was also seen that the size and tunability of block copolymers and the lack of processing needed to attain different structures makes them attractive for many applications, including microfluidic devices, surfaces to influence cellular signaling and growth, and as a nanopatterning surface for

  20. Visualization of Iron-Binding Micelles in Acidic Recombinant Biomineralization Protein, MamC

    DOE PAGES

    Kashyap, Sanjay; Woehl, Taylor; Valverde-Tercedor, Carmen; ...

    2014-01-01

    Biological macromolecules are utilized in low-temperature synthetic methods to exert precise control over nanoparticle nucleation and placement. They enable low-temperature formation of a variety of functional nanostructured materials with properties often not achieved via conventional synthetic techniques. Here we report on the in situ visualization of a novel acidic bacterial recombinant protein, MamC, commonly present in the magnetosome membrane of several magnetotactic bacteria, including Magnetococcus marinus , strain MC-1. Our findings provide an insight into the self-assembly of MamC and point to formation of the extended protein surface, which is assumed to play an important role in the formationmore » of biotemplated inorganic nanoparticles. The self-organization of MamC is compared to the behavior of another acidic recombinant iron-binding protein, Mms6.« less

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

    SciTech Connect

    Khan, Abdul Hamid; Chu, Fuliang; Feng, Youjun; Zhang, Qinagmin; Qi, Jianxun; Gao, George Fu

    2008-08-01

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

  2. 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

  3. Isolation of the gene encoding the Hin recombinational enhancer binding protein.

    PubMed Central

    Johnson, R C; Ball, C A; Pfeffer, D; Simon, M I

    1988-01-01

    In vitro DNA inversion mediated by the protein Hin requires the presence of a recombinational enhancer sequence located in cis relative to the recombination sites and a protein, Fis, which binds to the enhancer. We have cloned and determined the primary sequence of the gene encoding Fis. The deduced amino acid sequence of Fis indicates that the protein is 98 amino acids long and contains a potential helix-turn-helix DNA binding motif at its carboxyl terminus. The gene encoding Fis maps at 72 min on the Escherichia coli chromosome. The construction of mutant strains of E. coli that lack a functional fis gene demonstrates that Fis is not essential for cell growth under laboratory conditions but is required for high rates of Hin-mediated site-specific inversion in vivo. PMID:2835774

  4. Rapid and sensitive detection of recombinant soluble proteins in the supernatant of transfected mammalian cells.

    PubMed

    Gregorini, A; Cinti, C; Pigliapoco, F; Deaglio, S; Ferrero, E; Papa, S; Palma, F

    2002-01-01

    Cloning and expression of recombinant soluble proteins could be quite a difficult task, especially when it comes to reliably detect minute amounts of the soluble protein in the supernatant of transfected mammalian cells. Timing and sensitivity are of the essence in order to optimise the benefits/costs balance and to decide which clones to grow further and which ones to discard. Here we propose a modified inhibition assay. The key feature of this approach is the development of a sensitive and quantitative test to detect the presence of the recombinant soluble protein by exploiting its ability to compete with the binding of a specific monoclonal antibody to a target cell. The described procedure is a sensitive, efficient, dependable and low cost method.

  5. DNA vaccines expressing pneumococcal surface protein A (PspA) elicit protection levels comparable to recombinant protein.

    PubMed

    Ferreira, Daniela M; Miyaji, Eliane N; Oliveira, Maria Leonor S; Darrieux, Michelle; Arêas, Ana Paula M; Ho, Paulo L; Leite, Luciana C C

    2006-04-01

    Pneumococcal surface protein A (PspA) is a promising candidate for the development of cost-effective vaccines against Streptococcus pneumoniae. In the present study, BALB/c mice were immunized with DNA vaccine vectors expressing the N-terminal region of PspA. Animals immunized with a vector expressing secreted PspA developed higher levels of antibody than mice immunized with the vector expressing the antigen in the cytosol. However, both immunogens elicited similar levels of protection against intraperitoneal challenge. Furthermore, immunization with exactly the same fragment in the form of a recombinant protein, with aluminium hydroxide as an adjuvant, elicited even higher antibody levels, but this increased humoral response did not correlate with enhanced protection. These results show that DNA vaccines expressing PspA are able to elicit protection levels comparable to recombinant protein, even though total anti-PspA IgG response is considerably lower.

  6. Lack of protective efficacy in buffaloes vaccinated with Fasciola gigantica leucine aminopeptidase and peroxiredoxin recombinant proteins.

    PubMed

    Raina, O K; Nagar, Gaurav; Varghese, Anju; Prajitha, G; Alex, Asha; Maharana, B R; Joshi, P

    2011-06-01

    Gene coding for leucine aminopeptidase (LAP), a metalloprotease, was identified in the tropical liver fluke, Fasciola gigantica; that on sequence analysis showed a close homology (98.6%) with leucine aminopeptidase of the temperate liver fluke, Fasciola hepatica. The recombinant leucine aminopeptidase protein was expressed in Escherichia coli. F. gigantica peroxiredoxin, a hydrogen peroxide scavenger and an immunomodulating protein, was also cloned and expressed in E. coli. A vaccination trial in buffaloes was conducted with these two recombinant proteins, with 150 and 300 μg of leucine aminopeptidase and a cocktail of 150 μg each of recombinant leucine aminopeptidase and peroxiredoxin in three groups, respectively. Both Th1- and Th2-associated humoral immune responses were elicited to immunization with these antigens. A challenge study with 400 metacercariae did not show a significant protection in terms of reduction in the worm burden (8.4%) or anti-fecundity/embryonation effect in the immunized groups, as to the non-immunized control animals. Our observations in this buffalo vaccination trial are contrary to the earlier promise shown by leucine aminopeptidase of F. hepatica as a leading candidate vaccine molecule. Identification of leucine aminopeptidase gene and evaluation of the protein for its protective efficacy in buffaloes is the first scientific report on this protein in F. gigantica.

  7. Reflectometric interference spectroscopy-based immunosensing using immobilized antibody via His-tagged recombinant protein A.

    PubMed

    Choi, Hyung Woo; Sakata, Yasuhiko; Ooya, Tooru; Takeuchi, Toshifumi

    2015-02-01

    The proposed approach demonstrated in this study provides an immunosensing system based on reflectometric interference spectroscopy (RIfS) in combination with an antibody immobilization method using histidine-tagged recombinant protein A. Carboxymethyldextran (CMD) was immobilized on a 3-aminopropyltriethoxysilane-treated a silicon nitride-coated silicon wafer, followed by chelating histidine-tagged recombinant protein A with copper (II) ions. The CMD-layer was found to be advantageous in terms of not only immobilization of histidine-tagged recombinant protein A-mediated an antibody against myoglobin (anti-Myo) but also prevention of non-specific binding of myoglobin. Myoglobin was repeatedly detected, and the apparent detection limit was 0.1 μg mL(-1). The proposed RIfS-based protein sensing system, in conjunction with the easy preparation of silicon-based inexpensive immunosensing chips, is expected to be applicable for label-free optical detection for other proteins in various fields. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  8. Recombinant TNF-binding protein from variola virus as a novel potential TNF antagonist.

    PubMed

    Gileva, I P; Nepomnyashchikh, T S; Ryazankin, I A; Shchelkunov, S N

    2009-12-01

    Gel-filtration chromatographic separation of the lysate of Sf21 insect cells infected with recombinant baculovirus BVi67 containing the gene for TNF-binding protein (CrmB) of variola virus (VARV) revealed that hTNF-cytotoxicity neutralization activity is associated with a fraction corresponding mainly to high molecular weight proteins (above 500 kDa) and less with fractions corresponding to proteins of 270 or 90 kDa. The recombinant VARV-CrmB protein has been purified by affinity chromatography. Difference in the experimentally determined and estimated (according to amino acid composition) VARV-CrmB molecular weight is due to glycosylation of the recombinant protein expressed in the insect cells. VARV-CrmB neutralizes in vitro the cytotoxic effect of hTNF and hLTalpha, and its TNF-neutralizing activity is two to three orders of magnitude higher compared to the analogous effects of type I and II soluble TNF receptors, comparable with the activity of mAb MAK195, and somewhat lower than the effect of the commercial drug Remicade.

  9. Replication Checkpoint Kinase Cds1 Regulates Recombinational Repair Protein Rad60

    PubMed Central

    Boddy, Michael N.; Shanahan, Paul; Hayes McDonald, W.; Lopez-Girona, Antonia; Noguchi, Eishi; Yates III, John R.; Russell, Paul

    2003-01-01

    Genome integrity is protected by Cds1 (Chk2), a checkpoint kinase that stabilizes arrested replication forks. How Cds1 accomplishes this task is unknown. We report that Cds1 interacts with Rad60, a protein required for recombinational repair in fission yeast. Cds1 activation triggers Rad60 phosphorylation and nuclear delocalization. A Rad60 mutant that inhibits regulation by Cds1 renders cells specifically sensitive to replication fork arrest. Genetic and biochemical studies indicate that Rad60 functions codependently with Smc5 and Smc6, subunits of an SMC (structural maintenance of chromosomes) complex required for recombinational repair. These studies indicate that regulation of Rad60 is an important part of the replication checkpoint response controlled by Cds1. We propose that control of Rad60 regulates recombination events at stalled forks. PMID:12897162

  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. A generalised module for the selective extracellular accumulation of recombinant proteins

    PubMed Central

    2012-01-01

    Background It is widely believed that laboratory strains of Escherichia coli, including those used for industrial production of proteins, do not secrete proteins to the extracellular milieu. Results Here, we report the development of a generalised module, based on an E. coli autotransporter secretion system, for the production of extracellular recombinant proteins. We demonstrate that a wide variety of structurally diverse proteins can be secreted as soluble proteins when linked to the autotransporter module. Yields were comparable to those achieved with other bacterial secretion systems. Conclusions The advantage of this module is that it relies on a relatively simple and easily manipulated secretion system, exhibits no apparent limitation to the size of the secreted protein and can deliver proteins to the extracellular environment at levels of purity and yields sufficient for many biotechnological applications. PMID:22640772

  12. Refolding and simultaneous purification by three-phase partitioning of recombinant proteins from inclusion bodies

    PubMed Central

    Raghava, Smita; Barua, Bipasha; Singh, Pradeep K.; Das, Mili; Madan, Lalima; Bhattacharyya, Sanchari; Bajaj, Kanika; Gopal, B.; Varadarajan, Raghavan; Gupta, Munishwar N.

    2008-01-01

    Many recombinant eukaryotic proteins tend to form insoluble aggregates called inclusion bodies, especially when expressed in Escherichia coli. We report the first application of the technique of three-phase partitioning (TPP) to obtain correctly refolded active proteins from solubilized inclusion bodies. TPP was used for refolding 12 different proteins overexpressed in E. coli. In each case, the protein refolded by TPP gave either higher refolding yield than the earlier reported method or succeeded where earlier efforts have failed. TPP-refolded proteins were characterized and compared to conventionally purified proteins in terms of their spectral characteristics and/or biological activity. The methodology is scaleable and parallelizable and does not require subsequent concentration steps. This approach may serve as a useful complement to existing refolding strategies of diverse proteins from inclusion bodies. PMID:18780821

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

    PubMed

    Sarpong, Kwabena; Bose, Ron

    2017-03-15

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

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

    PubMed Central

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

    1996-01-01

    Two myxoma virus-rabbit hemorrhagic disease virus (RHDV) recombinant viruses were constructed with the SG33 strain of myxoma virus to protect rabbits against myxomatosis and rabbit viral hemorrhagic disease. These recombinant viruses expressed the RHDV capsid protein (VP60). The recombinant protein, which is 60 kDa in size, was antigenic, as revealed by its reaction in immunoprecipitation with antibodies raised against RHDV. Both recombinant viruses induced high levels of RHDV- and myxoma virus-specific antibodies in rabbits after immunization. Inoculations by the intradermal route protected animals against virulent RHDV and myxoma virus challenges. PMID:8764013

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

    PubMed

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

    1996-08-01

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

  16. 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

  17. 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.

  18. Immunoproteomics using polyclonal antibodies and stable isotope-labeled affinity-purified recombinant proteins.

    PubMed

    Edfors, Fredrik; Boström, Tove; Forsström, Björn; Zeiler, Marlis; Johansson, Henrik; Lundberg, Emma; Hober, Sophia; Lehtiö, Janne; Mann, Matthias; Uhlen, Mathias

    2014-06-01

    The combination of immuno-based methods and mass spectrometry detection has great potential in the field of quantitative proteomics. Here, we describe a new method (immuno-SILAC) for the absolute quantification of proteins in complex samples based on polyclonal antibodies and stable isotope-labeled recombinant protein fragments to allow affinity enrichment prior to mass spectrometry analysis and accurate quantification. We took advantage of the antibody resources publicly available from the Human Protein Atlas project covering more than 80% of all human protein-coding genes. Epitope mapping revealed that a majority of the polyclonal antibodies recognized multiple linear epitopes, and based on these results, a semi-automated method was developed for peptide enrichment using polyclonal antibodies immobilized on protein A-coated magnetic beads. A protocol based on the simultaneous multiplex capture of more than 40 protein targets showed that approximately half of the antibodies enriched at least one functional peptide detected in the subsequent mass spectrometry analysis. The approach was further developed to also generate quantitative data via the addition of heavy isotope-labeled recombinant protein fragment standards prior to trypsin digestion. Here, we show that we were able to use small amounts of antibodies (50 ng per target) in this manner for efficient multiplex analysis of quantitative levels of proteins in a human HeLa cell lysate. The results suggest that polyclonal antibodies generated via immunization of recombinant protein fragments could be used for the enrichment of target peptides to allow for rapid mass spectrometry analysis taking advantage of a substantial reduction in sample complexity. The possibility of building up a proteome-wide resource for immuno-SILAC assays based on publicly available antibody resources is discussed. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. 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

  20. Chromosome rearrangements, recombination suppression, and limited segregation distortion in hybrids between Yellowstone cutthroat trout (Oncorhynchus clarkii bouvieri) and rainbow trout (O. mykiss).

    PubMed

    Ostberg, Carl O; Hauser, Lorenz; Pritchard, Victoria L; Garza, John C; Naish, Kerry A

    2013-08-22

    Introgressive hybridization is an important evolutionary process that can lead to the creation of novel genome structures and thus potentially new genetic variation for selection to act upon. On the other hand, hybridization with introduced species can threaten native species, such as cutthroat trout (Oncorhynchus clarkii) following the introduction of rainbow trout (O. mykiss). Neither the evolutionary consequences nor conservation implications of rainbow trout introgression in cutthroat trout is well understood. Therefore, we generated a genetic linkage map for rainbow-Yellowstone cutthroat trout (O. clarkii bouvieri) hybrids to evaluate genome processes that may help explain how introgression affects hybrid genome evolution. The hybrid map closely aligned with the rainbow trout map (a cutthroat trout map does not exist), sharing all but one linkage group. This linkage group (RYHyb20) represented a fusion between an acrocentric (Omy28) and a metacentric chromosome (Omy20) in rainbow trout. Additional mapping in Yellowstone cutthroat trout indicated the two rainbow trout homologues were fused in the Yellowstone genome. Variation in the number of hybrid linkage groups (28 or 29) likely depended on a Robertsonian rearrangement polymorphism within the rainbow trout stock. Comparison between the female-merged F₁ map and a female consensus rainbow trout map revealed that introgression suppressed recombination across large genomic regions in 5 hybrid linkage groups. Two of these linkage groups (RYHyb20 and RYHyb25_29) contained confirmed chromosome rearrangements between rainbow and Yellowstone cutthroat trout indicating that rearrangements may suppress recombination. The frequency of allelic and genotypic segregation distortion varied among parents and families, suggesting few incompatibilities exist between rainbow and Yellowstone cutthroat trout genomes. Chromosome rearrangements suppressed recombination in the hybrids. This result supports several previous

  1. Chromosome rearrangements, recombination suppression, and limited segregation distortion in hybrids between Yellowstone cutthroat trout (Oncorhynchus clarkii bouvieri) and rainbow trout (O. mykiss)

    PubMed Central

    2013-01-01

    Background Introgressive hybridization is an important evolutionary process that can lead to the creation of novel genome structures and thus potentially new genetic variation for selection to act upon. On the other hand, hybridization with introduced species can threaten native species, such as cutthroat trout (Oncorhynchus clarkii) following the introduction of rainbow trout (O. mykiss). Neither the evolutionary consequences nor conservation implications of rainbow trout introgression in cutthroat trout is well understood. Therefore, we generated a genetic linkage map for rainbow-Yellowstone cutthroat trout (O. clarkii bouvieri) hybrids to evaluate genome processes that may help explain how introgression affects hybrid genome evolution. Results The hybrid map closely aligned with the rainbow trout map (a cutthroat trout map does not exist), sharing all but one linkage group. This linkage group (RYHyb20) represented a fusion between an acrocentric (Omy28) and a metacentric chromosome (Omy20) in rainbow trout. Additional mapping in Yellowstone cutthroat trout indicated the two rainbow trout homologues were fused in the Yellowstone genome. Variation in the number of hybrid linkage groups (28 or 29) likely depended on a Robertsonian rearrangement polymorphism within the rainbow trout stock. Comparison between the female-merged F1 map and a female consensus rainbow trout map revealed that introgression suppressed recombination across large genomic regions in 5 hybrid linkage groups. Two of these linkage groups (RYHyb20 and RYHyb25_29) contained confirmed chromosome rearrangements between rainbow and Yellowstone cutthroat trout indicating that rearrangements may suppress recombination. The frequency of allelic and genotypic segregation distortion varied among parents and families, suggesting few incompatibilities exist between rainbow and Yellowstone cutthroat trout genomes. Conclusions Chromosome rearrangements suppressed recombination in the hybrids. This result

  2. BTK gene targeting by homologous recombination using a helper-dependent adenovirus/adeno-associated virus hybrid vector.

    PubMed

    Yamamoto, H; Ishimura, M; Ochiai, M; Takada, H; Kusuhara, K; Nakatsu, Y; Tsuzuki, T; Mitani, K; Hara, T

    2016-02-01

    X-linked agammaglobulinemia (XLA) is one of the most common humoral immunodeficiencies, which is caused by mutations in Bruton's tyrosine kinase (BTK) gene. To examine the possibility of using gene therapy for XLA, we constructed a helper-dependent adenovirus/adeno-associated virus BTK targeting vector (HD-Ad.AAV BTK vector) composed of a genomic sequence containing BTK exons 6-19 and a green fluorescence protein-hygromycin cassette driven by a cytomegalovirus promoter. We first used NALM-6, a human male pre-B acute lymphoblastic leukemia cell line, as a recipient to measure the efficiency of gene targeting by homologous recombination. We identified 10 clones with the homologous recombination of the BTK gene among 107 hygromycin-resistant stable clones isolated from two independent experiments. We next used cord blood CD34⁺ cells as the recipient cells for the gene targeting. We isolated colonies grown in medium containing cytokines and hygromycin. We found that the targeting of the BTK gene occurred in four of the 755 hygromycin-resistant colonies. Importantly, the gene targeting was also observed in CD19⁺ lymphoid progenitor cells that were differentiated from the homologous recombinant CD34⁺ cells during growth in selection media. Our study shows the potential for the BTK gene therapy using the HD-Ad.AAV BTK vector via homologous recombination in hematopoietic stem cells.

  3. Aligning multiple protein sequences by parallel hybrid genetic algorithm.

    PubMed

    Nguyen, Hung Dinh; Yoshihara, Ikuo; Yamamori, Kunihito; Yasunaga, Moritoshi

    2002-01-01

    This paper presents a parallel hybrid genetic algorithm (GA) for solving the sum-of-pairs multiple protein sequence alignment. A new chromosome representation and its corresponding genetic operators are proposed. A multi-population GENITOR-type GA is combined with local search heuristics. It is then extended to run in parallel on a multiprocessor system for speeding up. Experimental results of benchmarks from the BAliBASE show that the proposed method is superior to MSA, OMA, and SAGA methods with regard to quality of solution and running time. It can be used for finding multiple sequence alignment as well as testing cost functions.

  4. 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.

  5. Polar Fixation of Plasmids during Recombinant Protein Production in Bacillus megaterium Results in Population Heterogeneity.

    PubMed

    Münch, Karin M; Müller, Johannes; Wienecke, Sarah; Bergmann, Simone; Heyber, Steffi; Biedendieck, Rebekka; Münch, Richard; Jahn, Dieter

    2015-09-01

    During the past 2 decades, Bacillus megaterium has been systematically developed for the gram-per-liter scale production of recombinant proteins. The plasmid-based expression systems employed use a xylose-controlled promoter. Protein production analyses at the single-cell level using green fluorescent protein as a model product revealed cell culture heterogeneity characterized by a significant proportion of less productive bacteria. Due to the enormous size of B. megaterium, such bistable behavior seen in subpopulations was readily analyzed by time lapse microscopy and flow cytometry. Cell culture heterogeneity was not caused simply by plasmid loss: instead, an asymmetric distribution of plasmids during cell division was detected during the exponential-growth phase. Multicopy plasmids are generally randomly distributed between daughter cells. However, in vivo and in vitro experiments demonstrated that under conditions of strong protein production, plasmids are retained at one of the cell poles. Furthermore, it was found that cells with accumulated plasmids and high protein production ceased cell division. As a consequence, the overall protein production of the culture was achieved mainly by the subpopulation with a sufficient plasmid copy number. Based on our experimental data, we propose a model whereby the distribution of multicopy plasmids is controlled by polar fixation under protein production conditions. Thereby, cell lines with fluctuating plasmid abundance arise, which results in population heterogeneity. Our results provide initial insights into the mechanism of cellular heterogeneity during plasmid-based recombinant protein production in a Bacillus species.

  6. Optical determination of Shockley-Read-Hall and interface recombination currents in hybrid perovskites

    PubMed Central

    Sarritzu, Valerio; Sestu, Nicola; Marongiu, Daniela; Chang, Xueqing; Masi, Sofia; Rizzo, Aurora; Colella, Silvia; Quochi, Francesco; Saba, Michele; Mura, Andrea; Bongiovanni, Giovanni

    2017-01-01

    Metal-halide perovskite solar cells rival the best inorganic solar cells in power conversion efficiency, providing the outlook for efficient, cheap devices. In order for the technology to mature and approach the ideal Shockley-Queissier efficiency, experimental tools are needed to diagnose what processes limit performances, beyond simply measuring electrical characteristics often affected by parasitic effects and difficult to interpret. Here we study the microscopic origin of recombination currents causing photoconversion losses with an all-optical technique, measuring the electron-hole free energy as a function of the exciting light intensity. Our method allows assessing the ideality factor and breaks down the electron-hole recombination current into bulk defect and interface contributions, providing an estimate of the limit photoconversion efficiency, without any real charge current flowing through the device. We identify Shockley-Read-Hall recombination as the main decay process in insulated perovskite layers and quantify the additional performance degradation due to interface recombination in heterojunctions. PMID:28317883

  7. Recombination sites on hybrid chromosomes in Saccharomyces pastorianus share common sequence motifs and define a complex evolutionary relationship between group I and II lager yeasts.

    PubMed

    Monerawela, Chandre; Bond, Ursula

    2017-08-01

    Saccharomyces pastorianus, referred to as lager yeasts, are hybrids of S. cerevisiae and S. eubayanus. Isolates within the species are divided into two groups (I and II) based on chromosome structure and composition. Following the hybridisation, the parental chromosomes underwent homeologous recombination, generating a set of hybrid chromosomes unique to the species. Here, we assessed the recombination events in seven lager yeast genomes to more clearly define the evolutionary route of lager yeasts. Meta-analysis of the recombination epicentres, as well as a detailed analysis of recombination events at the MAT locus, reveals a more complex evolutionary relationship between the group I and II lager yeasts than previously considered and identifies several divergent routes of evolution leading to the current S. pastorianus strains. We show that recombination epicentres contain sequential runs of pyrimidines, often flanked by purines, on one strand of the DNA, and identify two common sequence motifs present in >80% of the recombination epicentres, indicating that a common mechanism might account for the recombination events. Taken together, the data support a sequential hybridisation model of evolution for the two types of lager yeasts and suggest that the genomes of this newly emerged species are highly dynamic and continually evolving. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  8. Transgenic rabbits for the production of biologically-active recombinant proteins in the milk.

    PubMed

    Castro, F O; Limonta, J; Rodriguez, A; Aguirre, A; de la Fuente, J; Aguilar, A; Ramos, B; Hayes, O

    1999-11-01

    The use of live bioreactors for the expression of human genes in the mammary gland of transgenic animals is one of the most cost-effective ways for the production of valuable recombinant therapeutic proteins. Among the transgenic species used so far, rabbits are good candidates for the expression of tens to hundreds of grams of complex proteins in the milk during lactation. The lactating mammary gland of rabbits has proven to be effective in the processing of complex proteins. In this work. the potential use of rabbits as bioreactors is discussed based on our results and the published data.

  9. A facile method for reversibly linking a recombinant protein to DNA.

    PubMed

    Goodman, Russell P; Erben, Christoph M; Malo, Jonathan; Ho, Wei M; McKee, Mireya L; Kapanidis, Achillefs N; Turberfield, Andrew J

    2009-06-15

    We present a facile method for linking recombinant proteins to DNA. It is based on the nickel-mediated interaction between a hexahistidine tag (His(6)-tag) and DNA functionalized with three nitrilotriacetic acid (NTA) groups. The resulting DNA-protein linkage is site-specific. It can be broken quickly and controllably by the addition of a chelating agent that binds nickel. We have used this new linker to bind proteins to a variety of DNA motifs commonly used in the fabrication of nanostructures by DNA self-assembly.

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

    PubMed

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

    2017-04-01

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

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

    PubMed Central

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

    2017-01-01

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

  12. 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.

  13. Purification and characterization of recombinant supersweet protein thaumatin II from tomato fruit.

    PubMed

    Firsov, Aleksey; Shaloiko, Lyubov; Kozlov, Oleg; Vinokurov, Leonid; Vainstein, Alexander; Dolgov, Sergey

    2016-07-01

    Thaumatin, a supersweet protein from the African plant katemfe (Thaumatococcus daniellii Benth.), is a promising zero-calorie sweetener for use in the food and pharmaceutical industries. Due to limited natural sources of thaumatin, its production using transgenic plants is an advantageous alternative. We report a simple protocol for purification of recombinant thaumatin II from transgenic tomato. Thaumatin was extracted from ripe tomato fruit in a low-salt buffer and purified on an SP-Sephacryl column. Recombinant thaumatin yield averaged 50 mg/kg fresh fruit. MALDI-MS analysis showed correct processing of thaumatin in tomato plants. The recombinant thaumatin was indistinguishable from the native protein in a taste test. The purified tomato-derived thaumatin had an intrinsic sweetness with a threshold value in taste tests of around 50 nM. These results demonstrate the potential of an expression system based on transgenic tomato plants for production of recombinant thaumatin for the food and pharmaceutical industries. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. 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.

  15. 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

  16. 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

  17. The extent and position of homoeologous recombination in a distant hybrid of Alstroemeria: a molecular cytogenetic assessment of first generation backcross progenies.

    PubMed

    Kamstra, S A; Kuipers, A G; De Jeu, M J; Ramanna, M S; Jacobsen, E

    1999-04-01

    To estimate the extent and position of homoeologous recombination during meiosis in an interspecific hybrid between two distantly related Alstroemeria species, the chromosome constitution of six first generation backcross (BC1) plants was analysed using sequential fluorescent in situ hybridization (FISH) and genomic in situ hybridization (GISH) analysis. Four different probes were used for the FISH analysis: two species-specific and two rDNA probes. The six BC1 plants were obtained from crosses between the hybrid A. aurea x A. inodora with its parent A. inodora. GISH clearly identified all chromosomes of both parental genomes as well as recombinant chromosomes. The sequential GISH and FISH analysis enabled the accurate identification of all individual chromosomes in the BC1 plants, resulting in the construction of detailed karyotypes of the plants. The identification of the recombinant chromosomes provided evidence which chromosomes of the two species are homoeologous. Two of the BC1 plants were aneuploid (2n=2x+1=17) and four triploid (2n=3x=24), indicating that both n and 2n gametes were functional in the F1 hybrid. Using GISH, it was possible to estimate homeologous recombination in two different types of gametes in the F1 hyrid. The positions of the crossover points ranged from highly proximal to distal and the maximum number of crossover points per chromosome arm was three. Compared with the aneuploid plants, the triploid plants (which received 2n gametes) clearly possessed fewer crossovers per chromosome, indicating reduced chromosome pairing/recombination prior to the formation of the 2n gametes. Besides homeologous recombination, evidence was found for the presence of structural rearrangements (inversion and translocation) between the chromosomes of the parental species. The presence of the ancient translocation was confirmed through FISH analysis of mitotic and meiotic chromosomes.

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

    PubMed

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

    2015-12-01

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

  19. Hepatoma-derived growth factor-related protein 2 promotes DNA repair by homologous recombination

    PubMed Central

    Baude, Annika; Aaes, Tania Løve; Zhai, Beibei; Al-Nakouzi, Nader; Oo, Htoo Zarni; Daugaard, Mads; Rohde, Mikkel; Jäättelä, Marja

    2016-01-01

    We have recently identified lens epithelium-derived growth factor (LEDGF/p75, also known as PSIP1) as a component of the homologous recombination DNA repair machinery. Through its Pro-Trp-Trp-Pro (PWWP) domain, LEDGF/p75 binds to histone marks associated with active transcription and promotes DNA end resection by recruiting DNA endonuclease retinoblastoma-binding protein 8 (RBBP8/CtIP) to broken DNA ends. Here we show that the structurally related PWWP domain-containing protein, hepatoma-derived growth factor-related protein 2 (HDGFRP2), serves a similar function in homologous recombination repair. Its depletion compromises the survival of human U2OS osteosarcoma and HeLa cervix carcinoma cells and impairs the DNA damage-induced phosphorylation of replication protein A2 (RPA2) and the recruitment of DNA endonuclease RBBP8/CtIP to DNA double strand breaks. In contrast to LEDGF/p75, HDGFRP2 binds preferentially to histone marks characteristic for transcriptionally silent chromatin. Accordingly, HDGFRP2 is found in complex with the heterochromatin-binding chromobox homologue 1 (CBX1) and Pogo transposable element with ZNF domain (POGZ). Supporting the functionality of this complex, POGZ-depleted cells show a similar defect in DNA damage-induced RPA2 phosphorylation as HDGFRP2-depleted cells. These data suggest that HDGFRP2, possibly in complex with POGZ, recruits homologous recombination repair machinery to damaged silent genes or to active genes silenced upon DNA damage. PMID:26721387

  20. Towards a More Precise Serological Diagnosis of Human Tegumentary Leishmaniasis Using Leishmania Recombinant Proteins

    PubMed Central

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

    2013-01-01

    Background Exposure to Leishmania induces a humoral immune response that can be used as a marker of parasite exposure. Methodology/Principal Findings Herein, ELISA was used to screen sera from patients with Tegumentary Leishmaniasis (TL) against different L. infantum-chagasi-derived recombinant proteins (rHSP70, rH2A, rH2B, rH3, rH4 and rKMP11). Among the recombinant proteins, rHSP70 and rH2A showed the best reactivity against human sera obtained from endemic areas of TL. Receiver-Operator Characteristics (ROC) curve analysis was used to identify the effectiveness of these proteins for serodiagnosis of TL. ROC curves confirmed the superior performance of rHSP70 and rH2A, in comparison to the other tested recombinant proteins. Additionally, we evaluated the specificity of the response to rHSP70 and rH2A by testing sera obtained from patients with Chagas' disease, Tuberculosis, Leprosy or Systemic Lupus Erythematosus. In this case, rHSP70 displayed an increased ability to discriminate diseases, in comparison to SLA. Conclusion Our results raise possibility of using rHSP70 for the serodiagnosis of TL PMID:23776617

  1. Recombinant Protein-Stabilized Monodisperse Microbubbles with Tunable Size Using a Valve-Based Microfluidic Device

    PubMed Central

    2015-01-01

    Microbubbles are used as contrast enhancing agents in ultrasound sonography and more recently have shown great potential as theranostic agents that enable both diagnostics and therapy. Conventional production methods lead to highly polydisperse microbubbles, which compromise the effectiveness of ultrasound imaging and therapy. Stabilizing microbubbles with surfactant molecules that can impart functionality and properties that are desirable for specific applications would enhance the utility of microbubbles. Here we generate monodisperse microbubbles with a large potential for functionalization by combining a microfluidic method and recombinant protein technology. Our microfluidic device uses an air-actuated membrane valve that enables production of monodisperse microbubbles with narrow size distribution. The size of microbubbles can be precisely tuned by dynamically changing the dimension of the channel using the valve. The microbubbles are stabilized by an amphiphilic protein, oleosin, which provides versatility in controlling the functionalization of microbubbles through recombinant biotechnology. We show that it is critical to control the composition of the stabilizing agents to enable formation of highly stable and monodisperse microbubbles that are echogenic under ultrasound insonation. Our protein-shelled microbubbles based on the combination of microfluidic generation and recombinant protein technology provide a promising platform for ultrasound-related applications. PMID:25265041

  2. Recombination between Streptococcus suis ICESsu32457 and Streptococcus agalactiae ICESa2603 yields a hybrid ICE transferable to Streptococcus pyogenes.

    PubMed

    Marini, Emanuela; Palmieri, Claudio; Magi, Gloria; Facinelli, Bruna

    2015-07-09

    Integrative conjugative elements (ICEs) are mobile genetic elements that reside in the chromosome but retain the ability to undergo excision and to transfer by conjugation. Genes involved in drug resistance, virulence, or niche adaptation are often found among backbone genes as cargo DNA. We recently characterized in Streptococcus suis an ICE (ICESsu32457) carrying resistance genes [tet(O/W/32/O), tet(40), erm(B), aphA, and aadE] in the 15K unstable genetic element, which is flanked by two ∼1.3kb direct repeats. Remarkably, ∼1.3-kb sequences are conserved in ICESa2603 of Streptococcus agalactiae 2603V/R, which carry heavy metal resistance genes cadC/cadA and mer. In matings between S. suis 32457 (donor) and S. agalactiae 2603V/R (recipient), transconjugants were obtained. PCR experiments, PFGE, and sequence analysis of transconjugants demonstrated a tandem array between ICESsu32457 and ICESa2603. Matings between tandem array-containing S. agalactiae 2603V/R (donor) and Streptococcus pyogenes RF12 (recipient) yielded a single transconjugant containing a hybrid ICE, here named ICESa2603/ICESsu32457. The hybrid formed by recombination of the left ∼1.3-kb sequence of ICESsu32457 and the ∼1.3-kb sequence of ICESa2603. Interestingly, the hybrid ICE was transferable between S. pyogenes strains, thus demonstrating that it behaves as a conventional ICE. These findings suggest that both tandem arrays and hybrid ICEs may contribute to the evolution of antibiotic resistance in streptococci, creating novel mobile elements capable of disseminating new combinations of antibiotic resistance genes.

  3. Recombination-induced tag exchange to track old and new proteins

    PubMed Central

    Verzijlbergen, Kitty F.; Menendez-Benito, Victoria; van Welsem, Tibor; van Deventer, Sjoerd J.; Lindstrom, Derek L.; Ovaa, Huib; Neefjes, Jacques; Gottschling, Daniel E.; van Leeuwen, Fred

    2009-01-01

    The dynamic behavior of proteins is critical for cellular homeostasis. However, analyzing dynamics of proteins and protein complexes in vivo has been difficult. Here we describe recombination-induced tag exchange (RITE), a genetic method that induces a permanent epitope-tag switch in the coding sequence after a hormone-induced activation of Cre recombinase. The time-controlled tag switch provides a unique ability to detect and separate old and new proteins in time and space, which opens up opportunities to investigate the dynamic behavior of proteins. We validated the technology by determining exchange of endogenous histones in chromatin by biochemical methods and by visualizing and quantifying replacement of old by new proteasomes in single cells by microscopy. RITE is widely applicable and allows probing spatiotemporal changes in protein properties by multiple methods. PMID:20018668

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

    NASA Astrophysics Data System (ADS)

    Borjigin, Jimo; Nathans, Jeremy

    1993-01-01

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

  5. Engineering formation of multiple recombinant Eut protein nanocompartments in E. coli

    PubMed Central

    Held, Mark; Kolb, Alexander; Perdue, Sarah; Hsu, Szu-Yi; Bloch, Sarah E.; Quin, Maureen B.; Schmidt-Dannert, Claudia

    2016-01-01

    Compartmentalization of designed metabolic pathways within protein based nanocompartments has the potential to increase reaction efficiency in multi-step biosynthetic reactions. We previously demonstrated proof-of-concept of this aim by targeting a functional enzyme to single cellular protein nanocompartments, which were formed upon recombinant expression of the Salmonella enterica LT2 ethanolamine utilization bacterial microcompartment shell proteins EutS or EutSMNLK in Escherichia coli. To optimize this system, increasing overall encapsulated enzyme reaction efficiency, factor(s) required for the production of more than one nanocompartment per cell must be identified. In this work we report that the cupin domain protein EutQ is required for assembly of more than one nanocompartment per cell. Overexpression of EutQ results in multiple nanocompartment assembly in our recombinant system. EutQ specifically interacts with the shell protein EutM in vitro via electrostatic interactions with the putative cytosolic face of EutM. These findings lead to the theory that EutQ could facilitate multiple nanocompartment biogenesis by serving as an assembly hub for shell proteins. This work offers insights into the biogenesis of Eut bacterial microcompartments, and also provides an improved platform for the production of protein based nanocompartments for targeted encapsulation of enzyme pathways. PMID:27063436

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

    PubMed

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

    2017-01-01

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

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

    PubMed

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

    2013-05-01

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

  8. [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.

  9. [Recombinant expression of hantaan virus protein N with application of Western-blot in detecting anti-hantavirus antibody].

    PubMed

    Yao, P P; Xu, F; Sun, Y S; Yang, Z R; Zhang, Y; Yue, M; Zhu, H P

    2017-04-10

    Objective: S gene of hantavirus(HV) was expressed in insect cells by genetic engineering technology. The expression product of S gene was used as antigen to detect anti-HV specific antibody IgG in serum. Methods: Gene encoding NP of the strain HV-Z10 was amplified by PCR and then its eukaryotic expression system rBAC-Z10S-TN was constructed by using the routine genetic engineering method. SDS-PAGE was applied to measure the expression of rNP.Ion-exchange plus Ni-NTA-affinity chromatography was performed to purify the recombinant product. Indirect immuno-fluorescence assay (IFA) was used to determine the specific immune-reactivity of rNP. WB assay was established to detect the serum samples from 95 confirmed HFRS patients. Parameters related to the outcomes of detection were compared with the routine HV-IgG IFA method. Results: rBAC-Z10S-TN was able to express rNP with high efficiency. The purified rNP only showed a single protein fragment in the gel after SDS-PAGE. HV IgG could efficiently recognize rNP and hybridize with the recombinant protein. 97.67% of the serum samples from the HFRS patients were positive confirmed by WB. Conclusions: We successfully constructed a high efficient prokaryotic expression system of NP encoding gene from hantavirus strain HV-Z10. WB assay which was established in this study could be used as a new serological test for HFRS diagnosis, thanks to the simplicity, safety, sensitivity and specificity of this method.

  10. Antimicrobial activity of snakin-defensin hybrid protein in tobacco and potato plants

    USDA-ARS?s Scientific Manuscript database

    To augment plant protection against phytopathogens, we constructed a fusion gene for the simultaneous expression of snakin-1 (SN1) and defensin-1 (PTH1) antimicrobial proteins as a hybrid protein (SAP) in plant cells. Prior to in vivo evaluation of SAP phytoprotective activity, the hybrid protein ex...

  11. 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.

  12. Novel protein interactions with an actin homolog (MreB) of Helicobacter pylori determined by bacterial two-hybrid system.

    PubMed

    Zepeda Gurrola, Reyna Cristina; Fu, Yajuan; Rodríguez Luna, Isabel Cristina; Benítez Cardoza, Claudia Guadalupe; López López, María de Jesús; López Vidal, Yolanda; Gutíerrez, Germán Rubén Aguilar; Rodríguez Pérez, Mario A; Guo, Xianwu

    2017-08-01

    The bacterium Helicobacter pylori infects more than 50% of the world population and causes several gastroduodenal diseases, including gastric cancer. Nevertheless, we still need to explore some protein interactions that may be involved in pathogenesis. MreB, an actin homolog, showed some special characteristics in previous studies, indicating that it could have different functions. Protein functions could be realized via protein-protein interactions. In the present study, the MreB protein from H. pylori 26695 fused with two tags 10×His and GST in tandem was overexpressed and purified from Escherchia coli. The purified recombinant protein was used to perform a pull-down assay with H. pylori 26695 cell lysate. The pulled-down proteins were identified by mass spectrometry (MALDI-TOF), in which the known important proteins related to morphogenesis were absent but several proteins related to pathogenesis process were observed. The bacterial two-hybrid system was further used to evaluate the protein interactions and showed that new interactions of MreB respectively with VacA, UreB, HydB, HylB and AddA were confirmed but the interaction MreB-MreC was not validated. These results indicated that the protein MreB in H. pylori has a distinct interactome, does not participate in cell morphogenesis via MreB-MreC but could be related to pathogenesis. Copyright © 2017 Elsevier GmbH. All rights reserved.

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

    PubMed Central

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

    2006-01-01

    The process of recombinational repair is crucial for maintaining genomic integrity and generating biological diversity. In association with RuvB and RuvC, RuvA plays a central role in processing and resolving Holliday junctions, which are a critical intermediate in homologous recombination. Here, the cloning, purification and structure determination of the RuvA protein from Mycobacterium tuberculosis (MtRuvA) are reported. Analysis of the structure and comparison with other known RuvA proteins reveal an octameric state with conserved subunit–subunit interaction surfaces, indicating the requirement of octamer formation for biological activity. A detailed analysis of plasticity in the RuvA molecules has led to insights into the invariant and variable regions, thus providing a framework for understanding regional flexibility in various aspects of RuvA function. PMID:16880543

  14. Optimisation of expression and purification of the recombinant Yol066 (Rib2) protein from Saccharomyces cerevisiae.

    PubMed

    Urban, A; Ansmant, I; Motorin, Y

    2003-03-25

    Yeast protein Yol066 (encoded by YOL066 ORF, also known as Rib2) possesses two distinct sequence domains: C-terminal deaminase domain and N-terminal part related to RNA:pseudouridine (psi)-synthases. The deaminase domain is implicated in the riboflavine biosynthesis, while the exact function of the RNA:Psi-synthase domain remains obscure. Here we report the optimisation of growth conditions and purification scheme for recombinant His(6)-tagged Yol066 expressed in E. coli BL21(DE3) using pET28 plasmid. Production of soluble Yol066 protein is best at low temperature (18 degrees C) and IPTG concentration (50 micro M) and Yol066 purification was achieved using metal-affinity and ion-exchange chromatography. This optimised protocol yields about 10 mg of highly purified recombinant Yol066 from 3 l of E. coli culture.

  15. Fast quantification of recombinant protein inclusion bodies within intact cells by FT-IR spectroscopy.

    PubMed

    Gross-Selbeck, Sven; Margreiter, Gerd; Obinger, Christian; Bayer, Karl

    2007-01-01

    The accomplishment of the quantification of the recombinant protein content of whole bacterial cells by FT-IR spectroscopy by application of chemometrics is shown. Recombinant Escherichia coli cells expressing an inclusion body forming fusion protein were dried on a 96-well silicon plate for the analysis in a high-throughput FT-IR spectrometer. Acquired spectra of additionally conventionally quantified samples were used to establish a multivariate calibration. The obtained method was tested by predicting inclusion body contents of samples not used for the multivariate model. Results from FT-IR spectra coincided well with the data of universalized electrophoresis analysis. Hence FT-IR spectroscopy could prove as a fast and simple alternative to conventional quantification methods.

  16. Recombinant vaccinia/Venezuelan equine encephalitis (VEE) virus expresses VEE structural proteins.

    PubMed

    Kinney, R M; Esposito, J J; Johnson, B J; Roehrig, J T; Mathews, J H; Barrett, A D; Trent, D W

    1988-12-01

    cDNA molecules encoding the structural proteins of the virulent Trinidad donkey and the TC-83 vaccine strains of Venezuelan equine encephalitis (VEE) virus were inserted under control of the vaccinia virus 7.5K promoter into the thymidine kinase gene of vaccinia virus. Synthesis of the capsid protein and glycoproteins E2 and E1 of VEE virus was demonstrated by immunoblotting of lysates of CV-1 cells infected with recombinant vaccinia/VEE viruses. VEE glycoproteins were detected in recombinant virus-infected cells by fluorescent antibody (FA) analysis performed with a panel of VEE-specific monoclonal antibodies. Seven E2-specific epitopes and two of four E1-specific epitopes were demonstrated by FA.

  17. Structural and functional characterization of recombinant human cellular retinaldehyde-binding protein.

    PubMed Central

    Crabb, J. W.; Carlson, A.; Chen, Y.; Goldflam, S.;