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Sample records for active recombinant protein

  1. Recombinant protein production technology

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  2. Recombinant Human Peptidoglycan Recognition Proteins Reveal Antichlamydial Activity.

    PubMed

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

    2016-07-01

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

  3. 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.

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

    PubMed Central

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

    2015-01-01

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

  5. Complement receptor activity of recombinant porcine CR1-like protein expressed in a eukaryotic system.

    PubMed

    Yin, Wei; Wei, Xiaoming; Jiang, Junbing; Fan, Kuohai; Zhao, Junxing; Sun, Na; Wang, Zhiwei; Sun, Yaogui; Ma, Haili; Zhao, Xin; Li, Hongquan

    2016-08-01

    Primate complement receptor type 1 (CR1) protein, a single-chain transmembrane glycoprotein, plays an important role in immune adherence and clearing complement-opsonized immune complexes. Here, the mRNA of the porcine primate-like complement receptor (CR1-like) gene was analyzed, and two domain sequences with potential functions were cloned into the pwPICZalpha vector for expression in Pichia pastoris. The recombinant proteins were purified with both Protein Pure Ni-NTA resin and strong anion exchange resin. The activities of the purified recombinant proteins were evaluated by SDS-PAGE, western blotting, and complement receptor assays. The results indicated that two domains of the CR1-like protein, CCP36 and CCP811 with molecular weights of 29.8 kDa and 30 kDa, respectively, were successfully expressed in P. pastoris. These two recombinant proteins possess some of the functions of the primate CR1 protein. Using these two proteins coupled with an antibody blocking technique, we also showed that CR1-like is expressed on natural porcine erythrocytes. PMID:26903010

  6. AMP-activated protein kinase kinase: detection with recombinant AMPK alpha1 subunit.

    PubMed

    Hamilton, Stephen R; O'Donnell, John B; Hammet, Andrew; Stapleton, David; Habinowski, Susan A; Means, Anthony R; Kemp, Bruce E; Witters, Lee A

    2002-05-10

    The AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine protein kinase important for the responses to metabolic stress. It consists of a catalytic alpha subunit and two non-catalytic subunits, beta and gamma, and is regulated both by the allosteric action of AMP and by phosphorylation of the alpha and beta subunits catalyzed by AMPKK(s) and autophosphorylation. The Thr172 site on the alpha subunit has been previously characterized as an activating phosphorylation site. Using bacterially expressed AMPK alpha1 subunit proteins, we have explored the role of Thr172-directed AMPKKs in alpha subunit regulation. Recombinant alpha1 subunit proteins, representing the N-terminus, have been expressed as maltose binding protein (MBP) 6x His fusion proteins and purified to homogeneity by Ni(2+) chromatography. Both wild-type alpha1(1-312) and alpha1(1-312)T172D are inactive when expressed in bacteria, but the former can be fully phosphorylated (1 mol/mol) on Thr172 and activated by a surrogate AMPKK, CaMKKbeta. The corresponding AMPKalpha1(1-392), an alpha construct containing its autoinhibitory sequence, can be similarly phosphorylated, but it remains inactive. In an insulinoma cell line, either low glucose or 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) treatment leads to activation and T172 phosphorylation of endogenous AMPK. Under the same conditions of cell incubation, we have identified an AMPKK activity that both phosphorylates and activates the recombinant alpha1(1-312), but this Thr172-directed AMPKK activity is unaltered by low glucose or AICAR, indicating that it is constitutively active. PMID:12051742

  7. Expression and V(D)J recombination activity of mutated RAG-1 proteins.

    PubMed Central

    Sadofsky, M J; Hesse, J E; McBlane, J F; Gellert, M

    1993-01-01

    The products of the RAG-1 and RAG-2 genes are essential for the recombination of the DNA encoding the antigen receptors of the developing immune system. Little is known of the specific role these genes play. We have explored the sequences encoding mouse RAG-1 by deleting large parts of the gene and by introducing local sequence changes. We find that a RAG-1 gene with 40% of the coding region deleted still retains its recombination function. In addition, a series of small deletions within the strongly conserved remaining 60% of the coding region was tested. Nine out of ten of these prove unable to provide RAG-1 activity, but one is quite active. Certain peptide sequences were also specifically targeted for mutagenesis. The RAG-1 protein generated from this expression system is transported to the nucleus and is degraded with a 15 minute half-life. The fate of the proteins made by the deletion mutants were also assessed. Transport of RAG-1 protein to the nucleus was found even with the most extensive deletions studied. The functionality of the deleted proteins is discussed with relation to an alignment of RAG-1 sequences from five animal species. Images PMID:8284210

  8. Recombinant dengue type 1 virus NS5 protein expressed in Escherichia coli exhibits RNA-dependent RNA polymerase activity.

    PubMed

    Tan, B H; Fu, J; Sugrue, R J; Yap, E H; Chan, Y C; Tan, Y H

    1996-02-15

    The complete nonstructural NS5 gene of dengue type 1 virus, Singapore strain S275/90 (D1-S275/90) was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein (126 kDa). The GST-NS5 fusion protein was purified and the recombinant NS5 protein released from the fusion protein by thrombin cleavage. The recombinant NS5 had a predicted molecular weight of 100 kDa and reacted with antiserum against D1-S275/90 virus in Western blot analysis. The purified recombinant NS5 protein possessed RNA-dependent RNA polymerase activity which was inhibited (>99%) by antibodies against the recombinant NS5 protein. The polymerase product was shown to be a negative-stranded RNA molecule, of template size, which forms a double-stranded complex with the template RNA. PMID:8607261

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

    PubMed Central

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

    2014-01-01

    Many commercially available recombinant proteins are produced in Escherichia coli, and most suppliers guarantee contamination levels of less than 1 endotoxin unit (EU). When we analysed commercially available proteins for their endotoxin content, we found contamination levels in the same range as generally stated in the data sheets, but also some that were higher. To analyse whether these low levels of contamination have an effect on immune cells, we stimulated the monocytic cell line THP-1, primary human monocytes, in vitro differentiated human monocyte-derived dendritic cells, and primary human CD1c+ dendritic cells (DCs) with very low concentrations of lipopolysaccharide (LPS; ranging from 0.002–2 ng/ml). We show that CD1c+ DCs especially can be activated by minimal amounts of LPS, equivalent to the levels of endotoxin contamination we detected in some commercially available proteins. Notably, the enhanced endotoxin sensitivity of CD1c+ DCs was closely correlated with high CD14 expression levels observed in CD1c+ DCs that had been maintained in cell culture medium for 24 hours. When working with cells that are particularly sensitive to LPS, even low endotoxin contamination may generate erroneous data. We therefore recommend that recombinant proteins be thoroughly screened for endotoxin contamination using the limulus amebocyte lysate test, fluorescence-based assays, or a luciferase based NF-κB reporter assay involving highly LPS-sensitive cells overexpressing TLR4, MD-2 and CD14. PMID:25478795

  10. Improving recombinant protein purification yield

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  11. Porin activity of the native and recombinant outer membrane protein Oms28 of Borrelia burgdorferi.

    PubMed Central

    Skare, J T; Champion, C I; Mirzabekov, T A; Shang, E S; Blanco, D R; Erdjument-Bromage, H; Tempst, P; Kagan, B L; Miller, J N; Lovett, M A

    1996-01-01

    The outer membrane-spanning (Oms) proteins of Borrelia burgdorferi have been visualized by freeze-fracture analysis but, until recently, not further characterized. We developed a method for the isolation of B. burgdorferi outer membrane vesicles and described porin activities with single-channel conductances of 0.6 and 12.6 nS in 1 M KCI. By using both nondenaturing isoelectric focusing gel electrophoresis and fast-performance liquid chromatography separation after detergent solubilization, we found that the 0.6-nS porin activity resided in a 28-kDa protein, designated Oms28. The oms28 gene was cloned, and its nucleotide sequence was determined. The deduced amino acid sequence of Oms28 predicted a 257-amino-acid precursor protein with a putative 24-amino-acid leader peptidase I signal sequence. Processed Oms28 yielded a mature protein with a predicted molecular mass of 25,363 Da. When overproduced in Escherichia coli, the Oms28 porin fractionated in part to the outer membrane. Sodium dodecyl sulfate-polyacrylamide gel-purified recombinant Oms28 from E. coli retained functional activity as demonstrated by an average single-channel conductance of 1.1 nS in the planar lipid bilayer assay. These findings confirmed that Oms28 is a B. burgdorferi porin, the first to be described. As such, it is potential relevance to the pathogenesis of Lyme borreliosis and to the physiology of the spirochete. PMID:8759855

  12. Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL*S⃞

    PubMed Central

    Takaku, Motoki; Machida, Shinichi; Hosoya, Noriko; Nakayama, Shugo; Takizawa, Yoshimasa; Sakane, Isao; Shibata, Takehiko; Miyagawa, Kiyoshi; Kurumizaka, Hitoshi

    2009-01-01

    The RAD51 protein is a central player in homologous recombinational repair. The RAD51B protein is one of five RAD51 paralogs that function in the homologous recombinational repair pathway in higher eukaryotes. In the present study, we found that the human EVL (Ena/Vasp-like) protein, which is suggested to be involved in actin-remodeling processes, unexpectedly binds to the RAD51 and RAD51B proteins and stimulates the RAD51-mediated homologous pairing and strand exchange. The EVL knockdown cells impaired RAD51 assembly onto damaged DNA after ionizing radiation or mitomycin C treatment. The EVL protein alone promotes single-stranded DNA annealing, and the recombination activities of the EVL protein are further enhanced by the RAD51B protein. The expression of the EVL protein is not ubiquitous, but it is significantly expressed in breast cancer-derived MCF7 cells. These results suggest that the EVL protein is a novel recombination factor that may be required for repairing specific DNA lesions, and that may cause tumor malignancy by its inappropriate expression. PMID:19329439

  13. In Vitro and in Vivo Antistaphylococcal Activity Determination of the New Recombinant Lysostaphin Protein

    PubMed Central

    Abtahi, Hamid; Farhangnia, Leila; Ghaznavi-Rad, Ehsanollah

    2016-01-01

    Background: Bacterial infection by antibiotic-resistant Staphylococcus aureus strains is a worldwide concern and the development of novel antistaphylococcal agents is acutely needed. Lysostaphin, an example of such novel agents, is a bacteriocin secreted by S. simulans to kill S. aureus through proteolysis of the Staphylococcus cell wall. Objectives: The aim of this study was to evaluate the in vitro and in vivo antistaphylococcal activity of recombinant lysostaphin. Materials and Methods: The in vitro study of the recombinant lysostaphin activity against S. aureus was determined by turbidimetric assay. For in vivo investigation, two groups of rats were inoculated with 1.4 × 109 CFU S. aureus. Five days after the nasal instillation of S. aureus, treatment in one of the groups was performed with a single dose (200 μg/dose) of recombinant lysostaphin formulated in Eucerin-based cream. Results: Recombinant lysostaphin at 100 μg/mL concentration showed a significant decrease of the optical density compared to the control samples. The in vivo study demonstrated that a single dose (200 μg/dose) of recombinant lysostaphin cream significantly reduced nasal colonization in all the treated animals compared to the untreated ones. Conclusions: These results demonstrated that the recombinant lysostaphin produced in this study was able to kill nasal S. aureus in rats. It can be recommended for human clinical trial studies. PMID:27217919

  14. Sequence analysis, expression, and binding activity of recombinant major outer sheath protein (Msp) of Treponema denticola.

    PubMed Central

    Fenno, J C; Müller, K H; McBride, B C

    1996-01-01

    The gene encoding the major outer sheath protein (Msp) of the oral spirochete Treponema denticola ATCC 35405 was cloned, sequenced, and expressed in Escherichia coli. Preliminary sequence analysis showed that the 5' end of the msp gene was not present on the 5.5-kb cloned fragment described in a recent study (M. Haapasalo, K. H. Müller, V. J. Uitto, W. K. Leung, and B. C. McBride, Infect. Immun. 60:2058-2065,1992). The 5' end of msp was obtained by PCR amplification from a T. denticola genomic library, and an open reading frame of 1,629 bp was identified as the coding region for Msp by combining overlapping sequences. The deduced peptide consisted of 543 amino acids and had a molecular mass of 58,233 Da. The peptide had a typical prokaryotic signal sequence with a potential cleavage site for signal peptidase 1. Northern (RNA) blot analysis showing the msp transcript to be approximately 1.7 kb was consistent with the identification of a promoter consensus sequence located optimally upstream of msp and a transcription termination signal found downstream of the stop codon. The entire msp sequence was amplified from T. denticola genomic DNA and cloned in E. coli by using a tightly regulated T7 RNA polymerase vector system. Expression of Msp was toxic to E. coli when the entire msp gene was present. High levels of Msp were produced as inclusion bodies when the putative signal peptide sequence was deleted and replaced by a vector-encoded T7 peptide sequence. Recombinant Msp purified to homogeneity from a clone containing the full-length msp gene adhered to immobilized laminin and fibronectin but not to bovine serum albumin. Attachment of recombinant Msp was decreased in the presence of soluble substrate. Attachment of T. denticola to immobilized laminin and fibronectin was increased by pretreatment of the substrate with recombinant Msp. These studies lend further support to the hypothesis that Msp mediates the extracellular matrix binding activity of T. denticola. PMID

  15. 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. PMID:10596760

  16. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, C.W.; Mangel, W.F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

  17. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, Carl W.; Mangel, Walter F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

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

    PubMed Central

    Tufail, Soban; Ismat, Fouzia; Imran, Muhammad; Iqbal, Mazhar; Mirza, Osman; Rhaman, Moazur

    2016-01-01

    Proteases are widely used to remove affinity and solubility tags from recombinant proteins to avoid potential interference of these tags with the structure and function of the fusion partner. In recent years, great interest has been seen in use of the human rhinovirus 3C protease owing to its stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally employed for purification and stabilization of proteins, hence, necessitate their removal by tedious and time-consuming procedures before proteolysis can occur. To address this issue, we examined the effect of elution buffers used for common affinity based purifications, salt ions, stability/solubility and reducing agents, and detergents on the activity of the human rhinovirus 3C protease using three different fusion proteins at 4°C, a temperature of choice for purification of many proteins. The results show that the human rhinovirus 3C protease performs better at 4°C than the frequently used tobacco etch virus protease and its activity was insensitive to most of the experimental conditions tested. Though number of fusion proteins tested is limited, we expect that these finding will facilitate the use of the human rhinovirus 3C protease in recombinant protein production for pharmaceutical and biotechnological applications. PMID:27093053

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

    PubMed

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

    2016-01-01

    Proteases are widely used to remove affinity and solubility tags from recombinant proteins to avoid potential interference of these tags with the structure and function of the fusion partner. In recent years, great interest has been seen in use of the human rhinovirus 3C protease owing to its stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally employed for purification and stabilization of proteins, hence, necessitate their removal by tedious and time-consuming procedures before proteolysis can occur. To address this issue, we examined the effect of elution buffers used for common affinity based purifications, salt ions, stability/solubility and reducing agents, and detergents on the activity of the human rhinovirus 3C protease using three different fusion proteins at 4°C, a temperature of choice for purification of many proteins. The results show that the human rhinovirus 3C protease performs better at 4°C than the frequently used tobacco etch virus protease and its activity was insensitive to most of the experimental conditions tested. Though number of fusion proteins tested is limited, we expect that these finding will facilitate the use of the human rhinovirus 3C protease in recombinant protein production for pharmaceutical and biotechnological applications. PMID:27093053

  20. Safety and prolonged activity of recombinant factor VIII Fc fusion protein in hemophilia A patients

    PubMed Central

    Josephson, Neil C.; Quon, Doris; Ragni, Margaret V.; Cheng, Gregory; Li, Ella; Jiang, Haiyan; Li, Lian; Dumont, Jennifer A.; Goyal, Jaya; Zhang, Xin; Sommer, Jurg; McCue, Justin; Barbetti, Margaret; Luk, Alvin

    2012-01-01

    Current factor VIII (FVIII) products display a half-life (t1/2) of ∼ 8-12 hours, requiring frequent intravenous injections for prophylaxis and treatment of patients with hemophilia A. rFVIIIFc is a recombinant fusion protein composed of a single molecule of FVIII covalently linked to the Fc domain of human IgG1 to extend circulating rFVIII t1/2. This first-in-human study in previously treated subjects with severe hemophilia A investigated safety and pharmacokinetics of rFVIIIFc. Sixteen subjects received a single dose of rFVIII at 25 or 65 IU/kg followed by an equal dose of rFVIIIFc. Most adverse events were unrelated to study drug. None of the study subjects developed anti-rFVIIIFc antibodies or inhibitors. Across dose levels, compared with rFVIII, rFVIIIFc showed 1.54- to 1.70-fold longer elimination t1/2, 1.49- to 1.56-fold lower clearance, and 1.48- to 1.56-fold higher total systemic exposure. rFVIII and rFVIIIFc had comparable dose-dependent peak plasma concentrations and recoveries. Time to 1% FVIII activity above baseline was ∼ 1.53- to 1.68-fold longer than rFVIII across dose levels. Each subject showed prolonged exposure to rFVIIIFc relative to rFVIII. Thus, rFVIIIFc may offer a viable therapeutic approach to achieve prolonged hemostatic protection and less frequent dosing in patients with hemophilia A. This trial was registered at www.clinicaltrials.gov as NCT01027377. PMID:22223821

  1. Recombinant snake venom prothrombin activators.

    PubMed

    Lövgren, Ann

    2013-01-01

    Three prothrombin activators; ecarin, which was originally isolated from the venom of the saw-scaled viper Echis carinatus, trocarin from the rough-scaled snake Tropidechis carinatus, and oscutarin from the Taipan snake Oxyuranus scutellatus, were expressed in mammalian cells with the purpose to obtain recombinant prothrombin activators that could be used to convert prothrombin to thrombin. We have previously reported that recombinant ecarin can efficiently generate thrombin without the need for additional cofactors, but does not discriminate non-carboxylated prothrombin from biologically active γ-carboxylated prothrombin. Here we report that recombinant trocarin and oscutarin could not efficiently generate thrombin without additional protein co-factors. We confirm that both trocarin and oscutarin are similar to human coagulation Factor X (FX), explaining the need for additional cofactors. Sequencing of a genomic fragment containing 7 out of the 8 exons coding for oscutarin further confirmed the similarity to human FX. PMID:23111318

  2. Extraction of recombinant protein from Escherichia coli by using a novel cell autolysis activity of VanX.

    PubMed

    Kamioka, Tetsuya; Sohya, Shihori; Wu, Nan; Maki, Tei; Matsuda, Tomoki; Ikegami, Takahisa; Nakamura, Haruki; Kuroda, Yutaka

    2013-08-15

    Escherichia coli is a versatile, low-cost, and popular host for expressing recombinant proteins. However, extracting recombinant proteins from E. coli requires cell wall breakage, which is both time- and effort-consuming. Here we report a novel cell breakage method based on our recent finding that VanX, which is a d-Ala-d-Ala dipeptidase encoded in a vancomycin-resistant VanA gene cluster, exhibits a strong cell lysis activity when expressed in isolation in E. coli. In our strategy, we coexpress VanX with the target protein, causing cell autolysis and release of the cellular content into the culture medium. We demonstrated this strategy for two model proteins, a green fluorescent protein variant (GFPuv) and Gaussia luciferase, and optimized the autolysis conditions and coexpression vectors. The fluorescence activity of GFPuv collected from the medium was identical to that of GFPuv purified by conventional methods. Cell breakage by VanX-mediated autolysis is very simple to implement and will efficiently complement traditional methods. PMID:23624113

  3. Recombinant dengue virus type 1 NS3 protein exhibits specific viral RNA binding and NTPase activity regulated by the NS5 protein.

    PubMed

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

    1998-07-01

    The full-length dengue virus NS3 protein has been successfully expressed as a 94-kDa GST fusion protein in Escherichia coli. Treatment of the purified fusion protein with thrombin released a 68-kDa protein which is the expected molecular mass for the DEN1 NS3 protein. The identity of this protein was confirmed by Western blotting using dengue virus antisera. Two related activities of the recombinant NS3 protein were characterized, which were the binding of the protein to the 3'-noncoding region of the dengue virus RNA genome and NTPase activity. We demonstrated using a band shift assay that the DEN1 NS3 protein could form a complex with the stem-loop structure in the 3'-noncoding region (3'-NCR), although sites outside the stem-loop may also participate in binding. Using various unlabeled homopolymeric and heteropolymeric RNAs as competitors for binding, it was further shown that the DEN1 NS3 protein exhibits preferential binding to a 94-nt RNA transcript from the 3'-NCR of the dengue virus. The NTPase activity of the recombinant DEN1 NS3 protein was characterized using a thin-layer chromatography assay. We found that the DEN1 NS3 protein possesses some aspects of NTPase activity, which are distinct from those found in other flaviviruses. Although the NS3 protein was able to utilize all four ribonucleoside triphosphates as its substrates, the NS3 protein showed a distinct preference for purine triphosphates (i.e., ATP and GTP). The addition of poly(U) did not stimulate NTPase activity in DEN1 NS3 protein, which contrasts with the reports for other flaviviral NS3 proteins. However, NTPase activity was specifically stimulated by the viral NS5 protein, which was manifested by a more than twofold increase in the rate of ATP hydrolysis and a 25% increase in the yield of ADP at the end of a 120-min reaction. These data suggest that the NTPase activity of the NS3 protein may be regulated by the viral NS5 protein during virus replication. PMID:9657959

  4. Differential subcellular targeting of recombinant human α₁-proteinase inhibitor influences yield, biological activity and in planta stability of the protein in transgenic tomato plants.

    PubMed

    Jha, Shweta; Agarwal, Saurabh; Sanyal, Indraneel; Jain, G K; Amla, D V

    2012-11-01

    The response of protein accumulation site on yield, biological activity and in planta stability of therapeutic recombinant human proteinase inhibitor (α₁-PI) was analyzed via targeting to different subcellular locations, like endoplasmic reticulum (ER), apoplast, vacuole and cytosol in leaves of transgenic tomato plants. In situ localization of the recombinant α₁-PI protein in transgenic plant cells was monitored by immunohistochemical staining. Maximum accumulation of recombinant α₁-PI in T₀ and T₁ transgenic tomato plants was achieved from 1.5 to 3.2% of total soluble protein (TSP) by retention in ER lumen, followed by vacuole and apoplast, whereas cytosolic targeting resulted into degradation of the protein. The plant-derived recombinant α₁-PI showed biological activity for elastase inhibition, as monitored by residual porcine pancreatic elastase (PPE) activity assay and band-shift assay. Recombinant α₁-PI was purified from transgenic tomato plants with high yield, homogeneity and biological activity. Purified protein appeared as a single band of ∼48-50 kDa on SDS-PAGE with pI value ranging between 5.1 and 5.3. Results of mass spectrometry and optical spectroscopy of purified recombinant α₁-PI revealed the structural integrity of the recombinant protein comparable to native serum α₁-PI. Enzymatic deglycosylation and lectin-binding assays with the purified recombinant α₁-PI showed compartment-specific N-glycosylation of the protein targeted to ER, apoplast and vacuole. Conformational studies based on urea-induced denaturation and circular dichroism (CD) spectroscopy revealed relatively lower stability of the recombinant α₁-PI protein, compared to its serum counterpart. Pharmacokinetic evaluation of plant derived recombinant and human plasma-purified α₁-PI in rat, by intravenous route, revealed significantly faster plasma clearance and lower area under curve (AUC) of recombinant protein. Our data suggested significance of

  5. Construction of three different recombinant scorpion fusion proteins with bifunctional activity.

    PubMed

    Cui, Y; Guo, G L; Liu, Y F; Mao, Y Z; Zhang, R; Wu, C F; Zhang, J H

    2011-06-01

    This is the first report of three different fusion proteins with an antitumor-analgesic peptide obtained from Chinese scorpion Buthus martensii Karsch (BmKAGAP). The fusion proteins were constructed in the form of chimeric toxins, aiming to obtain bifunctional analgesic and antitumor activity. The fusion proteins consisted of luteinizing hormone-releasing hormone (LHRH), three different types of flexible linkers (L1, Ser-Ser-His-His-His-His-His-His-Ser-Ser-Gly-Leu-Val-Pro-Arg-Gly-Ser-His-Met; L2, Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser; L3, Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Gly-Ser-Ser-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser), and BmKAGAP. The genes coding three fusion proteins were cloned and expressed in E. coli in soluble form. Following two successive column chromatographic separations, purified fusion proteins were obtained. These fusion proteins exhibited analgesic activity in mice and were cytotoxic to a hepatocellular carcinoma cell line Hep3B. PMID:21793303

  6. Human T-lymphotropic virus tax activates human cytomegalovirus major-immediate early promoter and improves production of recombinant proteins in HEK293 cells.

    PubMed

    Lwa, Teng Rhui; Lee, Jialing; Ng, Chew Har; Lew, Qiao Jing; Hia, Hui Ching; Chao, Sheng-Hao

    2011-01-01

    The human cytomegalovirus (CMV) major immediate-early (MIE) promoter is widely used in mammalian cells for production of recombinant proteins. It is of great interest to further enhance protein production driven by the CMV promoter. Here, we report that the Tax protein of human T-lymphotropic virus stimulates the transgene expression under the control of CMV MIE promoter in HEK293 cells. At least threefold increases in transient production of recombinant proteins, including luciferase and two biopharmaceutical proteins (erythropoietin and interferon-γ), were detected. Furthermore, cyclic adenosine monophosphate (AMP)-response element binding protein 2 (CREB2) was identified as a cellular cofactor, which might be responsible for Tax transactivation of the CMV MIE promoter. Our results not only demonstrate the potential use of this novel expression strategy for improvement of recombinant protein production in HEK293 cells but also provide the molecular mechanism for Tax-mediated activation of CMV MIE promoter. PMID:21425252

  7. In Plant Activation: An Inducible, Hyperexpression Platform for Recombinant Protein Production in Plants[W][OPEN

    PubMed Central

    Dugdale, Benjamin; Mortimer, Cara L.; Kato, Maiko; James, Tess A.; Harding, Robert M.; Dale, James L.

    2013-01-01

    In this study, we describe a novel protein production platform that provides both activation and amplification of transgene expression in planta. The In Plant Activation (INPACT) system is based on the replication machinery of tobacco yellow dwarf mastrevirus (TYDV) and is essentially transient gene expression from a stably transformed plant, thus combining the advantages of both means of expression. The INPACT cassette is uniquely arranged such that the gene of interest is split and only reconstituted in the presence of the TYDV-encoded Rep/RepA proteins. Rep/RepA expression is placed under the control of the AlcA:AlcR gene switch, which is responsive to trace levels of ethanol. Transgenic tobacco (Nicotiana tabacum cv Samsun) plants containing an INPACT cassette encoding the β-glucuronidase (GUS) reporter had negligible background expression but accumulated very high GUS levels (up to 10% total soluble protein) throughout the plant, within 3 d of a 1% ethanol application. The GUS reporter was replaced with a gene encoding a lethal ribonuclease, barnase, demonstrating that the INPACT system provides exquisite control of transgene expression and can be adapted to potentially toxic or inhibitory compounds. The INPACT gene expression platform is scalable, not host-limited, and has been used to express both a therapeutic and an industrial protein. PMID:23839786

  8. Characterization of the highly active fragment of glyceraldehyde-3-phosphate dehydrogenase gene promoter for recombinant protein expression in Pleurotus ostreatus.

    PubMed

    Yin, Chaomin; Zheng, Liesheng; Zhu, Jihong; Chen, Liguo; Ma, Aimin

    2015-03-01

    Developing efficient native promoters is important for improving recombinant protein expression by fungal genetic engineering. The promoter region of glyceraldehyde-3-phosphate dehydrogenase gene in Pleurotus ostreatus (Pogpd) was isolated and optimized by upstream truncation. The activities of these promoters with different lengths were further confirmed by fluorescence, quantitative real-time PCR and Western blot analysis. A truncated Pogpd-P2 fragment (795 bp) drove enhanced green fluorescence protein (egfp) gene expression in P. ostreatus much more efficiently than full-length Pogpd-P1. Further truncating Pogpd-P2 to 603, 403 and 231 bp reduced the eGFP expression significantly. However, the 403-bp fragment between -356 bp and the start codon was the minimal but sufficient promoter element for eGFP expression. Compact native promoters for genetic engineering of P. ostreatus were successfully developed and validated in this study. This will broaden the preexisting repertoire of fungal promoters for biotechnology application. PMID:25743073

  9. Renaturation of Recombinant Treponema pallidum Rare Outer Membrane Protein 1 into a Trimeric, Hydrophobic, and Porin-Active Conformation

    PubMed Central

    Zhang, Hongwei H.; Blanco, David R.; Exner, Maurice M.; Shang, Ellen S.; Champion, Cheryl I.; Phillips, Martin L.; Miller, James N.; Lovett, Michael A.

    1999-01-01

    We have previously observed that while native Treponema pallidum rare outer membrane protein 1 (Tromp1) is hydrophobic and has porin activity, recombinant forms of Tromp1 do not possess these properties. In this study we show that these properties are determined by conformation and can be replicated by proper renaturation of recombinant Tromp1. Native Tromp1, but not the 47-kDa lipoprotein, extracted from whole organisms by using Triton X-114, was found to lose hydrophobicity after treatment in 8 M urea, indicating that Tromp1’s hydrophobicity is conformation dependent. Native Tromp1 was purified from 0.1% Triton X-100 extracts of whole organisms by fast-performance liquid chromatography (FPLC) and shown to have porin activity in planar lipid bilayers. Cross-linking studies of purified native Tromp1 with an 11 Å cross-linking agent showed oligomeric forms consistent with dimers and trimers. For renaturation studies of recombinant Tromp1 (rTromp1), a 31,109-Da signal-less construct was expressed in Escherichia coli and purified by FPLC. FPLC-purified rTromp1 was denatured in 8 M urea and then renatured in the presence of 0.5% Zwittergent 3,14 during dialysis to remove the urea. Renatured rTromp1 was passed through a Sephacryl S-300 gel exclusion column previously calibrated with known molecular weight standards. While all nonrenatured rTromp1 eluted from the column at approximately the position of the carbonic anhydrase protein standard (29 kDa), all renatured rTromp1 eluted at the position of the phosphorylase b protein standard (97 kDa), suggesting a trimeric conformation. Trimerization was confirmed by using an 11 Å cross-linking agent which showed both dimers and trimers similar to that of native Tromp1. Triton X-114 phase separations showed that all of renatured rTromp1, but none of nonrenatured rTromp1, phase separated exclusively into the hydrophobic detergent phase, similar to native Tromp1. Circular dichroism of nonrenatured and renatured rTromp1

  10. Recombinant Expression of a Novel Fungal Immunomodulatory Protein with Human Tumor Cell Antiproliferative Activity from Nectria haematococca

    PubMed Central

    Li, Shuying; Nie, Ying; Ding, Yang; Shi, Lijun; Tang, Xuanming

    2014-01-01

    To our best knowledge, all of the fungal immunomodulatory proteins (FIPs) have been successfully extracted and identified in Basidomycetes, with only the exception of FIP from ascomycete Nectria haematococca (FIP-nha) discovered through homology alignment most recently. In this work, a gene encoding FIP-nha was synthesized and recombinantly expressed in an Escherichia coli expression system. SDS-PAGE and MALDI-MS analyses of recombinant FIP-nha (rFIP-nha) indicated that the gene was successfully expressed. The yield of the bioactive FIP-nha protein was 42.7 mg/L. In vitro assays of biological activity indicated that the rFIP-nha caused hemagglutination of human and rabbit red blood cells, significantly stimulated mouse spleen lymphocyte proliferation, and enhanced expression of interleukin-2 (IL-2) released from mouse splenocytes, revealing a strong antitumor effect against HL60, HepG2 and MGC823. Through this work, we constructed a rapid and efficient method of FIP production, and suggested that FIP-nha is a valuable candidate for use in future medical care and pharmaceutical products. PMID:25272229

  11. Recombinant expression of a novel fungal immunomodulatory protein with human tumor cell antiproliferative activity from Nectria haematococca.

    PubMed

    Li, Shuying; Nie, Ying; Ding, Yang; Shi, Lijun; Tang, Xuanming

    2014-01-01

    To our best knowledge, all of the fungal immunomodulatory proteins (FIPs) have been successfully extracted and identified in Basidomycetes, with only the exception of FIP from ascomycete Nectria haematococca (FIP-nha) discovered through homology alignment most recently. In this work, a gene encoding FIP-nha was synthesized and recombinantly expressed in an Escherichia coli expression system. SDS-PAGE and MALDI-MS analyses of recombinant FIP-nha (rFIP-nha) indicated that the gene was successfully expressed. The yield of the bioactive FIP-nha protein was 42.7 mg/L. In vitro assays of biological activity indicated that the rFIP-nha caused hemagglutination of human and rabbit red blood cells, significantly stimulated mouse spleen lymphocyte proliferation, and enhanced expression of interleukin-2 (IL-2) released from mouse splenocytes, revealing a strong antitumor effect against HL60, HepG2 and MGC823. Through this work, we constructed a rapid and efficient method of FIP production, and suggested that FIP-nha is a valuable candidate for use in future medical care and pharmaceutical products. PMID:25272229

  12. Recombinant therapeutic proteins: production platforms and challenges.

    PubMed

    Dingermann, Theo

    2008-01-01

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

  13. Recombinant Ov-ASP-1, a Th1-biased protein adjuvant derived from the helminth Onchocerca volvulus, can directly bind and activate antigen-presenting cells.

    PubMed

    He, Yuxian; Barker, Sophie J; MacDonald, Angus J; Yu, Yu; Cao, Long; Li, Jingjing; Parhar, Ranjit; Heck, Susanne; Hartmann, Susanne; Golenbock, Douglas T; Jiang, Shibo; Libri, Nathan A; Semper, Amanda E; Rosenberg, William M; Lustigman, Sara

    2009-04-01

    We previously reported that rOv-ASP-1, a recombinant Onchocerca volvulus activation associated protein-1, was a potent adjuvant for recombinant protein or synthetic peptide-based Ags. In this study, we further evaluated the adjuvanticity of rOv-ASP-1 and explored its mechanism of action. Consistently, recombinant full-length spike protein of SARS-CoV or its receptor-binding domain in the presence of rOv-ASP-1 could effectively induce a mixed but Th1-skewed immune response in immunized mice. It appears that rOv-ASP-1 primarily bound to the APCs among human PBMCs and triggered Th1-biased proinflammatory cytokine production probably via the activation of monocyte-derived dendritic cells and the TLR, TLR2, and TLR4, thus suggesting that rOv-ASP-1 is a novel potent innate adjuvant. PMID:19299698

  14. Antiviral activity of recombinant porcine surfactant protein A against porcine reproductive and respiratory syndrome virus in vitro.

    PubMed

    Li, Lan; Zheng, Qisheng; Zhang, Yuanpeng; Li, Pengcheng; Fu, Yanfeng; Hou, Jibo; Xiao, Xilong

    2016-07-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) has caused significant economic losses in the swine industry worldwide. However, there is not an ideal vaccine to provide complete protection against PRRSV. Thus, the need for new antiviral strategies to control PRRSV still remains. Surfactant protein A (SP-A) belongs to the family of C-type lectins, which can exert antiviral activities. In this present study, we assessed the antiviral properties of recombinant porcine SP-A (RpSP-A) on PRRSV infection in Marc 145 cells and revealed its antiviral mechanism using a plaque assay, real-time qPCR, western blotting analysis and an attachment and penetration assay. Our results showed that RpSP-A could inhibit the infectivity of PRRSV in Marc 145 cells and could reduce the total RNA and protein level. The attachment assay indicated that RpSP-A in the presence of Ca(2+) could largely inhibit Marc 145 cell attachment; however, in the penetration assay, it was relatively inactive. Furthermore, our study suggested that virus progeny released from infected Marc145 cells were blocked by RpSP-A from infecting other cells. We conclude that RpSP-A has antiviral activity against PRRSV, most probably by blocking viral attachment and the cell-to-cell transmission pathway, and therefore, RpSP-A holds promise as a novel antiviral agent against PRRSV. PMID:27101074

  15. Heterologous Expression of MeLEA3: A 10 kDa Late Embryogenesis Abundant Protein of Cassava, Confers Tolerance to Abiotic Stress in Escherichia coli with Recombinant Protein Showing In Vitro Chaperone Activity.

    PubMed

    Barros, Nicolle L F; da Silva, Diehgo T; Marques, Deyvid N; de Brito, Fabiano M; dos Reis, Savio P; de Souza, Claudia R B

    2015-01-01

    Late embryogenesis abundant (LEA) proteins are small molecular weight proteins involved in acquisition of tolerance to drought, salinity, high temperature, cold, and freezing stress in many plants. Previous studies revealed a cDNA sequence coding for a 10 kDa atypical LEA protein, named MeLEA3, predicted to be located into mitochondria with potential role in salt stress response of cassava (Manihot esculenta Crantz). Here we aimed to produce the recombinant MeLEA3 protein by heterologous expression in Escherichia coli and evaluate the tolerance of bacteria expressing this protein under abiotic stress. Our result revealed that the recombinant MeLEA3 protein conferred a protective function against heat and salt stress in bacterial cells. Also, the recombinant MeLEA3 protein showed in vitro chaperone activity by protection of NdeI restriction enzyme activity under heat stress. PMID:25990084

  16. Heterologous recombinant protein with decapacitating activity prevents and reverts cryodamage in ram sperm: An emerging biotechnological tool for cryobiology.

    PubMed

    Zalazar, L; Ledesma, A; Hozbor, F; Cesari, A

    2016-01-01

    During the last decades fundamental and applied aspects of mammalian ram sperm cryopreservation have been increasingly explored by scientists and biotechnologists. Many works report modifications in the composition of the freezing extenders and explore the beneficial and detrimental effects of seminal plasma or seminal plasma components in cryopreservation. Seminal plasma is known to contain stabilizing proteins, thereby this is a good start point to study the maintenance of membrane stability based on the basic knowledge of sperm physiology. However, seminal plasma composition is variable among rams and also the introduction of exogenous seminal plasma or its fractions to commercial semen can be associated with the transmission of viral diseases. Our work shows that a mouse protein, called SPINK3 (Serine Protease Inhibitor Kazal type 3) with decapacitating activity interacts with heterologous ram sperm when it is produced as a recombinant molecule. By immunocytochemistry assays we demonstrate that this protein (naturally expressed by mouse seminal vesicle under androgenic control) binds to the apical portion of both fresh and frozen ram sperm, the same localization described in mouse homologous sperm. Furthermore, it significantly improves sperm progressive motility compared to non-treated samples when it is added to freezing extenders and to dilution media after thawing. On the contrary, addition of SPINK3 does not modify sperm viability. The percentage of sperm with intact acrosome after ionophore induction was also significantly higher in sperm frozen in the presence of SPINK3 compared to control samples and the addition of SPINK3 after thawing significantly reduced both induced and non induced acrosomal loss, indicating that heterologous SPINK3 might act as a calcium inhibitor transport as described in mouse. Based on our results SPINK3 may find a place as a desirable biotechnological tool to achieve a higher proportion of competent sperm to fertilize. PMID

  17. Recombinant protein production and streptomycetes.

    PubMed

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

    2012-04-30

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

  18. Prolonged activity of a recombinant factor VIII-Fc fusion protein in hemophilia A mice and dogs.

    PubMed

    Dumont, Jennifer A; Liu, Tongyao; Low, Susan C; Zhang, Xin; Kamphaus, George; Sakorafas, Paul; Fraley, Cara; Drager, Douglas; Reidy, Thomas; McCue, Justin; Franck, Helen W G; Merricks, Elizabeth P; Nichols, Timothy C; Bitonti, Alan J; Pierce, Glenn F; Jiang, Haiyan

    2012-03-29

    Despite proven benefits, prophylactic treatment for hemophilia A is hampered by the short half-life of factor VIII. A recombinant factor VIII-Fc fusion protein (rFVIIIFc) was constructed to determine the potential for reduced frequency of dosing. rFVIIIFc has an ∼ 2-fold longer half-life than rFVIII in hemophilia A (HemA) mice and dogs. The extension of rFVIIIFc half-life requires interaction of Fc with the neonatal Fc receptor (FcRn). In FcRn knockout mice, the extension of rFVIIIFc half-life is abrogated, and is restored in human FcRn transgenic mice. The Fc fusion has no impact on FVIII-specific activity. rFVIIIFc has comparable acute efficacy as rFVIII in treating tail clip injury in HemA mice, and fully corrects whole blood clotting time (WBCT) in HemA dogs immediately after dosing. Furthermore, consistent with prolonged half-life, rFVIIIFc shows 2-fold longer prophylactic efficacy in protecting HemA mice from tail vein transection bleeding induced 24-48 hours after dosing. In HemA dogs, rFVIIIFc also sustains partial correction of WBCT 1.5- to 2-fold longer than rFVIII. rFVIIIFc was well tolerated in both species. Thus, the rescue of FVIII by Fc fusion to provide prolonged protection presents a novel pathway for FVIII catabolism, and warrants further investigation. PMID:22246033

  19. Molecular cloning, expression of a galectin gene in Pacific white shrimp Litopenaeus vannamei and the antibacterial activity of its recombinant protein.

    PubMed

    Cha, Gui-Hong; Liu, Yuan; Peng, Ting; Huang, Ming-Zhu; Xie, Chen-Ying; Xiao, Yu-Chao; Wang, Wei-Na

    2015-10-01

    Galectins play crucial roles in innate immune responses in invertebrate by recognizing and eliminating microinvaders. In this study, a cDNA encoding a galectin in the Pacific white shrimp (L. vannamei) was identified and characterized. A recombinant variant of this lectin, rLvgalectin, was expressed in the model organism P. pastoris and its expression was confirmed by Western blot. Biochemical assays indicated that the recombinant protein antibacterial rLvgalectin activity and was expressed in all of the organism's tested tissues Injection of the bacterium V. alginolyticus into L. vannamei induced hemocytes upregulation of Lvgalectin. The recombinant Lvgalectin protein (rLvgalectin) could bind various microorganism including Gram-positive bacteria, Gram-negative bacteria and yeast. And it revealed antimicrobial activity against the test Gram-positive bacteria, Gram-negative bacteria, but did not inhibit the growth of fungus Pichia pastoris. Moreover, rLvgalectin could significantly enhance the clearance activity of V. alginolyticus in vivo. In vivo challenge experiments showed that the recombinant rLvgalectin protein can significantly reduce the mortalities of V. alginolyticus injection. Furthermore, Compared to their wild-type counterparts, Lvgalectin-silenced shrimp exhibited increased mortality and hemocyte apoptosis, decreased bacterial clearance ability and total hemocyte counts, and stronger expression of Lvp53, LvproPO, LvPEN3, and LvCrustin following V. alginolyticus challenge. Taken together, these results suggest that galectin is important in the innate immune response of shrimp to pathogens infection. PMID:26143399

  20. Production of Active Nonglycosylated Recombinant B-Chain of Type-2 Ribosome-Inactivating Protein from Viscum articulatum and Its Biological Effects on Peripheral Blood Mononuclear Cells

    PubMed Central

    Lu, Tzu-Li; Chuang, Jing-Yuan; Yang, Jai-Sing; Chiu, Shau-Ting; Hsiao, Nai-Wan; Wu, Mei-Chen; Wu, Shih-Hsiung; Hsu, Ching-Hsiang

    2011-01-01

    Type-2 ribosome-inactivating proteins, composed of a toxic A-chain and lectin-like B-chain, display various biological functions, including cytotoxicity and immunomodulation. We here cloned the lectin-like B-chain encoding fragment of a newly identified type-2 RIP gene, articulatin gene, from Viscum articulatum, into a bacterial expression vector to obtain nonglycosylated recombinant protein expressed in inclusion bodies. After purification and protein refolding, soluble refolded recombinant articulatin B-chain (rATB) showed lectin activity specific toward galactoside moiety and was stably maintained while stored in low ionic strength solution. Despite lacking glycosylation, rATB actively bound leukocytes with preferential binding to monocytes and in vitro stimulated PBMCs to release cytokines without obvious cytotoxicity. These results implicated such a B-chain fragment as a potential immunomodulator. PMID:21584270

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

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

  2. Identification of HUGT1 as a potential BiP activator and a cellular target for improvement of recombinant protein production using a cDNA screening system.

    PubMed

    Ku, Sebastian Chih Yuan; Lwa, Teng Rhui; Giam, Maybelline; Yap, Miranda Gek Sim; Chao, Sheng-Hao

    2009-05-31

    The development of a high-throughput functional genomic screening provides a novel and expeditious approach in identifying critical genes involved in specific biological processes. Here we describe a cell-based cDNA screening system to identify the transcription activators of BiP, an endoplasmic reticulum (ER) chaperone protein. BiP promoter contains the ER stress element which is commonly present in the genes involved in unfolded protein response (UPR) that regulates protein secretion in cells. Therefore, the positive regulators of BiP may also be utilized to improve the recombinant protein production through modulation of UPR. Four BiP activators, including human UDP-glucose:glycoprotein glucosyltransferase 1 (HUGT1), are identified by the cDNA screening. Overexpression of HUGT1 leads to a significant increase in the production of recombinant erythropoietin, interferon gamma, and monoclonal antibody in HEK293 cells. Our results demonstrate that the cDNA screening for BiP activators may be effective to identify the novel BiP regulators and HUGT1 may serve as an ideal target gene for improving the recombinant protein production in mammalian cells. PMID:19466607

  3. Immunoregulatory activities of human immunodeficiency virus (HIV) proteins: Effect of HIV recombinant and synthetic peptides on immunoglobulin synthesis and proliferative responses by normal lymphocytes

    SciTech Connect

    Nair, M.P.N.; Pottathil, R.; Heimer, E.P.; Schwartz, S.A.

    1988-09-01

    Recombinant and synthetic peptides corresponding to envelope proteins of the human immunodeficiency virus (HIV) were examined for their effects on the activities of lymphocytes from normal donors in vitro. Although lymphocytes cultured with env-gag peptides produced significant amounts of IgG, addition of env-gag peptides to a pokeweed mitogen-induced B-cell activation system resulted in suppression of immunoglobulin synthesis by normal lymphocytes. Recombinant antigens, env-gag and env-80 dihydrofolate reductase (DHFR), produced a substantial proliferative response by peripheral blood mononuclear cells (PBMC) as determined by (/sup 3/H)thymidine incorporation. PBMC precultured with HIV synthetic peptide env 578-608 also manifested significant proliferative responses as compared to control cultures. CD3/sup +/ lymphocytes precultured with recombinant HIV antigens, env-gag and env-80 DHFR, and synthetic HIV peptide, env 487-511, showed moderate but significant proliferative responses. Both recombinant antigens and synthetic peptides also produced a dose-dependent stimulatory effect on proliferation by CD3/sup /minus// lymphocytes. These studies demonstrate that recombinant and synthetic peptides of the HIV genome express immunoregulatory T- and B-cell epitopes. Identification of unique HIV epitopes with immunogenic and immunoregulatory activities is necessary for the development of an effective vaccine against HIV infection.

  4. Generation of active immunotoxins containing recombinant restrictocin.

    PubMed

    Rathore, D; Batra, J K

    1996-05-01

    Restrictocin, a toxin produced by the fungus Aspergillus restrictus, is a potent inhibitor of eukaryotic protein synthesis. Recombinant restrictocin was made in Escherichia coli and purified to homogeneity in large amounts. The recombinant protein was found to be poorly immunogenic in mice with low toxicity, when injected intraperitoneally. Two immunotoxins were constructed by coupling the recombinant restrictocin to an antibody to the human transferrin receptor, using a cleavable and a stable linkage. The immunotoxins so generated showed specific cytotoxic activity toward receptor bearing cells in tissue culture. Immunotoxin with a cleavable linkage, however, was more active than that containing a stable linkage. Restrictocin appears to be a promising candidate to be developed as a chimeric toxin for targeted therapy. PMID:8630074

  5. [The construction of recombinant adenovirus expressing bifunctional fusion protein sCAR-EGF and the detection of its activity].

    PubMed

    Ren, Peng-Kang; Wang, Feng; Li, Hui-Ming; Li, Zong-Hai; Huang, Qian

    2006-09-01

    To improve the targeting of adenovirus vector for gene therapy, a fusion gene sCAR-EGF, in which epidermal growth factor gene was fused to the 3' end of extracellular Coxsackie virus-adenovirus receptor gene, was constructed and cloned into shuttle plasmid pDC315 to obtain a recombinant plasmid pDC315-sCAR-EGF. With the AdMax system, AD-293 cells were co-transfected with pDC315-sCAR-EGF and adenovirus genomic plasmid pBHGloxdeltaE13cre. Through high efficiency site specific recombination, a replication-defective adenovirus Ad5-CMV-sCAR-EGF was constructed. The recombinant adenovirus was analyzed by PCR and Western blotting, the results indicated that Ad5-CMV-sCAR-EGF contained the fusion gene sCAR-EGF, and the adenovirus infected cells was induced to produce and secrete the fusion protein into the supernatant. We have demonstrated that the fusion protein sCAR-EGF is helpful for elevating the infection efficiency of Ad5-CMV-luc with the reporter gene in vitro, which providing a new approach to the gene therapy for tumors overexpressing EGFR. PMID:17037191

  6. 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

  7. Recombinant human activated protein C resets thrombin generation in patients with severe sepsis – a case control study

    PubMed Central

    de Pont, Anne-Cornélie JM; Bakhtiari, Kamran; Hutten, Barbara A; de Jonge, Evert; Vroom, Margreeth B; Meijers, Joost CM; Büller, Harry R; Levi, Marcel

    2005-01-01

    Introduction Recombinant human activated protein C (rhAPC) is the first drug for which a reduction of mortality in severe sepsis has been demonstrated. However, the mechanism by which this reduction in mortality is achieved is still not clearly defined. The aim of the present study was to evaluate the dynamics of the anticoagulant, anti-inflammatory and pro-fibrinolytic action of rhAPC in patients with severe sepsis, by comparing rhAPC-treated patients with case controls. Methods In this prospectively designed multicenter case control study, 12 patients who were participating in the ENHANCE study, an open-label study of rhAPC in severe sepsis, were treated intravenously with rhAPC at a constant rate of 24 μg/kg/h for a total of 96 h. Twelve controls with severe sepsis matching the inclusion criteria received standard therapy. The treatment was started within 48 h after the onset of organ failure. Blood samples were taken before the start of the infusion and at 4, 8, 24, 48, 96 and 168 h, for determination of parameters of coagulation and inflammation. Results Sepsis-induced thrombin generation as measured by thrombin-antithrombin complexes and prothrombin fragment F1+2, was reset by rhAPC within the first 8 h of infusion. The administration of rhAPC did not influence parameters of fibrinolysis and inflammation. There was no difference in outcome or occurrence of serious adverse events between the treatment group and the control group. Conclusion Sepsis-induced thrombin generation in severely septic patients is reset by rhAPC within the first 8 h of infusion without influencing parameters of fibrinolysis and inflammation. PMID:16277710

  8. Potent antitumor activities of recombinant human PDCD5 protein in combination with chemotherapy drugs in K562 cells

    SciTech Connect

    Shi, Lin; Song, Quansheng; Zhang, Yingmei; Lou, Yaxin; Wang, Yanfang; Tian, Linjie; Zheng, Yi; Ma, Dalong; Ke, Xiaoyan; Wang, Ying

    2010-05-28

    Conventional chemotherapy is still frequently used. Programmed cell death 5 (PDCD5) enhances apoptosis of various tumor cells triggered by certain stimuli and is lowly expressed in leukemic cells from chronic myelogenous leukemia patients. Here, we describe for the first time that recombinant human PDCD5 protein (rhPDCD5) in combination with chemotherapy drugs has potent antitumor effects on chronic myelogenous leukemia K562 cells in vitro and in vivo. The antitumor efficacy of rhPDCD5 protein with chemotherapy drugs, idarubicin (IDR) or cytarabine (Ara-C), was examined in K562 cells in vitro and K562 xenograft tumor models in vivo. rhPDCD5 protein markedly increased the apoptosis rates and decreased the colony-forming capability of K562 cells after the combined treatment with IDR or Ara-C. rhPDCD5 protein by intraperitoneal administration dramatically improved the antitumor effects of IDR treatment in the K562 xenograft model. The tumor sizes and cell proliferation were significantly decreased; and TUNEL positive cells were significantly increased in the combined group with rhPDCD5 protein and IDR treatment compared with single IDR treatment groups. rhPDCD5 protein, in combination with IDR, has potent antitumor effects on chronic myelogenous leukemia K562 cells and may be a novel and promising agent for the treatment of chronic myelogenous leukemia.

  9. Recombinant human bone morphogenetic protein induces bone formation.

    PubMed Central

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

    1990-01-01

    We have purified and characterized active recombinant human bone morphogenetic protein (BMP) 2A. Implantation of the recombinant protein in rats showed that a single BMP can induce bone formation in vivo. A dose-response and time-course study using the rat ectopic bone formation assay revealed that implantation of 0.5-115 micrograms of partially purified recombinant human BMP-2A resulted in cartilage by day 7 and bone formation by day 14. The time at which bone formation occurred was dependent on the amount of BMP-2A implanted; at high doses bone formation could be observed at 5 days. The cartilage- and bone-inductive activity of the recombinant BMP-2A is histologically indistinguishable from that of bone extracts. Thus, recombinant BMP-2A has therapeutic potential to promote de novo bone formation in humans. Images PMID:2315314

  10. Recombinant Treponema pallidum Protein Tp0965 Activates Endothelial Cells and Increases the Permeability of Endothelial Cell Monolayer

    PubMed Central

    Zhang, Rui-Li; Zhang, Jing-Ping; Wang, Qian-Qiu

    2014-01-01

    The recombinant Treponema pallidum protein Tp0965 (rTp0965), one of the many proteins derived from the genome of T. pallidum subsp. pallidum, shows strong immunogenicity and immunoreactivity. In this study, we investigated the effects of rTp0965 on the endothelial barrier. Treatment of human umbilical vein endothelial cells (HUVECs) with rTp0965 resulted in increased levels of ICAM-1, E-selectin, and MCP-1 mRNA and protein expression. These increases contributed to the adhesion and chemataxis of monocytes (THP-1 cells) to HUVECs preincubated with rTp0965. In addition, rTp0965 induced reorganization of F-actin and decreased expression of claudin-1 in HUVECs. Interestingly, inhibition of the RhoA/ROCK signal pathway protected against rTp0965-induced higher endothelial permeability as well as transendothelial migration of monocytes. These data indicate that Tp0965 protein may play an important role in the immunopathogenesis of syphilis. PMID:25514584

  11. Recombinant Treponema pallidum protein Tp0965 activates endothelial cells and increases the permeability of endothelial cell monolayer.

    PubMed

    Zhang, Rui-Li; Zhang, Jing-Ping; Wang, Qian-Qiu

    2014-01-01

    The recombinant Treponema pallidum protein Tp0965 (rTp0965), one of the many proteins derived from the genome of T. pallidum subsp. pallidum, shows strong immunogenicity and immunoreactivity. In this study, we investigated the effects of rTp0965 on the endothelial barrier. Treatment of human umbilical vein endothelial cells (HUVECs) with rTp0965 resulted in increased levels of ICAM-1, E-selectin, and MCP-1 mRNA and protein expression. These increases contributed to the adhesion and chemataxis of monocytes (THP-1 cells) to HUVECs preincubated with rTp0965. In addition, rTp0965 induced reorganization of F-actin and decreased expression of claudin-1 in HUVECs. Interestingly, inhibition of the RhoA/ROCK signal pathway protected against rTp0965-induced higher endothelial permeability as well as transendothelial migration of monocytes. These data indicate that Tp0965 protein may play an important role in the immunopathogenesis of syphilis. PMID:25514584

  12. The V(D)J recombination activating protein RAG2 consists of a six-bladed propeller and a PHD fingerlike domain, as revealed by sequence analysis.

    PubMed

    Callebaut, I; Mornon, J P

    1998-08-01

    The RAG1 and RAG2 proteins play a crucial role in V(D)J recombination by cooperating to make specific double-stranded DNA breaks at a pair of recombination signal sequences (RSSs). However, the exact function they perform has heretofore remained elusive. Using a combination of sensitive methods of sequence analysis, we show here that the active core region of the RAG2 protein, confined to the first three quarters of its sequence, is in fact composed of a six-fold repeat of a 50-residue motif which is related to the kelch/mipp motif. This motif, which forms a four-stranded twisted antiparallel beta sheet, is arranged in a circular formation like blades of a propeller or turbine. Given the known properties of the beta-propeller fold in mediating protein-protein interactions, it is proposed that this six-laded propeller structure of the RAG2 active core would play a crucial role in the tight complex formed by the RAG1 and RAG2 proteins and RSSs. Moreover, the presence of a plant homeodomain finger-like motif in the last quarter of the RAG2 sequence suggests a potential interaction of this domain with chromatin components. PMID:9760994

  13. Mapping and Quantitation of the Interaction between the Recombination Activating Gene Proteins RAG1 and RAG2.

    PubMed

    Zhang, Yu-Hang; Shetty, Keerthi; Surleac, Marius D; Petrescu, Andrei J; Schatz, David G

    2015-05-01

    The RAG endonuclease consists of RAG1, which contains the active site for DNA cleavage, and RAG2, an accessory factor whose interaction with RAG1 is critical for catalytic function. How RAG2 activates RAG1 is not understood. Here, we used biolayer interferometry and pulldown assays to identify regions of RAG1 necessary for interaction with RAG2 and to measure the RAG1-RAG2 binding affinity (KD ∼0.4 μM) (where RAG1 and RAG2 are recombination activating genes 1 or 2). Using the Hermes transposase as a guide, we constructed a 36-kDa "mini" RAG1 capable of interacting robustly with RAG2. Mini-RAG1 consists primarily of the catalytic center and the residues N-terminal to it, but it lacks a zinc finger region in RAG1 previously implicated in binding RAG2. The ability of Mini-RAG1 to interact with RAG2 depends on a predicted α-helix (amino acids 997-1008) near the RAG1 C terminus and a region of RAG1 from amino acids 479 to 559. Two adjacent acidic amino acids in this region (Asp-546 and Glu-547) are important for both the RAG1-RAG2 interaction and recombination activity, with Asp-546 of particular importance. Structural modeling of Mini-RAG1 suggests that Asp-546/Glu-547 lie near the predicted 997-1008 α-helix and components of the active site, raising the possibility that RAG2 binding alters the structure of the RAG1 active site. Quantitative Western blotting allowed us to estimate that mouse thymocytes contain on average ∼1,800 monomers of RAG1 and ∼15,000 molecules of RAG2, implying that nuclear concentrations of RAG1 and RAG2 are below the KD value for their interaction, which could help limit off-target RAG activity. PMID:25745109

  14. Mapping and Quantitation of the Interaction between the Recombination Activating Gene Proteins RAG1 and RAG2*♦

    PubMed Central

    Zhang, Yu-Hang; Shetty, Keerthi; Surleac, Marius D.; Petrescu, Andrei J.; Schatz, David G.

    2015-01-01

    The RAG endonuclease consists of RAG1, which contains the active site for DNA cleavage, and RAG2, an accessory factor whose interaction with RAG1 is critical for catalytic function. How RAG2 activates RAG1 is not understood. Here, we used biolayer interferometry and pulldown assays to identify regions of RAG1 necessary for interaction with RAG2 and to measure the RAG1-RAG2 binding affinity (KD ∼0.4 μm) (where RAG1 and RAG2 are recombination activating genes 1 or 2). Using the Hermes transposase as a guide, we constructed a 36-kDa “mini” RAG1 capable of interacting robustly with RAG2. Mini-RAG1 consists primarily of the catalytic center and the residues N-terminal to it, but it lacks a zinc finger region in RAG1 previously implicated in binding RAG2. The ability of Mini-RAG1 to interact with RAG2 depends on a predicted α-helix (amino acids 997–1008) near the RAG1 C terminus and a region of RAG1 from amino acids 479 to 559. Two adjacent acidic amino acids in this region (Asp-546 and Glu-547) are important for both the RAG1-RAG2 interaction and recombination activity, with Asp-546 of particular importance. Structural modeling of Mini-RAG1 suggests that Asp-546/Glu-547 lie near the predicted 997-1008 α-helix and components of the active site, raising the possibility that RAG2 binding alters the structure of the RAG1 active site. Quantitative Western blotting allowed us to estimate that mouse thymocytes contain on average ∼1,800 monomers of RAG1 and ∼15,000 molecules of RAG2, implying that nuclear concentrations of RAG1 and RAG2 are below the KD value for their interaction, which could help limit off-target RAG activity. PMID:25745109

  15. RAD6 Promotes Homologous Recombination Repair by Activating the Autophagy-Mediated Degradation of Heterochromatin Protein HP1

    PubMed Central

    Chen, Su; Wang, Chen; Sun, Luxi; Wang, Da-Liang; Chen, Lu; Huang, Zhuan; Yang, Qi; Gao, Jie; Yang, Xi-Bin; Chang, Jian-Feng; Chen, Ping; Lan, Li

    2014-01-01

    Efficient DNA double-strand break (DSB) repair is critical for the maintenance of genome stability. Unrepaired or misrepaired DSBs cause chromosomal rearrangements that can result in severe consequences, such as tumorigenesis. RAD6 is an E2 ubiquitin-conjugating enzyme that plays a pivotal role in repairing UV-induced DNA damage. Here, we present evidence that RAD6 is also required for DNA DSB repair via homologous recombination (HR) by specifically regulating the degradation of heterochromatin protein 1α (HP1α). Our study indicates that RAD6 physically interacts with HP1α and ubiquitinates HP1α at residue K154, thereby promoting HP1α degradation through the autophagy pathway and eventually leading to an open chromatin structure that facilitates efficient HR DSB repair. Furthermore, bioinformatics studies have indicated that the expression of RAD6 and HP1α exhibits an inverse relationship and correlates with the survival rate of patients. PMID:25384975

  16. Recombineering BAC transgenes for protein tagging.

    PubMed

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

    2011-02-01

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

  17. Interaction of 5-HT1B/D ligands with recombinant h 5-HT1A receptors: intrinsic activity and modulation by G-protein activation state.

    PubMed

    Pauwels, P J; Palmier, C; Dupuis, D S; Colpaert, F C

    1998-05-01

    Many 5-HT1B/D receptor ligands have affinity for 5-HT1A receptors. In the present study, the intrinsic activity of a series of 5-HT1B/D ligands was investigated at human 5-HT1A (h 5-HT1A) receptors by measuring G-protein activation in recombinant C6-glial and HeLa membranes, using agonist-stimulated [35S]GTPgammaS binding. In these two membrane preparations, the density of h 5-HT1A receptors (i.e., 246 to 320 fmol mg(-1) protein) and of their G-proteins, and the receptor: G-protein density ratio (0.08 to 0.18) appeared to be similar. It was found that: (i) the maximal [35S]GTPgammaS binding responses induced by the 5-HT1B/D receptor ligands in the HeLa preparation at 30 microM GDP were comparable to that of the native agonist 5-HT; (ii) as compared to 5-HT (1.00), similar potencies but lower maximal responses were observed in the C6-glial preparation at 0.3 microM GDP for zolmitriptan (0.89), dihydroergotamine (0.81), rizatriptan (0.71), CP122638 (0.69), naratriptan (0.60) and sumatriptan (0.53); and that (iii) maximal [35S]GTPgammaS binding responses induced by 5-HT1B/D ligands in the C6-glial preparation were either unaffected or significantly enhanced by increasing the GDP concentration from 0.3 to 30 microM and higher concentrations. These features differ from those observed with 5-HT1A receptor agonists; the latter display the same rank order of potency and efficacy in both membrane preparations, and increasing the amount of GDP with C6-glial membranes results in an attenuation of both the agonist's maximal effect and the apparent potency of partial agonists. The differential regulation of 5-HT1A and 5-HT1B/D agonist responses by GDP suggests that different G-protein subtypes are involved upon 5-HT1A receptor activation by 5-HT1A and 5-HT1B/D agonists. PMID:9650800

  18. Recombinant protein blends: silk beyond natural design.

    PubMed

    Dinjaski, Nina; Kaplan, David L

    2016-06-01

    Recombinant DNA technology and new material concepts are shaping future directions in biomaterial science for the design and production of the next-generation biomaterial platforms. Aside from conventionally used synthetic polymers, numerous natural biopolymers (e.g., silk, elastin, collagen, gelatin, alginate, cellulose, keratin, chitin, polyhydroxyalkanoates) have been investigated for properties and manipulation via bioengineering. Genetic engineering provides a path to increase structural and functional complexity of these biopolymers, and thereby expand the catalog of available biomaterials beyond that which exists in nature. In addition, the integration of experimental approaches with computational modeling to analyze sequence-structure-function relationships is starting to have an impact in the field by establishing predictive frameworks for determining material properties. Herein, we review advances in recombinant DNA-mediated protein production and functionalization approaches, with a focus on hybrids or combinations of proteins; recombinant protein blends or 'recombinamers'. We highlight the potential biomedical applications of fibrous protein recombinamers, such as Silk-Elastin Like Polypeptides (SELPs) and Silk-Bacterial Collagens (SBCs). We also discuss the possibility for the rationale design of fibrous proteins to build smart, stimuli-responsive biomaterials for diverse applications. We underline current limitations with production systems for these proteins and discuss the main trends in systems/synthetic biology that may improve recombinant fibrous protein design and production. PMID:26686863

  19. Recombinant activated protein C treatment improves tissue perfusion and oxygenation in septic patients measured by near-infrared spectroscopy

    PubMed Central

    2009-01-01

    Introduction The purpose was to test the hypothesis that muscle perfusion, oxygenation, and microvascular reactivity would improve in patients with severe sepsis or septic shock during treatment with recombinant activated protein C (rh-aPC) (n = 11) and to explore whether these parameters are related to macrohemodynamic indices, metabolic status or Sequential Organ Failure Assessment (SOFA) score. Patients with contraindications to rh-aPC were used as a control group (n = 5). Materials and methods Patients were sedated, intubated, mechanically ventilated, and hemodynamically monitored with the PiCCO system. Tissue oxygen saturation (StO2) was measured using near-infrared spectroscopy (NIRS) during the vascular occlusion test (VOT). Baseline StO2 (StO2 baseline), rate of decrease in StO2 during VOT (StO2 downslope), and rate of increase in StO2 during the reperfusion phase were (StO2 upslope) determined. Data were collected before (T0), during (24 hours (T1a), 48 hours (T1b), 72 hours (T1c) and 96 hours (T1d)) and 6 hours after stopping rh-aPC treatment (T2) and at the same times in the controls. At every assessment, hemodynamic and metabolic parameters were registered and the SOFA score calculated. Results The mean ± standard deviation Acute Physiology and Chronic Health Evaluation II score was 26.3 ± 6.6 and 28.6 ± 5.3 in rh-aPC and control groups, respectively. There were no significant differences in macrohemodynamic parameters between the groups at all the time points. In the rh-aPC group, base excess was corrected (P < 0.01) from T1a until T2, and blood lactate was significantly decreased at T1d and T2 (2.8 ± 1.3 vs. 1.9 ± 0.7 mmol/l; P < 0.05). In the control group, base excess was significantly corrected at T1a, T1b, T1c, and T2 (P < 0.05). The SOFA score was significantly lower in the rh-aPC group compared with the controls at T2 (7.9 ± 2.2 vs. 12.2 ± 3.2; P < 0.05). There were no differences between groups in StO2 baseline. StO2 downslope in the rh

  20. Refolding and purification of recombinant L-asparaginase from inclusion bodies of E. coli into active tetrameric protein.

    PubMed

    Upadhyay, Arun K; Singh, Anupam; Mukherjee, K J; Panda, Amulya K

    2014-01-01

    A tetrameric protein of therapeutic importance, Escherichia coli L-asparaginase-II was expressed in Escherichia coli as inclusion bodies (IBs). Asparaginase IBs were solubilized using low concentration of urea and refolded into active tetrameric protein using pulsatile dilution method. Refolded asparaginase was purified in two steps by ion-exchange and gel filtration chromatographic techniques. The recovery of bioactive asparaginase from IBs was around 50%. The melting temperature (Tm) of the purified asparaginase was found to be 64°C. The specific activity of refolded, purified asparaginase was found to be comparable to the commercial asparaginase (190 IU/mg). Enzymatic activity of the refolded asparaginase was high even at four molar urea solutions, where the IB aggregates are completely solubilized. From the comparison of chemical denaturation data and activity at different concentrations of guanidine hydrochloride, it was observed that dissociation of monomeric units precedes the complete loss of helical secondary structures. Protection of the existing native-like protein structure during solubilization of IB aggregates with 4 M urea improved the propensity of monomer units to form oligomeric structure. Our mild solubilization technique retaining native-like structures, improved recovery of asparaginase in bioactive tetrameric form. PMID:25309524

  1. Refolding and purification of recombinant L-asparaginase from inclusion bodies of E. coli into active tetrameric protein

    PubMed Central

    Upadhyay, Arun K.; Singh, Anupam; Mukherjee, K. J.; Panda, Amulya K.

    2014-01-01

    A tetrameric protein of therapeutic importance, Escherichia coli L-asparaginase-II was expressed in Escherichia coli as inclusion bodies (IBs). Asparaginase IBs were solubilized using low concentration of urea and refolded into active tetrameric protein using pulsatile dilution method. Refolded asparaginase was purified in two steps by ion-exchange and gel filtration chromatographic techniques. The recovery of bioactive asparaginase from IBs was around 50%. The melting temperature (Tm) of the purified asparaginase was found to be 64°C. The specific activity of refolded, purified asparaginase was found to be comparable to the commercial asparaginase (190 IU/mg). Enzymatic activity of the refolded asparaginase was high even at four molar urea solutions, where the IB aggregates are completely solubilized. From the comparison of chemical denaturation data and activity at different concentrations of guanidine hydrochloride, it was observed that dissociation of monomeric units precedes the complete loss of helical secondary structures. Protection of the existing native-like protein structure during solubilization of IB aggregates with 4 M urea improved the propensity of monomer units to form oligomeric structure. Our mild solubilization technique retaining native-like structures, improved recovery of asparaginase in bioactive tetrameric form. PMID:25309524

  2. Cultivating Insect Cells To Produce Recombinant Proteins

    NASA Technical Reports Server (NTRS)

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

    1996-01-01

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

  3. 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.

  4. 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.

  5. Recombinant prolylcarboxypeptidase activates plasma prekallikrein.

    PubMed

    Shariat-Madar, Zia; Mahdi, Fakhri; Schmaier, Alvin H

    2004-06-15

    The serine protease prolylcarboxypeptidase (PRCP), isolated from human umbilical vein endothelial cells (HUVECs), is a plasma prekallikrein (PK) activator. PRCP cDNA was cloned in pMT/BIP/V5-HIS-C, transfected into Schneider insect (S2) cells, and purified from serum-free media. Full-length recombinant PRCP (rPRCP) activates PK when bound to high-molecular-weight kininogen (HK). Recombinant PRCP is inhibited by leupeptin, angiotensin II, bradykinin, anti-PRCP, diisopropyl-fluorophosphonate (DFP), phenylmethylsulfonyl fluoride (PMSF), and Z-Pro-Proaldehyde-dimethyl acetate, but not by 1 mM EDTA (ethylenediaminetetraacetic acid), bradykinin 1-5, or angiotensin 1-7. Corn trypsin inhibitor binds to prekallikrein to prevent rPRCP activation, but it does not directly inhibit the active site of either enzyme. Unlike factor XIIa, the ability of rPRCP to activate PK is blocked by angiotensin II, not by neutralizing antibody to factor XIIa. PRCP antigen is detected on HUVEC membranes using flow cytometry and laser scanning confocal microscopy. PRCP antigen does not colocalize with LAMP1 on nonpermeabilized HUVECs, but it partially colocalizes in permeabilized cells. PRCP colocalizes with all the HK receptors, gC1qR, uPAR, and cytokeratin 1 antigen, on nonpermeabilized HUVECs. PRCP activity and antigen expression on cultured HUVECs are blocked by a morpholino antisense oligonucleotide. These investigations indicate that rPRCP is functionally identical to isolated HUVEC PRCP and is a major HUVEC membrane-expressed, PK-activating enzyme detected in the intravascular compartment. PMID:14996700

  6. Production and characterization of active recombinant interleukin-12/eGFP fusion protein in stably-transfected DF1 chicken cells.

    PubMed

    Wu, Hsing Chieh; Chen, Yu San; Shen, Pin Chun; Shien, Jui Hung; Lee, Long Huw; Chiu, Hua Hsien

    2015-01-01

    The adjuvant activity of chicken interleukin-12 (chIL-12) protein has been described as similar to that of mammalian IL-12. Recombinant chIL-12 can be produced using several methods, but chIL-12 production in eukaryotic cells is lower than that in prokaryotic cells. Stimulating compounds, such as dimethyl sulfoxide (DMSO), can be added to animal cell cultures to overcome this drawback. In this study, we constructed a cell line, DF1/chIL-12 which stably expressed a fusion protein, chIL-12 and enhanced green fluorescent protein (eGFP) connected by a (G4 S)3 linker sequence. Fusion protein production was increased when cells were cultured in the presence of DMSO. When 1 × 10(6) DF1/chIL-12 cells were inoculated in a T-175 flask containing 30 mL of media, incubated for 15 h, and further cultivated in the presence of 4% DMSO for 48 h, the production of total fusion protein was mostly enhanced compared with the production of total fusion protein by using cell lysates induced with DMSO at other concentrations. The concentrations of the unpurified and purified total fusion proteins in cell lysates were 2,781 ± 2.72 ng mL(-1) and 2,207 ± 3.28 ng mL(-1) , respectively. The recovery rate was 79%. The fusion protein stimulated chicken splenocytes to produce IFN-γ, which was measured using an enzyme-linked immunosorbent assay, in the culture supernatant, indicating that treating DF1/chIL-12 cells with DMSO or producing chIL-12 in a fusion protein form does not have adverse effects on the bioactivity of chIL-12. PMID:25583174

  7. 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

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

    PubMed

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

    2014-01-01

    A metabolic engineering perspective which views recombinant protein expression as a multistep pathway allows us to move beyond vector design and identify the downstream rate limiting steps in expression. In E.coli these are typically at the translational level and the supply of precursors in the form of energy, amino acids and nucleotides. Further recombinant protein production triggers a global cellular stress response which feedback inhibits both growth and product formation. Countering this requires a system level analysis followed by a rational host cell engineering to sustain expression for longer time periods. Another strategy to increase protein yields could be to divert the metabolic flux away from biomass formation and towards recombinant protein production. This would require a growth stoppage mechanism which does not affect the metabolic activity of the cell or the transcriptional or translational efficiencies. Finally cells have to be designed for efficient export to prevent buildup of proteins inside the cytoplasm and also simplify downstream processing. The rational and the high throughput strategies that can be used for the construction of such improved host cell platforms for recombinant protein expression is the focus of this review. PMID:25523647

  9. Recombinant protein scaffolds for tissue engineering.

    PubMed

    Werkmeister, Jerome A; Ramshaw, John A M

    2012-02-01

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

  10. Posttranslational modifications and activity of natural and recombinant tissue factor

    PubMed Central

    Butenas, Saulius; Krudysz-Amblo, Jolanta; Mann, Kenneth G

    2010-01-01

    Tissue factor is a membrane protein, which in a complex with factor VIIa initiates in vivo blood coagulation. Due to the scarcity of natural tissue factor protein, most studies have relied upon recombinant tissue factor forms. However, there have been only cursory experimental comparisons of natural and recombinant tissue factor proteins. Our preliminary data suggested that placental tissue factor in a complex with factor VIIa was more efficient activator of factor X than the recombinant protein. After deglycosylation, both forms of tissue factor showed almost an identical activity in the extrinsic factor Xase. Analyses using tryptic digestion and mass-spectrometry revealed that the levels of glycosylation and the composition of carbohydrates present in natural placental tissue factor were different than those in its recombinant counterpart. These data indicate that natural and recombinant tissue factor proteins differ in their posttranslational modifications and that these differences translate into different cofactor activity. Thus the use of recombinant tissue factor proteins for the quantitation of natural tissue factor is misleading. PMID:20138335

  11. Expression of recombinant AccMRJP1 protein from royal jelly of Chinese honeybee in Pichia pastoris and its proliferation activity in an insect cell line

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Main royal jelly protein 1 (MRJP1) is the most abundant member of the main royal jelly protein (MRJP) family among honeybees. Mature MRJP1 cDNA of the Chinese honeybee (Apis cerana cerana MRJP1, or AccMRJP1) was expressed in Pichia pastoris. SDS-PAGE showed that recombinant AccMRJP1 was identical in...

  12. Soluble FasR ligand-binding domain: high-yield production of active fusion and non-fusion recombinant proteins using the baculovirus/insect cell system.

    PubMed

    Mahiou, J; Abastado, J P; Cabanie, L; Godeau, F

    1998-03-01

    We used the recombinant baculovirus/insect cell system to express two soluble forms of the mouse Fas receptor (mFasR) extracellular domain (ECD): a monomer comprising the entire ligand-binding portion of mFasR followed by a carboxy-terminal hexa-histidine extension aiding purification by immobilized metal affinity chromatography and an immunoadhesin in which the same 148 residues were fused to the Fc portion of a truncated human IgG1 immunoglobulin heavy chain. Both constructs harboured a 24 base pairs insertion placed upstream of the initiating ATG [Peakman, Charles, Sydenham, Gewert, Page, and Makoff (1992) Nucleic Acids Res. 20, 6111-6112]. Despite its hexa-histidine extension, the monovalent recombinant protein from crude culture media failed to bind immobilized Ni2+ unless proteins were first precipitated twice by ammonium sulphate. The overall procedure then yielded approximately 10mg/l of protein which could be purified to near homogeneity using two additional chromatographic steps. The glycosylated polypeptide migrated as a band of Mr=(21-31) x 10(3) in SDS/PAGE and was monomeric in physiological buffers. Under non-reducing conditions, denaturation in 6 M guanidinium chloride was reversible after slow removal of the denaturing agent. The mFasR immunoadhesin was secreted (approximately 5-10 mg/l) as a disulphide-linked homodimer, and endowed with ligand-binding activity since it could bind FasL on the surface of D11S, FasL-expressing cells. When tested for their ability to inhibit FasR-dependent cell lysis, the soluble dimeric immunoadhesin markedly inhibited FasL-mediated cytotoxicity (IC50 approximately 30 nM), and was approximately 6 times as effective as its monomeric counterpart. PMID:9480929

  13. Ca2+ store-independent augmentation of [Ca2+]i responses to G-protein coupled receptor activation in recombinantly TRPC5-expressed rat pheochromocytoma (PC12) cells.

    PubMed

    Ohta, Toshio; Morishita, Masataka; Mori, Yasuo; Ito, Shigeo

    2004-04-01

    Mammalian homologues of the Drosophila canonical transient receptor potential (trp) protein (TRPC) have been implicated to function as receptor-operated Ca(2+) channels (ROCs) or store-operated Ca(2+) channels (SOCs). To determine the role of TRPC5 protein in neural cells, TRPC5 was recombinantly expressed in rat pheochromocytoma cells (PC12) and changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) and Na(+) concentration ([Na(+)](i)) were analyzed. TRPC1 and TRPC3 mRNAs were endogenously expressed in PC12 cells. In TRPC5-expressed cells (TRPC5-cells), the resting [Ca(2+)](i) and [Na(+)](i) were significantly higher than those in control cells. The [Ca(2+)](i) increases induced by bradykinin and uridine 5'-triphosphate were significantly larger in TRPC5-cells. TRPC5 expression did not change in store-operated Ca(2+) entry elicited by thapsigarigin. TRPC5-cells showed larger inward current and increase of [Na(+)](i) in response to BK than control cells. These results suggest that TRPC5 channels expressed in PC12 cells function as ROCs activated by G-protein/phospholipase C coupled receptors, but not as SOCs. PMID:15039106

  14. Recombinant Treponema pallidum rare outer membrane protein 1 (Tromp1) expressed in Escherichia coli has porin activity and surface antigenic exposure.

    PubMed Central

    Blanco, D R; Champion, C I; Exner, M M; Shang, E S; Skare, J T; Hancock, R E; Miller, J N; Lovett, M A

    1996-01-01

    We recently reported the cloning and sequencing of the gene encoding a 31-kDa Treponema pallidum subsp. pallidum rare outer membrane porin protein, designated Tromp1 (D. R. Blanco, C. I. Champion, M. M. Exner, H. Erdjument-Bromage, R. E. W. Hancock, P. Tempst, J. N. Miller, and M. A. Lovett, J. Bacteriol. 177:3556-3562, 1995). Here, we report the stable expression of recombinant Tromp1 (rTromp1) in Escherichia coli. rTromp1 expressed without its signal peptide and containing a 22-residue N-terminal fusion resulted in high-level accumulation of a nonexported soluble protein that was purified to homogeneity by fast protein liquid chromatography (FPLC). Specific antiserum generated to the FPLC-purified rTromp1 fusion identified on immunoblots of T. pallidum the native 31-kDa Tromp1 protein and two higher-molecular-mass oligomeric forms of Tromp1 at 55 and 80 kDa. rTromp1 was also expressed with its native signal peptide by using an inducible T7 promoter. Under these conditions, rTromp1 fractionated predominantly with the E. coli soluble and outer membrane fractions, but not with the inner membrane fraction. rTromp1 isolated from the E. coli outer membrane and reconstituted into planar lipid bilayers showed porin activity based on average single-channel conductances of 0.4 and 0.8 nS in 1 M KCl. Whole-mount immunoelectron microscopy using infection-derived immune serum against T. pallidum indicated that rTromp1 was surface exposed when expressed in E. coli. These findings demonstrate that rTromp1 can be targeted to the E. coli outer membrane, where it has both porin activity and surface antigenic exposure. PMID:8955283

  15. Cytotoxic Effects and Osteogenic Activity of Calcium Sulfate with and without Recombinant Human Bone Morphogenetic Protein 2 and Nano-Hydroxyapatite Adjacent to MG-63 Cell Line

    PubMed Central

    Ghorbanzadeh, Abdollah; Aminsobhani, Mohsen; Khoshzaban, Ahad; Abbaszadeh, Armin; Ghorbanzadeh, Atiyeh; Shamshiri, Ahmad Reza

    2015-01-01

    Objectives: The aim of this study was to assess the cytotoxic effects and osteogenic activity of recombinant human bone morphogenetic protein (rhBMP2) and nano-hydroxyapatite (n-HA) adjacent to MG-63 cell line. Materials and Methods: To assess cytotoxicity, the 4,5-dimethyl thiazolyl-2,5-diphenyl tetrazolium bromide (MTT) assay was used. Alkaline phosphatase (ALP) activity and osteogenic activity were evaluated using Alizarin red and the von Kossa staining and analyzed by one-way ANOVA followed by Tukey’s post hoc test. Results: The n-HA/calcium sulfate (CS) mixture significantly promoted cell growth in comparison to pure CS. Moreover, addition of rhBMP2 to CS (P=0.02) and also mixing CS with n-HA led to further increase in extracellular calcium production and ALP activity (P=0.03). Conclusion: This in vitro study indicates that a scaffold material in combination with an osteoinductive material is effective for bone matrix formation. PMID:26877731

  16. Overview of the Purification of Recombinant Proteins

    PubMed Central

    Wingfield, Paul T.

    2015-01-01

    When the first version of this unit was written in 1995 protein purification of recombinant proteins was based on a variety of standard chromatographic methods and approaches many of which were described and mentioned in this unit and elsewhere in the book. In the interim there has been a shift towards an almost universal usage of the affinity or fusion tag. This may not be the case for biotechnology manufacture where affinity tags can complicate producing proteins under regulatory conditions. Regardless of the protein expression system, questions are asked as to which and how many affinity tags to use, where to attach them in the protein and whether to engineer a self cleavage system or simply leave them on. We will briefly address some of these issues. Also although this overview focuses on E.coli, protein expression and purification from the other commonly used expression systems are mentioned and apart from cell breakage methods, the protein purification methods and strategies are essentially the same. PMID:25829302

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

    PubMed Central

    2014-01-01

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

  18. 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

  19. Immune Response to Recombinant Capsid Proteins of Adenovirus in Humans: Antifiber and Anti-Penton Base Antibodies Have a Synergistic Effect on Neutralizing Activity

    PubMed Central

    Gahéry-Ségard, Hanne; Farace, Françoise; Godfrin, Dominique; Gaston, Jesintha; Lengagne, Renée; Tursz, Thomas; Boulanger, Pierre; Guillet, Jean-Gérard

    1998-01-01

    Replication-deficient adenovirus used in humans for gene therapy induces a strong immune response to the vector, resulting in transient recombinant protein expression and the blocking of gene transfer upon a second administration. Therefore, in this study we examined in detail the capsid-specific humoral immune response in sera of patients with lung cancer who had been given one dose of a replication-defective adenovirus. We analyzed the immune response to the three major components of the viral capsid, hexon (Hx), penton base (Pb), and fiber (Fi). A longitudinal study of the humoral response assayed on adenovirus particle-coated enzyme-linked immunosorbent assay plates showed that patients had preexisting immunity to adenovirus prior to the administration of adenovirus–β-gal. The level of the response increased in three patients after adenovirus administration and remained at a maximum after three months. One patient had a strong immune response to adenovirus prior to treatment, and this response was unaffected by adenovirus administration. Sera collected from the patients were assayed for recognition of each individual viral capsid protein to determine more precisely the molecular basis of the humoral immune response. Clear differences existed in the humoral response to the three major components of the viral capsid in serum from humans. Sequential appearance of these antibodies was observed: anti-Fi antibodies appeared first, followed by anti-Pb antibodies and then by anti-Hx antibodies. Moreover, anti-Fi antibodies preferentially recognized the native trimeric form of Fi protein, suggesting that they recognized conformational epitopes. Our results showed that sera with no neutralizing activity contained only anti-Fi antibodies. In contrast, neutralizing activity was only obtained with sera containing anti-Fi and anti-Pb antibodies. More importantly, we showed that anti-native Fi and anti-Pb antibodies had a synergistic effect on neutralization. The

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

    PubMed Central

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

    2015-01-01

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

  1. Activation of Escherichia coli heat-labile enterotoxins by native and recombinant adenosine diphosphate-ribosylation factors, 20-kD guanine nucleotide-binding proteins.

    PubMed Central

    Lee, C M; Chang, P P; Tsai, S C; Adamik, R; Price, S R; Kunz, B C; Moss, J; Twiddy, E M; Holmes, R K

    1991-01-01

    Escherichia coli heat-labile enterotoxins (LT) are responsible in part for "traveler's diarrhea" and related diarrheal illnesses. The family of LTs comprises two serogroups termed LT-I and LT-II; each serogroup includes two or more antigenic variants. The effects of LTs result from ADP ribosylation of Gs alpha, a stimulatory component of adenylyl cyclase; the mechanism of action is identical to that of cholera toxin (CT). The ADP-ribosyltransferase activity of CT is enhanced by 20-kD guanine nucleotide-binding proteins, known as ADP-ribosylation factors or ARFs. These proteins directly activate the CTA1 catalytic unit and stimulate its ADP ribosylation of Gs alpha, other proteins, and simple guanidino compounds (e.g., agmatine). Because of the similarities between CT and LTs, we investigated the effects of purified bovine brain ARF and a recombinant form of bovine ARF synthesized in Escherichia coli on LT activity. ARF enhanced the LT-I-, LT-IIa-, and LT-IIb-catalyzed ADP ribosylation of agmatine, as well as the auto-ADP ribosylation of the toxin catalytic unit. Stimulation of ADP-ribosylagmatine formation by LTs and CT in the presence of ARF was GTP dependent and enhanced by sodium dodecyl sulfate. With agmatine as substrate, LT-IIa and LT-IIb exhibited less than 1% the activity of CT and LT-Ih. CT and LTs catalyzed ADP-ribosyl-Gs alpha formation in a reaction dependent on ARF, GTP, and dimyristoyl phosphatidylcholine/cholate. With Gs alpha as substrate, the ADP-ribosyltransferase activities of the toxins were similar, although CT and LT-Ih appeared to be slightly more active than LT-IIa and LT-IIb. Thus, LT-IIa and LT-IIb appear to differ somewhat from CT and LT-Ih in substrate specificity. Responsiveness to stimulation by ARF, GTP, and phospholipid/detergent as well as the specificity of ADP-ribosyltransferase activity are functions of LTs from serogroups LT-I and LT-II that are shared with CT. Images PMID:1902492

  2. The Characterization of Escherichia coli CpdB as a Recombinant Protein Reveals that, besides Having the Expected 3´-Nucleotidase and 2´,3´-Cyclic Mononucleotide Phosphodiesterase Activities, It Is Also Active as Cyclic Dinucleotide Phosphodiesterase

    PubMed Central

    López-Villamizar, Iralis; Cabezas, Alicia; Pinto, Rosa María; Canales, José; Ribeiro, João Meireles; Cameselle, José Carlos; Costas, María Jesús

    2016-01-01

    Endogenous cyclic diadenylate phosphodiesterase activity was accidentally detected in lysates of Escherichia coli BL21. Since this kind of activity is uncommon in Gram-negative bacteria, its identification was undertaken. After partial purification and analysis by denaturing gel electrophoresis, renatured activity correlated with a protein identified by fingerprinting as CpdB (cpdB gene product), which is annotated as 3´-nucleotidase / 2´,3´-cyclic-mononucleotide phosphodiesterase, and it is synthesized as a precursor protein with a signal sequence removable upon export to the periplasm. It has never been studied as a recombinant protein. The coding sequence of mature CpdB was cloned and expressed as a GST fusion protein. The study of the purified recombinant protein, separated from GST, confirmed CpdB annotation. The assay of catalytic efficiencies (kcat/Km) for a large substrate set revealed novel CpdB features, including very high efficiencies for 3´-AMP and 2´,3´-cyclic mononucleotides, and previously unknown activities on cyclic and linear dinucleotides. The catalytic efficiencies of the latter activities, though low in relative terms when compared to the major ones, are far from negligible. Actually, they are perfectly comparable to those of the ‘average’ enzyme and the known, bona fide cyclic dinucleotide phosphodiesterases. On the other hand, CpdB differs from these enzymes in its extracytoplasmic location and in the absence of EAL, HD and DHH domains. Instead, it contains the domains of the 5´-nucleotidase family pertaining to the metallophosphoesterase superfamily, although CpdB lacks 5´-nucleotidase activity. The possibility that the extracytoplasmic activity of CpdB on cyclic dinucleotides could have physiological meaning is discussed. PMID:27294396

  3. Co-factor activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, Carl W.; Mangel, Walter F.

    1996-08-06

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  4. Co-factor activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, C.W.; Mangel, W.F.

    1996-08-06

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying the peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

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

    DOEpatents

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

    1996-01-01

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

  6. A Novel Recombinant Anti-CD22 Immunokinase Delivers Proapoptotic Activity of Death-Associated Protein Kinase (DAPK) and Mediates Cytotoxicity in Neoplastic B Cells.

    PubMed

    Lilienthal, Nils; Lohmann, Gregor; Crispatzu, Giuliano; Vasyutina, Elena; Zittrich, Stefan; Mayer, Petra; Herling, Carmen Diana; Tur, Mehmet Kemal; Hallek, Michael; Pfitzer, Gabriele; Barth, Stefan; Herling, Marco

    2016-05-01

    The serine/threonine death-associated protein kinases (DAPK) provide pro-death signals in response to (oncogenic) cellular stresses. Lost DAPK expression due to (epi)genetic silencing is found in a broad spectrum of cancers. Within B-cell lymphomas, deficiency of the prototypic family member DAPK1 represents a predisposing or early tumorigenic lesion and high-frequency promoter methylation marks more aggressive diseases. On the basis of protein studies and meta-analyzed gene expression profiling data, we show here that within the low-level context of B-lymphocytic DAPK, particularly CLL cells have lost DAPK1 expression. To target this potential vulnerability, we conceptualized B-cell-specific cytotoxic reconstitution of the DAPK1 tumor suppressor in the format of an immunokinase. After rounds of selections for its most potent cytolytic moiety and optimal ligand part, a DK1KD-SGIII fusion protein containing a constitutive DAPK1 mutant, DK1KD, linked to the scFv SGIII against the B-cell-exclusive endocytic glyco-receptor CD22 was created. Its high purity and large-scale recombinant production provided a stable, selectively binding, and efficiently internalizing construct with preserved robust catalytic activity. DK1KD-SGIII specifically and efficiently killed CD22-positive cells of lymphoma lines and primary CLL samples, sparing healthy donor- or CLL patient-derived non-B cells. The mode of cell death was predominantly PARP-mediated and caspase-dependent conventional apoptosis as well as triggering of an autophagic program. The notoriously high apoptotic threshold of CLL could be overcome by DK1KD-SGIII in vitro also in cases with poor prognostic features, such as therapy resistance. The manufacturing feasibility of the novel CD22-targeting DAPK immunokinase and its selective antileukemic efficiency encourage intensified studies towards specific clinical application. Mol Cancer Ther; 15(5); 971-84. ©2016 AACR. PMID:26826117

  7. Antitumor activity of recombinant Bacille Calmette-Guérin secreting interleukin-15-Ag85B fusion protein against bladder cancer.

    PubMed

    Takeuchi, Ario; Eto, Masatoshi; Tatsugami, Katsunori; Shiota, Masaki; Yamada, Hisakata; Kamiryo, Yoriyuki; Dejima, Takashi; Kashiwagi, Eiji; Kiyoshima, Keijiro; Inokuchi, Junichi; Takahashi, Ryosuke; Yokomizo, Akira; Ohara, Naoya; Yoshikai, Yasunobu

    2016-06-01

    Mycobacterium bovis Bacillus Calmette-Guérin (BCG) is used for the treatment of bladder cancer. The recruitment of neutrophlis to the bladder after BCG instillation exerts anti-tumor activity against bladder tumor. We have recently demonstrated that interleukin (IL)-17A produced by γδ T cells played a role in the recruitment of neutrophlis to the bladder after BCG instillation. IL-15 is known to play an important role in neutrophil migration during inflammation. We previously constructed a recombinant BCG strain expressing the fusion protein of IL-15 and Ag85B (BCG-IL-15) for prevention of Mycobacterium tuberculosis infection. Here we compared the efficacy of the BCG-IL-15 in protection against bladder cancer with that of rBCG-Ag85B (BCG). Six-week-old female C57BL/6 mice were inoculated with MB49 bladder tumor cells in the bladder and subsequently intravesically inoculated with BCG or BCG-IL-15. BCG-IL-15 treatment significantly prolonged survival of mice inoculated with bladder cancer cells compared with BCG treatment. Infiltration of neutrophils was significantly elevated in BCGB-IL-15 treated mice accompanied by increased chemokines (MIP-2 and MIP-1α) in the bladder. Thus, BCG-IL-15 exerted additive effect on Infiltration of neutrophils in the bladder. BCG-IL-15 may be a promising drug for non-muscle invasive bladder cancer. PMID:27093372

  8. Multivalent Recombinant Protein Vaccine against Coccidioidomycosis

    PubMed Central

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

    2006-01-01

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

  9. Recombinant protein vaccines produced in insect cells.

    PubMed

    Cox, Manon M J

    2012-02-27

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

  10. Expression of the immediate early IE180 protein under the control of the hTERT and CEA tumor-specific promoters in recombinant pseudorabies viruses: Effects of IE180 protein on promoter activity and apoptosis induction.

    PubMed

    Lerma, L; Alcalá, S; Piñero, C; Torres, M; Martin, B; Lim, F; Sainz, B; Tabarés, E

    2016-01-15

    Since the pseudorabies virus (PRV) genome encodes for a single immediate-early protein, IE180, we reasoned that this strong transactivating protein could represent a key regulatory switch that could be genetically manipulated in order to alter its tropism towards cancer cells. We therefore initiated studies to test whether the human telomerase reverse transcriptase (hTERT) and carcinoembryonic antigen (CEA) tumor promoters could functionally replace the IE180 promoter. We show that both promoters can functionally substitute the IE180 promoter in plasmid constructs and recombinant viruses, and observed that IE180 differentially auto-regulated each promoter tested, with PRV IE180 negatively regulating the hTERT promoter but positively hyper-activating the CEA promoter. Interestingly, we also observed that the recombinant PRV-TER and PRV-CEA viruses preferentially replicated in diverse cancer cell lines compared to control non-cancer cells, and the PRV-CEA was capable of additionally inducing a profound apoptotic phenotype which we correlated to the overexpression of IE180. PMID:26590793

  11. Integrated continuous production of recombinant therapeutic proteins.

    PubMed

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

    2012-12-01

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

  12. Daedalus: a robust, turnkey platform for rapid production of decigram quantities of active recombinant proteins in human cell lines using novel lentiviral vectors

    PubMed Central

    Bandaranayake, Ashok D.; Correnti, Colin; Ryu, Byoung Y.; Brault, Michelle; Strong, Roland K.; Rawlings, David J.

    2011-01-01

    A key challenge for the academic and biopharmaceutical communities is the rapid and scalable production of recombinant proteins for supporting downstream applications ranging from therapeutic trials to structural genomics efforts. Here, we describe a novel system for the production of recombinant mammalian proteins, including immune receptors, cytokines and antibodies, in a human cell line culture system, often requiring <3 weeks to achieve stable, high-level expression: Daedalus. The inclusion of minimized ubiquitous chromatin opening elements in the transduction vectors is key for preventing genomic silencing and maintaining the stability of decigram levels of expression. This system can bypass the tedious and time-consuming steps of conventional protein production methods by employing the secretion pathway of serum-free adapted human suspension cell lines, such as 293 Freestyle. Using optimized lentiviral vectors, yields of 20–100 mg/l of correctly folded and post-translationally modified, endotoxin-free protein of up to ~70 kDa in size, can be achieved in conventional, small-scale (100 ml) culture. At these yields, most proteins can be purified using a single size-exclusion chromatography step, immediately appropriate for use in structural, biophysical or therapeutic applications. PMID:21911364

  13. The generation of germline transgenic silkworms for the production of biologically active recombinant fusion proteins of fibroin and human basic fibroblast growth factor.

    PubMed

    Hino, Rika; Tomita, Masahiro; Yoshizato, Katsutoshi

    2006-11-01

    We generated germline transgenic silkworms bearing a fibroin light chain (FL) promoter-driven FL gene whose 3'-end was flanked with human basic fibroblast growth factor (bFGF) gene, FL/bFGF gene. The cocoons from transgenic worms were trypsinized to remove sericin layers, and treated with solution containing CaCl(2), ethanol, and water at a molar ratio of 1:2:8 (CaCl(2)/ethanol/water) to solubilize fibroin layers. Western blot analysis showed that the recombinant protein, r(FL/bFGF), was solubilized with CaCl(2)/ethanol/water, but not with trypsin, indicating that r(FL/bFGF) was in fibroin layers. Thus, it was concluded that the worms spun cocoons whose fibroin layers were composed of the inherent gene-derived natural fibroin (nF) and r(FL/bFGF). The mixture of nF and r(FL/bFGF) was dubbed r(FL/bFGF)nF. The solubilized r(FL/bFGF)nF was refolded using the glutathione redox system. Human umbilical vein endothelial cells (HUVECs) grew in the refolded r(FL/bFGF)nF-containing culture media, showing that bFGF in r(FL/bFGF) was biologically active. r(FL/bFGF)nF immobilized on a culture dish also supported the growth of HUVECs in bFGF-free media, suggesting the usefulness of r(FL/bFGF)nF as a new biomaterial for tissue engineering. The currently developed transgenic silkworms will be suitable for mass production of fibroins bearing a variety of biological activities. PMID:16905183

  14. Protection of immunocompromised mice against lethal infection with Pseudomonas aeruginosa by active or passive immunization with recombinant P. aeruginosa outer membrane protein F and outer membrane protein I fusion proteins.

    PubMed Central

    von Specht, B U; Knapp, B; Muth, G; Bröker, M; Hungerer, K D; Diehl, K D; Massarrat, K; Seemann, A; Domdey, H

    1995-01-01

    Recombinant outer membrane proteins (Oprs) of Pseudomonas aeruginosa were expressed in Escherichia coli as glutathione S-transferase (GST)-linked fusion proteins. GST-linked Oprs F and I (GST-OprF190-350 [GST linked to OprF spanning amino acids 190 to 350] and GST-OprI21-83, respectively) and recombinant hybrid Oprs (GST-OprF190-342-OprI21-83 and GST-OprI21-83-OprF190-350) were isolated and tested for their efficacy as vaccines in immunodeficient mice. GST-OprF-OprI protected the mice against a 975-fold 50% lethal dose of P. aeruginosa. Expression of GST-unfused OprF-OprI failed in E. coli, although this hybrid protein has been expressed without a fusion part in Saccharomyces cerevisiae and used for immunizing rabbits. The immune rabbit sera protected severe combined deficient (SCID) mice against a 1,000-fold 50% lethal dose of P. aeruginosa. Evidence is provided to show that the most C-terminal part of OprF (i.e., amino acids 332 to 350) carries an important protective epitope. Opr-based hybrid proteins may have implications for a clinical vaccine against P. aeruginosa. PMID:7729895

  15. 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.

  16. Chemical Polysialylation of Recombinant Human Proteins.

    PubMed

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

    2015-01-01

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

  17. Production of biologically active recombinant human lactoferrin in Aspergillus oryzae.

    PubMed

    Ward, P P; Lo, J Y; Duke, M; May, G S; Headon, D R; Conneely, O M

    1992-07-01

    We report the production of recombinant human lactoferrin in Aspergillus oryzae. Expression of human lactoferrin (hLF), a 78 kD glycoprotein, was achieved by placing the cDNA under the control of the A. oryzae alpha-amylase promoter and the 3' flanking region of the A. niger glucoamylase gene. Using this system, hLF is expressed and secreted into the growth medium at levels up to 25 mg/l. The recombinant lactoferrin is indistinguishable from human milk lactoferrin with respect to its size, immunoreactivity, and iron-binding capacity. The recombinant protein appears to be appropriately N-linked glycosylated and correctly processed at the N-terminus by the A. oryzae secretory apparatus. Lactoferrin is the largest heterologous protein and the first mammalian glycoprotein expressed in the Aspergillus system to date. Hence, this expression system appears suitable for the large-scale production and secretion of biologically active mammalian glycoproteins. PMID:1368268

  18. Sustained in vivo inhibition of protein domains using single-chain Fv recombinant antibodies and its application to dissect RGMa activity on axonal outgrowth.

    PubMed

    Tassew, Nardos G; Charish, Jason; Chestopalova, Larisa; Monnier, Philippe P

    2009-01-28

    Antibodies are powerful tools for delineating the specific function of protein domains, yet several limitations restrict their in vivo applicability. Here we present a new method to obtain sustained in vivo inhibition of specific protein domains using recombinant antibodies. We show that long term in vivo expression of single-chain Fv (scFv) fragments in the developing CNS can be achieved through retroviral transduction. Moreover, specific scFvs generated against the N- and C-terminal domains of the repulsive guidance molecule, RGMa, prevent proper axon targeting in the visual system. This work reveals a previously unappreciated role for the RGMa N-terminal domain in axon guidance, and provides a novel, broadly applicable and rapid procedure to functionally antagonize any protein domain in vivo. PMID:19176821

  19. Rabbits immunized with Epstein-Barr virus gH/gL or gB recombinant proteins elicit higher serum virus neutralizing activity than gp350.

    PubMed

    Cui, Xinle; Cao, Zhouhong; Chen, Quanyi; Arjunaraja, Swadhinya; Snow, Andrew L; Snapper, Clifford M

    2016-07-25

    Epstein-Barr virus (EBV) is the primary cause of infectious mononucleosis and has been strongly implicated in the etiology of multiple epithelial and lymphoid cancers, such as nasopharyngeal carcinoma, gastric carcinoma, Hodgkin lymphoma, Burkitt lymphoma, non-Hodgkin lymphoma and post-transplant lymphoproliferative disorder. There is currently no licensed prophylactic vaccine for EBV. Most efforts to develop prophylactic vaccines have focused on EBV gp350, which binds to CD21/CD35 to gain entry into B cells, and is a major target of serum neutralizing antibody in EBV seropositive humans. However, a recombinant monomeric gp350 protein failed to prevent EBV infection in a phase II clinical trial. Thus, alternative or additional target antigens may be necessary for a successful prophylactic vaccine. EBV gH/gL and gB proteins coordinately mediate EBV fusion and entry into B cells and epithelial cells, strongly suggesting that vaccination with these proteins might elicit antibodies that will prevent EBV infection. We produced recombinant trimeric and monomeric EBV gH/gL heterodimeric proteins and a trimeric EBV gB protein, in addition to tetrameric and monomeric gp350(1-470) proteins, in Chinese hamster ovary cells. We demonstrated that vaccination of rabbits with trimeric and monomeric gH/gL, trimeric gB, and tetrameric gp350(1-470) induced serum EBV-neutralizing titers, using cultured human B cells, that were >100-fold, 20-fold, 18-fold, and 4-fold higher, respectively, than monomeric gp350(1-470). These data strongly suggest a role for testing EBV gH/gL and EBV gB in a future prophylactic vaccine to prevent EBV infection of B cells, as well as epithelial cells. PMID:27291087

  20. A systematic analysis of recombination activity and genotype-phenotype correlation in human recombination-activating gene 1 deficiency

    PubMed Central

    Lee, Yu Nee; Frugoni, Francesco; Dobbs, Kerry; Walter, Jolan E.; Giliani, Silvia; Gennery, Andrew R.; Al-Herz, Waleed; Haddad, Elie; LeDeist, Francoise; Bleesing, Jack H.; Henderson, Lauren A.; Pai, Sung-Yun; Nelson, Robert P.; El-Ghoneimy, Dalia H.; El-Feky, Reem A.; Reda, Shereen M.; Hossny, Elham; Soler-Palacin, Pere; Fuleihan, Ramsay L.; Patel, Niraj C.; Massaad, Michel J.; Geha, Raif S.; Puck, Jennifer M.; Palma, Paolo; Cancrini, Caterina; Chen, Karin; Vihinen, Mauno; Alt, Frederick W.; Notarangelo, Luigi D.

    2014-01-01

    Background The recombination-activating gene (RAG) 1/2 proteins play a critical role in the development of T and B cells by initiating the VDJ recombination process that leads to generation of a broad T-cell receptor (TCR) and B-cell receptor repertoire. Pathogenic mutations in the RAG1/2 genes result in various forms of primary immunodeficiency, ranging from T−B− severe combined immune deficiency to delayed-onset disease with granuloma formation, autoimmunity, or both. It is not clear what contributes to such heterogeneity of phenotypes. Objective We sought to investigate the molecular basis for phenotypic diversity presented in patients with various RAG1 mutations. Methods We have developed a flow cytometry–based assay that allows analysis of RAG recombination activity based on green fluorescent protein expression and have assessed the induction of the Ighc locus rearrangements in mouse Rag1−/− pro-B cells reconstituted with wild-type or mutant human RAG1 (hRAG1) using deep sequencing technology. Results Here we demonstrate correlation between defective recombination activity of hRAG1 mutant proteins and severity of the clinical and immunologic phenotype and provide insights on the molecular mechanisms accounting for such phenotypic diversity. Conclusions Using a sensitive assay to measure the RAG1 activity level of 79 mutations in a physiologic setting, we demonstrate correlation between recombination activity of RAG1 mutants and the severity of clinical presentation and show that RAG1 mutants can induce specific abnormalities of the VDJ recombination process. PMID:24290284

  1. 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.

  2. 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.

  3. 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.

  4. A recombinant Sp185/333 protein from the purple sea urchin has multitasking binding activities towards certain microbes and PAMPs.

    PubMed

    Lun, Cheng Man; Schrankel, Catherine S; Chou, Hung-Yen; Sacchi, Sandro; Smith, L Courtney

    2016-08-01

    The purple sea urchin, Strongylocentrotus purpuratus, possesses a sophisticated innate immune system that responds to microbes effectively by swift expression of the highly diverse Sp185/333 gene family. The Sp185/333 proteins are predicted to have anti-pathogen functions based on inducible gene expression and their significant sequence diversity. Sp185/333 proteins are all predicted to be intrinsically disordered and do not exhibit sequence similarities to other known proteins. To test the anti-pathogen hypothesis, a recombinant Sp185/333 protein, rSp0032, was evaluated and found to exhibit specific binding to marine Vibrio diazotrophicus and to Saccharomyces cerevisiae, but not to two Bacillus species. rSp0032 also binds to LPS, β-1,3-glucan and flagellin but not to peptidoglycan. rSp0032 binding to LPS can be competed by LPS, β-1,3-glucan and flagellin but not by peptidoglycan. We speculate that the predicted intrinsically disordered structure of rSp0032 may adapt to different conformations in binding to a limited number of PAMPs and pathogens. Given that rSp0032 binds to a range of targets, and that up to 260 different Sp185/333 proteins can be expressed per individual sea urchin, this family of immune response proteins may facilitate effective host protection against a broad array of potential pathogens encountered in the marine environment. PMID:27020848

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

    PubMed

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

    1994-06-01

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

  6. Data on the evolutionary history of the V(D)J recombination-activating protein 1 - RAG1 coupled with sequence and variant analyses.

    PubMed

    Kumar, Abhishek; Bhandari, Anita; Sarde, Sandeep J; Muppavarapu, Sekhar; Tandon, Ravi

    2016-09-01

    RAG1 protein is one of the key component of RAG complex regulating the V(D)J recombination. There are only few studies for RAG1 concerning evolutionary history, detailed sequence and mutational hotspots. Herein, we present out datasets used for the recent comprehensive study of RAG1 based on sequence, phylogenetic and genetic variant analyses (Kumar et al., 2015) [1]. Protein sequence alignment helped in characterizing the conserved domains and regions of RAG1. It also aided in unraveling ancestral RAG1 in the sea urchin. Human genetic variant analyses revealed 751 mutational hotspots, located both in the coding and the non-coding regions. For further analysis and discussion, see (Kumar et al., 2015) [1]. PMID:27284568

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

    PubMed

    Xu, Hanfu

    2014-10-01

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

  8. Production of soluble recombinant proteins with Kell, Duffy and Lutheran blood group antigen activity, and their use in screening human sera for Kell, Duffy and Lutheran antibodies.

    PubMed

    Ridgwell, K; Dixey, J; Scott, M L

    2007-10-01

    The aim of this study was to show that soluble recombinant (sr) proteins can mimic blood group antigens and be used to screen human sera for blood-group-specific antibodies. The blood of all pregnant women and pretransfusion patients should be screened for blood-group-specific antibodies to identify and monitor pregnancies at risk of haemolytic disease of the foetus and newborn (HDFN), and to prevent haemolytic transfusion reactions. Current antibody screening and identification methods use human red blood cell panels, which can complicate antibody identification if more than one antibody specificity is present. COS-7 cells were transfected to produce sr forms of the extracellular domains of the red blood cell membrane proteins that express Kell, Duffy or Lutheran blood group antigens. These sr proteins were used to screen for and identify anti-Kell, anti-Duffy or anti-Lutheran blood-group-specific allo-antibodies in human sera by haemagglutination inhibition and in solid-phase enzyme-linked immunosorbent assays (ELISAs). There is a positive correlation (correlation coefficient 0.605, P value 0.002) between antibody titre by standard indirect antiglobulin test (IAT) and signal intensity in the ELISA test. This work shows that sr proteins can mimic blood group antigens and react with human allogeneic antibodies, and that such proteins could be used to develop solid-phase, high-throughput blood group antibody screening and identification platforms. PMID:17725551

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed

    Bonander, Nicklas; Bill, Roslyn M

    2012-01-01

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    2015-08-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed

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

    2013-12-01

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

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

    PubMed

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

    2008-05-01

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

  16. Secretion-dependent proteolysis of heterologous protein by recombinant Escherichia coli is connected to an increased activity of the energy-generating dissimilatory pathway.

    PubMed

    Schmidt, M; Viaplana, E; Hoffmann, F; Marten, S; Villaverde, A; Rinas, U

    1999-01-01

    The synthesis of a proteolytically unstable protein, originally designed for periplasmic export in recombinant Escherichia coli BL21(DE3), a strain naturally deficient for the ATP-dependent protease Lon (or La) and the outer membrane protease OmpT, is associated with a severe growth inhibition. This inhibition is not observed in BL21(DE3) synthesizing a closely related but proteolytically stable protein that is sequestered into inclusion bodies. It is shown that the growth inhibition is mainly caused by a slower cell division rate and a reduced growth yield and not by a general loss of cell division competence. Cells proceed with their normal growth characteristics when exposed again to conditions that do not sustain the expression of the heterologous gene. The performance of cells synthesizing either the stable or the degraded protein was also studied in high cell density cultures by employing a new method to calculate the actual specific growth rate, the biomass yield coefficient, and the dissimilated fraction of the carbon substrate in real-time. It is shown that the growth inhibition of cells synthesizing the proteolytically degraded protein is connected to an increased dissimilation of the carbon substrate resulting in a concomitant reduction of the growth rate and the biomass yield coefficient with respect to the carbon source. It is postulated that the increased dissimilation of the carbon substrate by lon-deficient Bl21(DE3) cells synthesizing the proteolytically unstable protein may result from a higher energy demand required for the in vivo degradation of this protein by ATP-dependent proteases different from the protease Lon. PMID:10556795

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

    PubMed

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

    1997-03-01

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

  18. Metabolic Adaptation in Transplastomic Plants Massively Accumulating Recombinant Proteins

    PubMed Central

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

    2011-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  20. Recombinant fusion protein of cholera toxin B subunit with YVAD secreted by Lactobacillus casei inhibits lipopolysaccharide-induced caspase-1 activation and subsequent IL-1 beta secretion in Caco-2 cells

    PubMed Central

    2014-01-01

    Background Lactobacillus species are used as bacterial vectors to deliver functional peptides to the intestine because they are delivered live to the intestine, colonize the mucosal surface, and continue to produce the desired protein. Previously, we generated a recombinant Lactobacillus casei secreting the cholera toxin B subunit (CTB), which can translocate into intestinal epithelial cells (IECs) through GM1 ganglioside. Recombinant fusion proteins of CTB with functional peptides have been used as carriers for the delivery of these peptides to IECs because of the high cell permeation capacity of recombinant CTB (rCTB). However, there have been no reports of rCTB fused with peptides expressed or secreted by Lactobacillus species. In this study, we constructed L. casei secreting a recombinant fusion protein of CTB with YVAD (rCTB–YVAD). YVAD is a tetrapeptide (tyrosine–valine–alanine–aspartic acid) that specifically inhibits caspase-1, which catalyzes the production of interleukin (IL)-1β, an inflammatory cytokine, from its inactive precursor. Here, we examined whether rCTB–YVAD secreted by L. casei binds to GM1 ganglioside and inhibits caspase-1 activation in Caco-2 cells used as a model of IECs. Results We constructed the rCTB–YVAD secretion vector pSCTB–YVAD by modifying the rCTB secretion vector pSCTB. L. casei secreting rCTB–YVAD was generated by transformation with pSCTB–YVAD. Both the culture supernatant of pSCTB–YVAD-transformed L. casei and purified rCTB–YVAD bound to GM1 ganglioside, as did the culture supernatant of pSCTB-transformed L. casei and purified rCTB. Interestingly, although both purified rCTB–YVAD and rCTB translocated into Caco-2 cells, regardless of lipopolysaccharide (LPS), only purified rCTB–YVAD but not rCTB inhibited LPS-induced caspase-1 activation and subsequent IL-1β secretion in Caco-2 cells, without affecting cell viability. Conclusions The rCTB protein fused to a functional peptide secreted by L. casei

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    PubMed

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

    2014-08-01

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

  3. 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

  4. 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

  5. Isolation and Purification of Recombinant Serine/Threonine Protein Kinases of the Strain Bifidobacterium longum B379M and Investigation of Their Activity.

    PubMed

    Alekseeva, M G; Mavletova, D A; Kolchina, N V; Nezametdinova, V Z; Danilenko, V N

    2015-10-01

    Previously, we identified six serine/threonine protein kinases (STPK) of Bifidobacterium and named them Pkb1-Pkb6. In the present study, we optimized methods for isolation of the six STPK catalytic domains proteins of B. longum B379M: a method for isolation of Pkb3 and Pkb4 in native conditions, a method for isolation of Pkb5 in denaturing conditions, and a method for isolation of Pkb1, Pkb2, and Pkb6 from inclusion bodies. The dialysis conditions for the renaturation of the proteins were optimized. All of the enzymes were isolated in quantities sufficient for study of the protein activity. The proteins were homogeneous according to SDS-PAGE. The autophosphorylation ability of Pkb1, Pkb3, Pkb4, and Pkb6 was investigated for the first time. Autophosphorylation was detected only for the Pkb3 catalytic domain. PMID:26567574

  6. Optimizing transient recombinant protein expression in mammalian cells.

    PubMed

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

    2012-01-01

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

  7. Expression of an active form of recombinant Ty1 reverse transcriptase in Escherichia coli: a fusion protein containing the C-terminal region of the Ty1 integrase linked to the reverse transcriptase-RNase H domain exhibits polymerase and RNase H activities.

    PubMed Central

    Wilhelm, M; Boutabout, M; Wilhelm, F X

    2000-01-01

    Replication of the Saccharomyces cerevisiae Ty1 retrotransposon requires a reverse transcriptase capable of synthesizing Ty1 DNA. The first description of an active form of a recombinant Ty1 enzyme with polymerase and RNase H activities is reported here. The Ty1 enzyme was expressed as a hexahistidine-tagged fusion protein in Escherichia coli to facilitate purification of the recombinant protein by metal-chelate chromatography. Catalytic activity of the recombinant protein was detected only when amino acid residues encoded by the integrase gene were added to the N-terminus of the reverse transcriptase-RNase H domain. This suggests that the integrase domain could play a role in proper folding of reverse transcriptase. Several biochemical properties of the Ty1 enzyme were analysed, including the effect of MgCl(2), NaCl, temperature and of the chain terminator dideoxy GTP on its polymerase activity. RNase H activity was examined by monitoring the cleavage of a RNA-DNA template-primer. Our results suggest that the distance between the RNase H and polymerase active sites corresponds to the length of a 14-nucleotide RNA-DNA heteroduplex. The recombinant protein produced in E. coli should be useful for further biochemical and structural analyses and for a better understanding of the role of integrase in the activation of reverse transcriptase. PMID:10816427

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

    PubMed

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

    2012-01-01

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

  9. High-yield production of biologically active recombinant protein in shake flask culture by combination of enzyme-based glucose delivery and increased oxygen transfer

    PubMed Central

    2011-01-01

    This report describes the combined use of an enzyme-based glucose release system (EnBase®) and high-aeration shake flask (Ultra Yield Flask™). The benefit of this combination is demonstrated by over 100-fold improvement in the active yield of recombinant alcohol dehydrogenase expressed in E. coli. Compared to Terrific Broth and ZYM-5052 autoinduction medium, the EnBase system improved yield mainly through increased productivity per cell. Four-fold increase in oxygen transfer by the Ultra Yield Flask contributed to higher cell density with EnBase but not with the other tested media, and consequently the product yield per ml of EnBase culture was further improved. PMID:22152005

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

    PubMed

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

    2015-02-01

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

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

    PubMed

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

    2009-10-01

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

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

    PubMed

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

    1993-08-01

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

  13. [Comparison of expression and antibacterial activities of recombinant porcine lactoferrin expressed in four Lactobacillus species].

    PubMed

    Yu, Hui; Jiang, Yanping; Cui, Wen; Wu, Xiao; He, Jia; Qiao, Xinyuan; Li, Yijing; Tang, Lijie

    2014-09-01

    The coding sequence for the mature peptide of porcine lactoferrin (Plf) was synthesized according to the codon usage of lactobacillus, to establish optimized porcine lactoferrin Lactobacillus expression system. The gene was ligated into the Xho I/BamH I site of Lactobacillus expression vector pPG612.1 and the recombinant plasmid pPG612.1-plf was transformed individually into Lactobacillus casei ATCC393, Lactobacillus pentosus KLDS1.0413, Lactobacillus plantarum KLDS1.0344 or Lactobacillus paracasei KLDS1.0652 by electroporation. After induction with xylose, expression of the recombinant proteins was detected by Western blotting and confocal laser scanning microscopy. Secretion of recombinant Plf proteins from four recombinant species was determined quantitatively by ELISA. The antibacterial activities of recombinant proteins were measured by agar diffusion method. The result shows that Plf was correctly expressed in four species of recombinant lactobacillus, with molecular weight of about 73 kDa. The expression levels in recombinant Lactobacillus casei, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus paracasei were 9.6 μg/mL, 10.8 μg/mL, 12.5 μg/mL and 9.9 μg/mL, respectively. Antimicrobial activity experiment shows that the recombinant proteins were active against E. coli, Staphylococcus aureus, Salmonella typhimurium, Listeria, Pasteurella. The recombinant Plf expressed by recombinant Lactobacillus plantarum showed the best antibacterial activity among all recombinant lactobacillus species. These data represent a basis for the development and application of porcine lactoferrin from recombinant lactobacillus. PMID:25720152

  14. 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.

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

    PubMed

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

    2004-11-01

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

  16. An optimized protocol for overproduction of recombinant protein expression in Escherichia coli.

    PubMed

    Bahreini, Elham; Aghaiypour, Khosrow; Abbasalipourkabir, Roghayeh; Goodarzi, Mohammad Taghi; Saidijam, Massoud; Safavieh, Sedigheh Sadat

    2014-01-01

    The gram-negative bacterium Escherichia coli (E. coli) offers a means for rapid, high-yield, and economical production of recombinant proteins. Here, a protocol for optimization of parameters involved in bacterial expression conditions is described. L-Asparaginase (ASNase II) was chosen as a model protein for our experiments. ASNase II gene (ansB) was cloned into the pAED4 plasmid and transformed into E. coli BL21pLysS (DE3)-competent cells. It was assumed that high cell density and high copy number of recombinant plasmid in the bacteria host could result in very high production of the recombinant protein. Circumstances for the overproduction of recombinant ASNase II including cell growth conditions, isopropyl β-D-1-thiogalactopyranoside (IPTG) level, ampicillin (Amp) concentration before and during IPTG induction, and cell density were optimized. Regarding the final optimization, overexpression of ASNase II was assessed on a large scale in LB medium. Periplasmic ASNase II was extracted using an alkaline lysis method. The extracted protein was purified by one-step DEAE-Sepharose fast-flow chromatography. ASNase II activity was considered an index for the protein expression. Applying the optimized practical protocol, protein production was significantly enhanced in comparison to the traditional IPTG induction method in the absence of a fermentor and can be applied for overexpression of other recombinant proteins. PMID:24219068

  17. Molecular identification and expression analysis of a natural killer cell enhancing factor (NKEF) from rock bream Oplegnathus fasciatus and the biological activity of its recombinant protein

    PubMed Central

    Kim, Ju-Won; Choi, Hye-Sung; Kwon, Mun-Gyeong; Park, Myoung-Ae; Hwang, Jee-Youn; Kim, Do-Hyung; Park, Chan-Il

    2011-01-01

    Natural killer cell enhancing factor (NKEF) belongs to the defined peroxiredoxin (Prx) family. Rock bream NKEF cDNA was identified by expressed sequence tag (EST) analysis of rock bream liver that was stimulated with the LPS. The full-length RbNKEF cDNA (1062 bp) contained an open reading frame (ORF) of 594 bp encoding 198 amino acids. RbNKEF was significantly expressed in the gill, liver, and intestine. mRNA expression of NKEF in the head kidney was examined under viral and bacterial challenge via real-time RT-PCR. Experimental challenge of rock bream with Edwardsiella tarda, Streptococcus iniae, and RSIV resulted in significant increases in RbNKEF mRNA in the head kidney. To obtain a recombinant NKEF, the RbNKEF ORF was expressed in Escherichia coli BL21 (DE3), and the purified soluble protein exhibited a single band corresponding to the predicted molecular mass. When kidney leucocytes were treated with a high concentration of rRbNKEF (10 μg/mL), they exhibited significantly enhanced cell proliferation and viability under oxidative stress. PMID:24371552

  18. A novel recombinant protein of ephrinA1-PE38/GM-CSF activate dendritic cells vaccine in rats with glioma.

    PubMed

    Li, Ming; Wang, Bin; Wu, Zhonghua; Zhang, Jiadong; Shi, Xiwen; Cheng, Wenlan; Han, Shuangyin

    2015-07-01

    Dendritic cells loaded with tumor-associated antigens can effectively stimulate the antitumor immune response of cytotoxic T lymphocytes in the body, which facilitates the development of novel and effective treatments for cancer. In this study, the adenovirus-mediated ephrinA1-PE38/GM-CSF was successfully constructed using the overlap extension method, and verified with sequencing analysis. HEK293 cells were infected with the adenovirus and the cellular expression of ephrinA1-PE38/GM-CSF was measured with an enzyme-linked immunosorbent assay. The recombinant adenovirus was then delivered into the tumor-bearing rats and the results showed that such treatment significantly reduced the volumes of gliomas and improved the survival of the transplanted rats. The results from immunohistochemistry and flow cytometry suggested that this immunomodulatory agent cause activation of dendritic cells. The findings that ephrinA1-PE38/GM-CSF had a high efficacy in the activation of the dendritic cells would facilitate the development of in vivo dendritic-cell vaccines for the treatment of gliomas in rats. Our new method of DC vaccine production induces not only a specific local antitumor immune response but also a systemic immunotherapeutic effect. In addition, this method completely circumvents the risk of contamination related to the in vitro culture of DCs, thus greatly improving the safety and feasibility of clinical application of the DC vaccines in glioma. PMID:25677907

  19. 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

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

    PubMed

    Chen, Qiang; Lai, Huafang

    2015-01-01

    Recombinant proteins are primarily produced from cultures of mammalian, insect, and bacteria cells. In recent years, the development of deconstructed virus-based vectors has allowed plants to become a viable platform for recombinant protein production, with advantages in versatility, speed, cost, scalability, and safety over the current production paradigms. In this paper, we review the recent progress in the methodology of agroinfiltration, a solution to overcome the challenge of transgene delivery into plant cells for large-scale manufacturing of recombinant proteins. General gene delivery methodologies in plants are first summarized, followed by extensive discussion on the application and scalability of each agroinfiltration method. New development of a spray-based agroinfiltration and its application on field-grown plants is highlighted. The discussion of agroinfiltration vectors focuses on their applications for producing complex and heteromultimeric proteins and is updated with the development of bridge vectors. Progress on agroinfiltration in Nicotiana and non-Nicotiana plant hosts is subsequently showcased in context of their applications for producing high-value human biologics and low-cost and high-volume industrial enzymes. These new advancements in agroinfiltration greatly enhance the robustness and scalability of transgene delivery in plants, facilitating the adoption of plant transient expression systems for manufacturing recombinant proteins with a broad range of applications. PMID:26075275

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

    PubMed

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

    2013-01-01

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

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

    PubMed

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

    2012-03-01

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

  3. Characterization of functionally active interleukin-18/eGFP fusion protein expression during cell cycle phases in recombinant chicken DF1 Cells.

    PubMed

    Wu, Hsing Chieh; Chen, Yu San; Shien, Jui Hung; Shen, Pin Chun; Lee, Long Huw

    2016-05-01

    The dependence of foreign gene expression on cell cycle phases in mammalian cells has been described. In this study, a DF1/chIL-18a cell line that stably expresses the fusion protein chIL-18 was constructed and the enhanced green fluorescence protein connected through a (G4 S)3 linker sequence investigated the relationship between cell cycle phases and fusion protein production. DF1/chIL-18a cells (1 × 10(5) ) were inoculated in 60-mm culture dishes containing 5 mL of media to achieve 50%-60% confluence and were cultured in the presence of the cycle-specific inhibitors 10058-F4, aphidicolin, and colchicine for 24 and 48 h. The percentage of cell density and mean fluorescence intensity in each cell cycle phase were assessed using flow cytometry. The inhibitors effectively arrested cell growth. The fusion protein production rate was higher in the S phase than in the G0/G1 and G2/M phases. When cell cycle progression was blocked in the G0/G1, S, and G2/M phases by the addition of 10058-F4, aphidicolin, and colchicine, respectively, the aphidicolin-induced single cells showed higher fusion protein levels than did the 10058-F4- or colchicine-induced phase cells and the uninduced control cells. Although the cells did not proliferate after the drug additions, the amount of total fusion protein accumulated in aphidicolin-treated cells was similar to that in the untreated cultures. Fusion protein is biologically active because it induces IFN-γ production in splenocyte cultures of chicken. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:581-591, 2016. PMID:26850993

  4. 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.

  5. Antibacterial activity of recombinant murine beta interferon.

    PubMed Central

    Fujiki, T; Tanaka, A

    1988-01-01

    Recombinant murine beta interferon was protective and therapeutic for mice against Listeria monocytogenes infection in vivo. The recombinant murine beta interferon caused enhanced H2O2 release by macrophages in vivo, but not in vitro. PMID:3343048

  6. Expression and Characterization of Recombinant Campylobacter jejuni Chemotactic Proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  7. Immune function of patients receiving recombinant human interleukin-6 (IL-6) in a phase I clinical study: induction of C-reactive protein and IgE and inhibition of natural killer and lymphokine-activated killer cell activity.

    PubMed

    Scheid, C; Young, R; McDermott, R; Fitzsimmons, L; Scarffe, J H; Stern, P L

    1994-02-01

    Interleukin-6 (IL-6) is a cytokine that acts on a variety of cell types, including myeloid progenitor cells and B and T lymphocytes. It has been found to activate cytotoxic T cells and natural killer (NK) cells and to induce T-cell-mediated antitumour effects in animal models. In a phase I clinical trial of recombinant human IL-6, 20 patients with advanced cancer were entered to receive daily subcutaneous injections of IL-6 over 7 days followed by a 2-week observation period and another 4 weeks of daily IL-6 injections. Doses varied between 0.5 microgram/kg and 20 micrograms/kg body weight and immune functions were monitored throughout. At all dose levels IL-6 administration led to a marked increase in serum levels of C-reactive protein and a moderate rise in complement factor C3. The proportions of CD4, CD8 or HLA-DR lymphocytes in peripheral blood did not alter with IL-6 treatment nor did the in vitro proliferation of peripheral blood mononuclear cells induced by either phytohaemagglutinin, pokeweed mitogen or fixed Staphylococcus aureus. By contrast, NK cell activity, lymphokine-activated killer (LAK) cell activity and proliferation induced by in vitro culture with interleukin-2 (IL-2) were suppressed at doses exceeding 2.5 micrograms/kg. Serum IgE levels were consistently elevated over the IL-6 dose range but IgM, IgG and IgA levels were unaffected. In summary there is a dose-dependent induction of acute-phase proteins by in vivo IL-6 treatment. At higher IL-6 doses there is a suppressive effect on NK and LAK activity measured in vitro. IL-6 may thus be useful in combination cytokine therapies that seek to suppress LAK and favour cytotoxic T lymphocyte responses. The rise in IgE levels in response to IL-6 was unexpected and suggests a more pivotal role than previously known for the control of IgE production; this could include IgE-related diseases. PMID:8306367

  8. Expression and purification of two recombinant sterol-carrier proteins: SCPX and SCP2.

    PubMed

    Manfra, D J; Baum, C L; Reschley, E; Lundell, D; Zavodny, P; Dalie, B

    1995-04-01

    We report the cloning, expression, and purification of the rat sterol carrier proteins SCPX and SCP2. The cDNA's encoding rat SCPX and SCP2 were isolated from a lambda gt11 rat liver cDNA library. To maximize expression and to facilitate the purification of the recombinant proteins, the SCPX and SCP2 proteins were expressed as carboxy-terminal fusion proteins to the glutathione S-transferase (GST). The GST-SCPX and GST-SCP2 fusion proteins contained a thrombin recognition site between the GST and SCPX or SCP2 polypeptides. The expression of the fusion proteins was controlled by the inducible tac promoter. Under optimal conditions, the approximately 85-kDa GST-SCPX and the approximately 41-kDa GST-SCP2 proteins represented approximately 1-2% of the total cell lysate. Both fusion proteins were easily purified under nondenaturing conditions from the soluble fraction of total cell lysate by glutathione-Sepharose 4B affinity chromatography. Thrombin cleavage resulted in the release of the SCPX and SCP2 proteins from the GST-SCPX and GST-SCP2 fusions, respectively. Amino terminal protein sequencing confirmed the authenticity of the recombinant proteins. Furthermore, functional assay revealed that recombinant SCP2 is highly active in facilitating the conversion of 7-dehydrocholesterol to cholesterol. Recombinant SCPX is also active in this assay but only 50% as active as SCP2. We anticipate that the preparation and purification techniques described in this study will facilitate further biochemical characterization of these proteins. PMID:7606169

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2004-01-01

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

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

    PubMed

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

    2011-05-01

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

  12. Protection of Mice from Fatal Measles Encephalitis by Vaccination with Vaccinia Virus Recombinants Encoding Either the Hemagglutinin or the Fusion Protein

    NASA Astrophysics Data System (ADS)

    Drillien, Robert; Spehner, Daniele; Kirn, Andre; Giraudon, Pascale; Buckland, Robin; Wild, Fabian; Lecocq, Jean-Pierre

    1988-02-01

    Vaccinia virus recombinants encoding the hemagglutinin or fusion protein of measles virus have been constructed. Infection of cell cultures with the recombinants led to the synthesis of authentic measles proteins as judged by their electrophoretic mobility, recognition by antibodies, glycosylation, proteolytic cleavage, and presentation on the cell surface. Mice vaccinated with a single dose of the recombinant encoding the hemagglutinin protein developed antibodies capable of both inhibiting hemagglutination activity and neutralizing measles virus, whereas animals vaccinated with the recombinant encoding the fusion protein developed measles neutralizing antibodies. Mice vaccinated with either of the recombinants resisted a normally lethal intracerebral inoculation of a cell-associated measles virus subacute sclerosing panencephalitis strain.

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

    PubMed

    Hacker, David L; Balasubramanian, Sowmya

    2016-06-01

    We highlight recent developments for the production of recombinant proteins from suspension-adapted mammalian cell lines. We discuss the generation of stable cell lines using transposons and lentivirus vectors (non-targeted transgene integration) and site-specific recombinases (targeted transgene integration). Each of these methods results in the generation of cell lines with protein yields that are generally superior to those achievable through classical plasmid transfection that depends on the integration of the transfected DNA by non-homologous DNA end-joining. This is the main reason why these techniques can also be used for the generation of stable cell pools, heterogenous populations of recombinant cells generated by gene delivery and genetic selection without resorting to single cell cloning. This allows the time line from gene transfer to protein production to be reduced. PMID:27322762

  14. Ribonuclease activity and RNA binding of recombinant human Dicer

    PubMed Central

    Provost, Patrick; Dishart, David; Doucet, Johanne; Frendewey, David; Samuelsson, Bengt; Rådmark, Olof

    2002-01-01

    RNA silencing phenomena, known as post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference (RNAi) in animals, are mediated by double-stranded RNA (dsRNA) and mechanistically intersect at the ribonuclease Dicer. Here, we report cloning and expression of the 218 kDa human Dicer, and characterization of its ribonuclease activity and dsRNA-binding properties. The recombinant enzyme generated ∼21–23 nucleotide products from dsRNA. Processing of the microRNA let-7 precursor by Dicer produced an apparently mature let-7 RNA. Mg2+ was required for dsRNase activity, but not for dsRNA binding, thereby uncoupling these reaction steps. ATP was dispensable for dsRNase activity in vitro. The Dicer·dsRNA complex formed at high KCl concentrations was catalytically inactive, suggesting that ionic interactions are involved in dsRNA cleavage. The putative dsRNA-binding domain located at the C-terminus of Dicer was demonstrated to bind dsRNA in vitro. Human Dicer expressed in mammalian cells colocalized with calreticulin, a resident protein of the endoplasmic reticulum. Availability of the recombinant Dicer protein will help improve our understanding of RNA silencing and other Dicer-related processes. PMID:12411504

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

    PubMed Central

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

    2016-01-01

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

  16. Association of 14-3-3 Proteins to β1-Adrenergic Receptors Modulates Kv11.1 K+ Channel Activity in Recombinant Systems

    PubMed Central

    Tutor, Antonio S.; Delpón, Eva; Caballero, Ricardo; Gómez, Ricardo; Núñez, Lucía; Vaquero, Miguel; Tamargo, Juan; Penela, Petronila

    2006-01-01

    We identify a new mechanism for the β1-adrenergic receptor (β1AR)-mediated regulation of human ether-a-go-go–related gene (HERG) potassium channel (Kv11.1). We find that the previously reported modulatory interaction between Kv11.1 channels and 14-3-3ε proteins is competed by wild type β1AR by means of a novel interaction between this receptor and 14-3-3ε. The association between β1AR and 14-3-3ε is increased by agonist stimulation in both transfected cells and heart tissue and requires cAMP-dependent protein kinase (PKA) activity. The β1AR/14-3-3ε association is direct, since it can be recapitulated using purified 14-3-3ε and β1AR fusion proteins and is abolished in cells expressing β1AR phosphorylation–deficient mutants. Biochemical and electrophysiological studies of the effects of isoproterenol on Kv11.1 currents recorded using the whole-cell patch clamp demonstrated that β1AR phosphorylation–deficient mutants do not recruit 14-3-3ε away from Kv11.1 and display a markedly altered agonist-mediated modulation of Kv11.1 currents compared with wild-type β1AR, increasing instead of inhibiting current amplitudes. Interestingly, such differential modulation is not observed in the presence of 14-3-3 inhibitors. Our results suggest that the dynamic association of 14-3-3 proteins to both β1AR and Kv11.1 channels is involved in the adrenergic modulation of this critical regulator of cardiac repolarization and refractoriness. PMID:16914520

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

    NASA Astrophysics Data System (ADS)

    Wilson, Donna Lucille

    The structure of silk proteins consists of alternating amorphous (glycine-rich) and ordered crystalline regions (poly(alanine) and poly(glycine-alanine) repeats), where the organized regions are typically beta-sheet assemblies. In collagen, the basic helical repeat (glycine-proline-hydroxyproline and variants on this repeat) drives hierarchical assembly. Three polypeptide chains form left-handed poly-proline II-like helices, these three chains then self-assemble to form a right-handed triple helix. The focus of this thesis is on these proteins and defined variations thereof to reveal features of fibrous protein self-assembly. The amino acid sequences of native silk and collagen and their respective assembly environments have been systematically manipulated. Spider silk protein, based on the consensus sequence of Nephila clavipes dragline-silk, was genetically engineered to include methionines flanking the beta-sheet forming polyalanine regions. These methionines could be selectively oxidized and reduced, altering the bulkiness and charge of a methionine-based sulfoxide group to control beta-sheet formation by steric hindrance. A second version of the sterical trigger included a recognition site for Protein Kinase A allowing for the selective phosphorylation of a serine. Patterning a monolayer of precursor "director" molecules on length scales ranging from nanometer- to micrometer-length scales simplifies the interpretation of supramolecular assembly. Utilizing the atomic force microscopy (AFM)-based technique of dip-pen nanolithography, thiolated collagen and a collagen-like peptide were patterned at 30--50 nm line widths on evaporated gold surfaces. These are the largest molecules thus far positively printed on a surface at such small-length scales. The method preserved the triple helical structure and biological activity of collagen and even fostered the formation of characteristic higher-levels of structural organization. Nanopatterns were also achieved for

  18. 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. PMID:26076028

  19. Pharmacological analysis of G-protein activation mediated by guinea-pig recombinant 5-HT1B receptors in C6-glial cells: similarities with the human 5-HT1B receptor.

    PubMed

    Pauwels, P J; Wurch, T; Palmier, C; Colpaert, F C

    1998-01-01

    1. The guinea-pig recombinant 5-hydroxytryptamine1B (gp 5-HT1B) receptor stably transfected in rat C6-glial cells was characterized by monitoring G-protein activation in a membrane preparation with agonist-stimulated [35S]-GTPgammaS binding. The intrinsic activity of 5-HT receptor ligands was compared with that determined previously at the human recombinant 5-HT1B (h 5-HT1B) receptor under similar experimental conditions. 2. Membrane preparations of C6-glial/gp 5-HT1B cells exhibited [3H]-5-carboxamidotryptamine (5-CT) and [3H]-N-[4-methoxy-3,4-methylpiperazin-1-yl) phenyl]-3-methyl-4-(4-pyridinyl)benzamide (GR 125743) binding sites with a pKd of 9.62 to 9.85 and a Bmax between 2.1 to 6.4 fmol mg(-1) protein. The binding affinities of a series of 5-HT receptor ligands determined with [3H]-5-CT and [3H]-GR 125743 were similar. Ligand affinities were comparable to and correlated (r2: 0.74, P<0.001) with those determined at the recombinant h 5-HT1B receptor. 3. [35S]-GTPgammaS binding to membrane preparations of C6-glial/gp 5-HT1B cells was stimulated by the 5-HT receptor agonists that were being investigated. The maximal responses of naratriptan, zolmitriptan, sumatriptan, N-methyl-3-[pyrrolidin-2(R)-ylmethyl]-1H-indol-5-ylmethyl sulphonamide (CP 122638), rizatriptan and dihydroergotamine were between 0.76 and 0.85 compared to 5-HT. The potency of these agonists showed a positive correlation (r2: 0.72, P=0.015) with their potency at the recombinant h 5-HT1B receptor. 1-naphthylpiperazine, (+/-)-cyanopindolol and (2'-methyl-4'-(5-methyl[1,2,4] oxadiazole-3-yl)biphenyl-4-carboxylic acid [4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]amide (GR 127935) elicited an even smaller response (Emax: 0.32 to 0.63). 4. The ligands 1'-methyl-5-(2'-methyl-4'-(5-methyl-1,2,4-oxadiazole-3-yl) biphenyl-4-carbonyl)-2,3,6,7tetrahydrospiro [furo[2,3-f]indole-3-spiro-4'-piperidine] (SB224289), methiothepin and ritanserin displayed inhibition of basal [35S]-GTPgammaS binding at concentrations

  20. Cellular immune responses to recombinant heat shock protein 70 from Histoplasma capsulatum.

    PubMed Central

    Allendoerfer, R; Maresca, B; Deepe, G S

    1996-01-01

    Heat shock protein (hsp) 70 from several microbes is antigenic in mammals. In this study we sequenced and expressed the gene encoding this protein from Histoplasma capsulatum to study its immunological activity. The deduced amino acid sequence of the gene demonstrated 71 and 76% identity to hsp7O from humans and Saccharomyces cerevisiae, respectively. A cDNA was synthesized by reverse transcription-PCR and was expressed in Escherichia coli. Recombinant protein reacted with a mouse monoclonal antibody raised against human hsp7O. Splenocytes from C57BL/6 mice immunized with recombinant hsp7O emulsified in adjuvant, but not yeast cells, reacted in vitro to the antigen. Recombinant hsp7O elicited a cutaneous delayed-type hypersensitivity response in mice immunized with protein or with viable yeast cells. Mice were injected with recombinant hsp7O and challenged intranasally with a sublethal inoculum of yeast cells. Vaccination did not confer protection in this model. Thus, recombinant hsp7O can induce a cell-mediated immune response but does not induce a protective response. PMID:8926078

  1. Detection of UV-induced activation of NF-kappaB in a recombinant human cell line by means of Enhanced Green Fluorescent Protein (EGFP).

    PubMed

    Hellweg, Christine E; Baumstark-Khan, Christa

    2007-08-01

    The cellular protection reaction known as ultraviolet (UV) response leads to increased transcription of several genes. Parts of this transcriptional response are transmitted via activation of the Nuclear factor kappaB (NF-kappaB). The contribution of different UV radiation qualities to this process is not yet known. In a previous work, a stably transfected human cell line was developed which indicates activation of the NF-kappaB pathway by fluorescence of the reporters Enhanced Green Fluorescent Protein (EGFP) and its destabilized variant (d2EGFP) thereby allowing a fast and reliable monitoring of UV effects on the NF-kappaB pathway. Cells were exposed to a mercury low-pressure lamp or to simulated sunlight of different wavelength ranges and subjected to flow cytometric analysis after different post-irradiation periods. Growth capacity of cells after UV irradiation was quantified using a luminance measurement of crystal violet stained cell layers. In contrast to UVC and UVB, UVA radiation induced d2EGFP expression and NF-kappaB activation in a non-cytotoxic dose range. These results show that NF-kappaB plays a role in the UVA-induced gene activation in a non-cytotoxic dose range in a human epithelial cell line. PMID:17429671

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

    PubMed

    Duong-Ly, Krisna C; Gabelli, Sandra B

    2014-01-01

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

  3. Characterization of a double-CRD-mutated Gal-8 recombinant protein that retains co-stimulatory activity on antigen-specific T-cell response.

    PubMed

    Schroeder, Matías Nicolás; Tribulatti, María Virginia; Carabelli, Julieta; André-Leroux, Gwenaëlle; Caramelo, Julio Javier; Cattaneo, Valentina; Campetella, Oscar

    2016-04-01

    Galectins (Gals) constitute a family of mammalian lectins with affinity for β-galactosides, characterized by the presence of conserved CRDs (carbohydrate-recognition domains). We have found previously that Gal-8, from the tandem-repeat group with two linked CRDs, exerts two separate actions on CD4(+)T-cells: antigen-independent proliferation and, at lower concentration, antigen-specific co-stimulation. Whereas proliferation can be ascribed to the pro-inflammatory role of Gal-8, the co-stimulatory activity of borderline T-cell-specific responses allows the proposal of Gal-8 as an adjuvant in vaccination. To study the relevance of glycan-lectin interaction to these T-cell activities, we generated a double-mutated protein (Gal-8mut) by replacing canonical arginine residues on each CRD, so as to abolish sugar-binding capacity. As expected, Gal-8mut was unable to bind to lactosyl-Sepharose, confirming that lactose recognition was precluded; however, preservation of lectin activity was still evident since Gal-8mut displayed haemoagglutinatory effects and binding capacity to the T-cell surface. To search for glycan affinity, a glycan microarray analysis was conducted which revealed that Gal-8mut lost most low- and intermediate-, but retained high-, affinity interactions, mainly to polylactosamines and blood group antigens. These findings were supported further by molecular modelling. Regarding biological activity, Gal-8mut was unable to induce T-cell proliferation, but efficiently co-stimulated antigen-specific responses, bothin vitroandin vivo.Therefore Gal-8mut represents a useful tool to dissect the specificities of lectin-glycan interactions underlying distinctive Gal-8 activities on T-cell biology. Moreover, given its distinguishing properties, Gal-8mut could be used to enhance borderline immune responses without the non-specific pro-inflammatory activity or other potential adverse effects. PMID:26795039

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

    PubMed

    Biedendieck, Rebekka

    2016-01-01

    For many years the Gram-positive bacterium Bacillus megaterium has been used for the production and secretion of recombinant proteins. For this purpose it was systematically optimized. Plasmids with different inducible promoter systems, with different compatible origins, with small tags for protein purification and with various specific signals for protein secretion were combined with genetically improved host strains. Finally, the development of appropriate cultivation conditions for the production strains established this organism as a bacterial cell factory even for large proteins. Along with the overproduction of individual proteins the organism is now also used for the simultaneous coproduction of up to 14 recombinant proteins, multiple subsequently interacting or forming protein complexes. Some of these recombinant strains are successfully used for bioconversion or the biosynthesis of valuable components including vitamins. The titers in the g per liter scale for the intra- and extracellular recombinant protein production prove the high potential of B. megaterium for industrial applications. It is currently further enhanced for the production of recombinant proteins and multi-subunit protein complexes using directed genetic engineering approaches based on transcriptome, proteome, metabolome and fluxome data. PMID:27165321

  5. Immunodiagnosis of Ehrlichia canis Infection with Recombinant Proteins

    PubMed Central

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

    2001-01-01

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

  6. 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

  7. Multiple biological activities of human recombinant interleukin 1.

    PubMed Central

    Dinarello, C A; Cannon, J G; Mier, J W; Bernheim, H A; LoPreste, G; Lynn, D L; Love, R N; Webb, A C; Auron, P E; Reuben, R C

    1986-01-01

    Complementary DNA coding for human monocyte interleukin 1 (IL-1), pI 7 form, was expressed in Escherichia coli. During purification, IL-1 activity on murine T cells was associated with the recombinant protein. Homogeneous human recombinant IL-1 (hrIL-1) was tested in several assays to demonstrate the immunological and inflammatory properties attributed to this molecule. hrIL-1 induced proliferative responses in a cloned murine T cell in the presence of suboptimal concentrations of mitogen, whereas no effect was observed with hrIL-1 alone. At concentrations of 0.05 ng/ml, hrIL-1 doubled the response to mitogen (5 X 10(6) half maximal units/mg). Human peripheral blood T cells depleted of adherent cells underwent a blastogenic response and released interleukin 2 in the presence of hrIL-1 and mitogen. hrIL-1 was a potent inflammatory agent by its ability to induce human dermal fibroblast prostaglandin E2 production in vitro and to produce monophasic (endogenous pyrogen) fever when injected into rabbits or endotoxin-resistant mice. These studies establish that the dominant pI 7 form of recombinant human IL-1 possesses immunological and inflammatory properties and acts on the central nervous system to produce fever. Images PMID:3519678

  8. Evaluation of adjuvant activity of fractions derived from Agaricus blazei, when in association with the recombinant LiHyp1 protein, to protect against visceral leishmaniasis.

    PubMed

    de Jesus Pereira, Nathália Cristina; Régis, Wiliam César Bento; Costa, Lourena Emanuele; de Oliveira, Jamil Silvano; da Silva, Alanna Gomes; Martins, Vivian Tamietti; Duarte, Mariana Costa; de Souza, José Roberto Rodrigues; Lage, Paula Sousa; Schneider, Mônica Santos; Melo, Maria Norma; Soto, Manuel; Soares, Sandra Aguiar; Tavares, Carlos Alberto Pereira; Chávez-Fumagalli, Miguel Angel; Coelho, Eduardo Antonio Ferraz

    2015-06-01

    The development of effective prophylactic strategies to prevent leishmaniasis has become a high priority. No less important than the choice of an antigen, the association of an appropriate adjuvant is necessary to achieve a successful vaccination, as the majority of the tested antigens contain limited immunogenic properties, and need to be supplemented with immune response adjuvants in order to boost their immunogenicity. However, few effective adjuvants that can be used against leishmaniasis exist on the market today; therefore, it is possible to speculate that the research aiming to identify new adjuvants could be considered relevant. Recently, Agaricus blazei extracts have proved to be useful in enhancing the immune response to DNA vaccines against some diseases. This was based on the Th1 adjuvant activity of the polysaccharide-rich fractions from this mushroom. In this context, the present study evaluated purified fractions derived from Agaricus blazei as Th1 adjuvants through in vitro assays of their immune stimulation of spleen cells derived from naive BALB/c mice. Two of the tested six fractions (namely F2 and F4) were characterized as polysaccharide-rich fractions, and were able to induce high levels of IFN-γ, and low levels of IL-4 and IL-10 in the spleen cells. The efficacy of adjuvant action against L. infantum was evaluated in BALB/c mice, with these fractions being administered together with a recombinant antigen, LiHyp1, which was previously evaluated as a vaccine candidate, associated with saponin, against visceral leishmaniasis (VL). The associations between LiHyp1/F2 and LiHyp1/F4 were able to induce an in vivo Th1 response, which was primed by high levels of IFN-γ, IL-12, and GM-CSF, by low levels of IL-4 and IL-10; as well as by a predominance of IgG2a antibodies in the vaccinated animals. After infection, the immune profile was maintained, and the vaccines proved to be effective against L. infantum. The immune stimulatory effects in the

  9. A Library of Functional Recombinant Cell-surface and Secreted P. falciparum Merozoite Proteins*

    PubMed Central

    Crosnier, Cécile; Wanaguru, Madushi; McDade, Brian; Osier, Faith H.; Marsh, Kevin; Rayner, Julian C.; Wright, Gavin J.

    2013-01-01

    Malaria, an infectious disease caused by parasites of the Plasmodium genus, is one of the world's major public health concerns causing up to a million deaths annually, mostly because of P. falciparum infections. All of the clinical symptoms are associated with the blood stage of the disease, an obligate part of the parasite life cycle, when a form of the parasite called the merozoite recognizes and invades host erythrocytes. During erythrocyte invasion, merozoites are directly exposed to the host humoral immune system making the blood stage of the parasite a conceptually attractive therapeutic target. Progress in the functional and molecular characterization of P. falciparum merozoite proteins, however, has been hampered by the technical challenges associated with expressing these proteins in a biochemically active recombinant form. This challenge is particularly acute for extracellular proteins, which are the likely targets of host antibody responses, because they contain structurally critical post-translational modifications that are not added by some recombinant expression systems. Here, we report the development of a method that uses a mammalian expression system to compile a protein resource containing the entire ectodomains of 42 P. falciparum merozoite secreted and cell surface proteins, many of which have not previously been characterized. Importantly, we are able to recapitulate known biochemical activities by showing that recombinant MSP1-MSP7 and P12-P41 directly interact, and that both recombinant EBA175 and EBA140 can bind human erythrocytes in a sialic acid-dependent manner. Finally, we use sera from malaria-exposed immune adults to profile the relative immunoreactivity of the proteins and show that the majority of the antigens contain conformational (heat-labile) epitopes. We envisage that this resource of recombinant proteins will make a valuable contribution toward a molecular understanding of the blood stage of P. falciparum infections and

  10. 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.

  11. Dielectronic Recombination In Active Galactic Nuclei

    NASA Technical Reports Server (NTRS)

    Lukic, D. V.; Schnell, M.; Savin, D. W.; Altun, Z.; Badnell, N.; Brandau, C.; Schmidt, E. W.; Mueller, A.; Schippers, S.; Sprenger, F.; Lestinsky, M.; Wolf, A.

    2006-01-01

    XMM-Newton and Chandra observations of active galactic nuclei (AGN) show rich spectra of X-ray absorption lines. These observations have detected a broad unresolved transition array (UTA) between approx. 15-17 A. This is attributed to inner-shell photoexcitation of M-shell iron ions. Modeling these UTA features is currently limited by uncertainties in the low-temperature dielectronic recombination (DR) data for M-shell iron. In order to resolve this issue, and to provide reliable iron M-shell DR data for plasma modeling, we are carrying out a series of laboratory measurements using the heavy-ion Test Storage Ring (TSR) at the Max-Plank-Institute for Nuclear Physics in Heidelberg, Germany. Currently, laboratory measurements of low temperature DR can only be performed at storage rings. We use the DR data obtained at TSR, to calculate rate coefficients for plasma modeling and to benchmark theoretical DR calculations. Here we report our recent experimental results for DR of Fe XIV forming Fe XIII.

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

    PubMed

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

    2013-01-01

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    2011-01-01

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

  15. The Use of Affinity Tags to Overcome Obstacles in Recombinant Protein Expression and Purification.

    PubMed

    Amarasinghe, Chinthaka; Jin, Jian-Ping

    2015-01-01

    Research and industrial demands for recombinant proteins continue to increase over time for their broad applications in structural and functional studies and as therapeutic agents. These applications often require large quantities of recombinant protein at desirable purity, which highlights the importance of developing and improving production approaches that provide high level expression and readily achievable purity of recombinant protein. E. coli is the most widely used host for the expression of a diverse range of proteins at low cost. However, there are common pitfalls that can severely limit the expression of exogenous proteins, such as stability, low solubility and toxicity to the host cell. To overcome these obstacles, one strategy that has found to be promising is the use of affinity tags or carrier peptide to aid in the folding of the target protein, increase solubility, lower toxicity and increase the level of expression. In the meantime, the tags and fusion proteins can be designed to facilitate affinity purification. Since the fusion protein may not exhibit the native conformation of the target protein, various strategies have been developed to remove the tag during or after purification to avoid potential complications in structural and functional studies and to obtain native biological activities. Despite extensive research and rapid development along these lines, there are unsolved problems and imperfect applications. This focused review compares and contrasts various strategies that employ affinity tags to improve bacterial expression and to facilitate purification of recombinant proteins. The pros and cons of the approaches are discussed for more effective applications and new directions of future improvement. PMID:26216265

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

    PubMed

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

    2010-01-01

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

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

    PubMed

    Walls, Dermot; Loughran, Sinéad T

    2011-01-01

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

  18. 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

  19. 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

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

    PubMed

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

    2004-06-01

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

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

    PubMed

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

    2014-11-01

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

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

    PubMed

    Prashad, Kamal; Mehra, Sarika

    2015-03-01

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

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

    PubMed

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

    1991-03-01

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

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

    PubMed

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

    2003-03-30

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

  5. [Recombinant expression and antibacterial activity of i-type lysozyme from sea cucumber Stichopus japonicus].

    PubMed

    Wang, Xiuxia; Cong, Lina; Wang, Dan; Yang, Xijian; Zhu, Beiwei

    2009-02-01

    The cDNA of an i type lysozyme was cloned from Stichopus japonicus (named as SjLys). The DNA fragment of the mature SjLys was subcloned into expression vector of pET-32a (+) to construct the recombinant plasmid of pET32a (+)-SjLys. The recombinant plasmid was then transformed into Escherichia coli BL21 (DE3) pLysS and induced by isopropylthio-beta-D-galactoside (IPTG). The recombinant protein expressed as inclusion bodies was denatured, partially purified and refolded to be an active form. The bacteriolytic activity of recombinant protein purified by the metal-chelating was 19.2 U/mg. The antibacterial activity of the purified recombinant SjLys (rSjLys) was analyzed. The rSjLys protein displayed inhibitive effect on the growth of the tested Gram-positive and Gram-negative bacteria. In particular, rSjLys had a strong inhibitive activity on Vibrio parahaemolyticus and Pseudomonas aeruginosa, both the most common pathogenic bacteria in the marine animals. The heat-treated rSjLys exhibited more potent activities against all tested bacteria. These results indicated that the S. japonicus lysozyme was the enzyme with combined enzymatic (glycosidase) and non-enzymatic antibacterial action, and it had a wide antibacterial spectrum. Therefore, it is suggested that the S. japonicus lysozyme should be one of the important molecules against pathogens in the innate immunity of sea cucumbers. PMID:19459322

  6. Recombinant sheep pox virus proteins elicit neutralizing antibodies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  7. 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

    Background A vaccine targeting Plasmodium vivax will be an essential component of any comprehensive malaria elimination program, but major gaps in our understanding of P. vivax biology, including the protein-protein interactions that mediate merozoite invasion of reticulocytes, hinder the search for candidate antigens. Only one ligand-receptor interaction has been identified, that between P. vivax Duffy Binding Protein (PvDBP) and the erythrocyte Duffy Antigen Receptor for Chemokines (DARC), and strain-specific immune responses to PvDBP make it a complex vaccine target. To broaden the repertoire of potential P. vivax merozoite-stage vaccine targets, we exploited a recent breakthrough in expressing full-length ectodomains of Plasmodium proteins in a functionally-active form in mammalian cells and initiated a large-scale study of P. vivax merozoite proteins that are potentially involved in reticulocyte binding and invasion. Methodology/Principal Findings We selected 39 P. vivax proteins that are predicted to localize to the merozoite surface or invasive secretory organelles, some of which show homology to P. falciparum vaccine candidates. Of these, we were able to express 37 full-length protein ectodomains in a mammalian expression system, which has been previously used to express P. falciparum invasion ligands such as PfRH5. To establish whether the expressed proteins were correctly folded, we assessed whether they were recognized by antibodies from Cambodian patients with acute vivax malaria. IgG from these samples showed at least a two-fold change in reactivity over naïve controls in 27 of 34 antigens tested, and the majority showed heat-labile IgG immunoreactivity, suggesting the presence of conformation-sensitive epitopes and native tertiary protein structures. Using a method specifically designed to detect low-affinity, extracellular protein-protein interactions, we confirmed a predicted interaction between P. vivax 6-cysteine proteins P12 and P41, further

  8. 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

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

    PubMed Central

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

    1991-01-01

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

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

    PubMed

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

    2012-01-01

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

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

    PubMed

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

    2016-03-18

    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

  12. 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

  13. Recombinant human bone morphogenetic protein-9 potently induces osteogenic differentiation of human periodontal ligament fibroblasts.

    PubMed

    Fuchigami, Sawako; Nakamura, Toshiaki; Furue, Kirara; Sena, Kotaro; Shinohara, Yukiya; Noguchi, Kazuyuki

    2016-04-01

    To accomplish effective periodontal regeneration for periodontal defects, several regenerative methods using growth and differentiation factors, including bone morphogenetic proteins (BMPs), have been developed. Bone morphogenetic protein-9 exhibits the most potent osteogenic activity of this growth factor family. However, it is unclear whether exogenous BMP-9 can induce osteogenic differentiation in human periodontal ligament (PDL) fibroblasts. Here, we examined the effects of recombinant human (rh) BMP-9 on osteoblastic differentiation in human PDL fibroblasts in vitro, compared with rhBMP-2. Recombinant human BMP-9 potently induced alkaline phosphatase (ALP) activity, mineralization, and increased expression of runt-related transcription factor-2/core binding factor alpha 1 (RUNX2/CBFA1), osterix, inhibitor of DNA binding/differentiation-1 (ID1), osteopontin, and bone sialoprotein genes, compared with rhBMP-2. The levels of rhBMP-9-induced osterix and ALP mRNA were significantly reduced in activin receptor-like kinase-1 and -2 small interfering RNA (siRNA)-transfected human PDL fibroblasts. Recombinant human BMP-9-induced ALP activity was not inhibited by noggin, in contrast to rhBMP-2 induced ALP activity, which was. Phosphorylation of SMAD1/5/8 in human PDL fibroblasts was induced by addition of rhBMP-9. Recombinant human BMP-9-induced ALP activity was suppressed by SB203580, SP600125, and U0126, which are inhibitors of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase 1/2 (ERK1/2), respectively. Our data suggest that rhBMP-9 is a potent inducer of the differentiation of human PDL fibroblasts into osteoblast-like cells and that this may be mediated by the SMAD and mitogen-activated protein kinase (p38, ERK1/2, and JNK) pathways. PMID:26879145

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

    PubMed

    Wiesler, Simone C; Weinzierl, Robert O J

    2015-01-01

    Affinity purification of recombinant proteins has become the method of choice to obtain good quantities and qualities of proteins for a variety of downstream biochemical applications. While manual or FPLC-assisted purification techniques are generally time-consuming and labor-intensive, the advent of high-throughput technologies and liquid handling robotics has simplified and accelerated this process significantly. Additionally, without the human factor as a potential source of error, automated purification protocols allow for the generation of large numbers of proteins simultaneously and under directly comparable conditions. The delivered material is ideal for activity comparisons of different variants of the same protein. Here, we present our strategy for the simultaneous purification of up to 24 affinity-tagged proteins for activity measurements in biochemical assays. The protocol described is suitable for the scale typically required in individual research laboratories. PMID:25749949

  15. Analysis of the cruciform binding activity of recombinant 14-3-3zeta-MBP fusion protein, its heterodimerization profile with endogenous 14-3-3 isoforms, and effect on mammalian DNA replication in vitro.

    PubMed

    Alvarez, David; Callejo, Mario; Shoucri, Rami; Boyer, Lee; Price, Gerald B; Zannis-Hadjopoulos, Maria

    2003-06-17

    The human cruciform binding protein (CBP), a member of the 14-3-3 protein family, has been recently identified as an origin of DNA replication binding protein and involved in DNA replication. Here, pure recombinant 14-3-3zeta tagged with maltose binding protein (r14-3-3zeta-MBP) at its N-terminus was tested for binding to cruciform DNA either in the absence or presence of F(TH), a CBP-enriched fraction, by electromobility shift assay (EMSA), followed by Western blot analysis of the electroeluted CBP-cruciform DNA complex. The r14-3-3zeta-MBP was found to have cruciform binding activity only after preincubation with F(TH). Anti-MBP antibody immunoprecipitation of F(TH) preincubated with r14-3-3zeta-MBP, followed by Western blot analysis with antibodies specific to the beta, gamma, epsilon, zeta, and sigma 14-3-3 isoforms showed that r14-3-3zeta-MBP heterodimerized with the endogenous beta, epsilon, and zeta isoforms present in the F(TH) but not with the gamma or sigma isoforms. Immunoprecipitation of endogenous 14-3-3zeta from nuclear extracts (NE) of HeLa cells that were either serum-starved (s-s) or blocked at the G(1)/S or G(2)/M phases of the cell cycle revealed that at G(1)/S and G(2)/M, the zeta isoform heterodimerized only with the beta and epsilon isoforms, while in s-s extracts, the 14-3-3zeta/epsilon heterodimer was never detected, and the 14-3-3zeta/beta heterodimer was seldom detected. Furthermore, addition of r14-3-3zeta-MBP to HeLa cell extracts used in a mammalian in vitro replication system increased the replication level of p186, a plasmid bearing the minimal 186-bp origin of the monkey origin of DNA replication ors8, by approximately 3.5-fold. The data suggest that specific dimeric combinations of the 14-3-3 isoforms have CBP activity and that upregulation of this activity leads to an increase in DNA replication. PMID:12795617

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

    PubMed

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

    2012-05-01

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

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

    PubMed

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

    2007-04-27

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

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed

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

    2010-03-15

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

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

    PubMed

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

    1997-12-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2015-11-01

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

  3. Versatile Recombinant SUMOylation System for the Production of SUMO-Modified Protein

    PubMed Central

    Weber, Alain R.; Schuermann, David; Schär, Primo

    2014-01-01

    Posttranslational modification by small ubiquitin-like modifiers (SUMO) is being associated with a growing number of regulatory functions in diverse cellular processes. The biochemical investigation into the underlying molecular mechanisms, however, has been lagging behind due to the difficulty to generate sufficient amounts of recombinant SUMOylated proteins. Here, we present two newly designed two-component vector systems for the expression and purification of SUMO-modified target proteins in Escherichia coli. One system consists of a vector for SUMO conjugation, expressing human SUMO-activating (SAE1/SAE2) and conjugating (Ubc9) enzymes together with His6-tagged SUMO1, 2 or 3, that can be combined with commonly used expression constructs for any gene of interest. To facilitate SUMOylation of targets normally requiring a SUMO-E3 ligase for efficient modification, a second system is designed to express the target protein as a fusion with the human SUMO-conjugating enzyme Ubc9, thus compensating the absence of a potential SUMO ligase. We demonstrate the proficiency of these systems by SUMOylation of two DNA repair proteins, the thymine DNA glycosylase (TDG) and XRCC1, and describe purification schemes for SUMOylated proteins in native and active form. This SUMO toolbox facilitates “in-cell” and “in-extract” production and purification of recombinant SUMO-modified target proteins for functional and structural analysis. PMID:25007328

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

    PubMed

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

    2015-05-01

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

  5. 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.

  6. Construction and characterization of a recombinant human adenovirus vector expressing bone morphogenetic protein 2.

    PubMed

    Zhang, Zheng; Wang, Guoxian; Li, Chen; Liu, Danping

    2013-08-01

    The aim of this study was to construct and characterize a novel recombinant human adenovirus vector expressing bone morphogenetic protein 2 (BMP2) and green fluorescent protein (GFP). The BMP2 gene in the plasmid pcDNA3-BMP2 was sequenced and the restriction enzyme recognition sites were analyzed. Following mutagenesis using polymerase chain reaction (PCR), the gene sequence after the translation termination codon was removed and new restriction sites were added. The mutated BMP2 gene (BMP2(+) gene) was cloned into an adenovirus shuttle vector to obtain pShuttle cytomegalovirus (CMV)-BMP2(+)-internal ribosome entry site (IRES)-hrGFP-1. The adenovirus plasmid pAd CMV-BMP2(+)-IRES-hrGFP-1 was constructed by homologous recombination and was transfected into HEK293A cells, followed by adenovirus packaging. pAd CMV-BMP2 was used as the control. The two types of adenovirus were transfected into marrow stromal cells (MSCs). The expression of BMP2 and GFP, as well as the alkaline phosphatase (ALP) activity of expressed BMP2 were detected. Following mutagenesis, the BMP2 gene sequence and recombinant adenovirus vector were as predicted. The novel adenovirus vector expressed both BMP2 and GFP, indicating that a novel recombinant human adenovirus vector expressing BMP2 had been successfully constructed. PMID:24137184

  7. Expression and activity of recombinant proaerolysin derived from Aeromonas hydrophila cultured from diseased channel catfish

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proaerolysin-coding gene was cloned from the genomic DNA of A. hydrophila and heterologously expressed in E. coli. The purified recombinant proaerolysin was inactive and could be activated by treatment with proteases, furin and trypsin, and extra-cellular proteins (ECPs, the cell-free supernatant of...

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

    PubMed

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

    1991-04-23

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

  9. The insecticidal activity of recombinant garlic lectins towards aphids.

    PubMed

    Fitches, Elaine; Wiles, Duncan; Douglas, Angela E; Hinchliffe, Gareth; Audsley, Neil; Gatehouse, John A

    2008-10-01

    The heterodimeric and homodimeric garlic lectins ASAI and ASAII were produced as recombinant proteins in the yeast Pichia pastoris. The proteins were purified as functional dimeric lectins, but underwent post-translational proteolysis. Recombinant ASAII was a single homogenous polypeptide which had undergone C-terminal processing similar to that occurring in planta. The recombinant ASAI was glycosylated and subject to variable and heterogenous proteolysis. Both lectins showed insecticidal effects when fed to pea aphids (Acyrthosiphon pisum) in artificial diet, ASAII being more toxic than ASAI at the same concentration. Acute toxicity (mortality at < or =48 h exposure; similar timescale to starvation) was only apparent at the highest lectin concentrations tested (2.0 mg ml(-)1), but dose-dependent chronic toxicity (mortality at >3d exposure) was observed over the concentration range 0.125-2.0 mg ml(-1). The recombinant lectins caused mortality in both symbiotic and antibiotic-treated aphids, showing that toxicity is not dependent on the presence of the bacterial symbiont (Buchnera aphidicola), or on interaction with symbiont proteins, such as the previously identified lectin "receptor" symbionin. A pull-down assay coupled with peptide mass fingerprinting identified two abundant membrane-associated aphid gut proteins, alanyl aminopeptidase N and sucrase, as "receptors" for lectin binding. PMID:18707000

  10. Collaboration of RAG2 with RAG1-like proteins during the evolution of V(D)J recombination.

    PubMed

    Carmona, Lina Marcela; Fugmann, Sebastian D; Schatz, David G

    2016-04-15

    The recombination-activating gene 1 (RAG1) and RAG2 proteins initiate V(D)J recombination, the process that assembles the B- and T-lymphocyte antigen receptor genes of jawed vertebrates. RAG1 and RAG2 are thought to have arisen from a transposable element, but the origins of this element are not understood. We show that two ancestral RAG1 proteins, Transib transposase and purple sea urchin RAG1-like, have a latent ability to initiate V(D)J recombination when coexpressed with RAG2 and that in vitro transposition by Transib transposase is stimulated by RAG2. Conversely, we report low levels of V(D)J recombination by RAG1 in the absence of RAG2. Recombination by RAG1 alone differs from canonical V(D)J recombination in having lost the requirement for asymmetric DNA substrates, implicating RAG2 in the origins of the "12/23 rule," a fundamental regulatory feature of the reaction. We propose that evolution of RAG1/RAG2 began with a Transib transposon whose intrinsic recombination activity was enhanced by capture of an ancestral RAG2, allowing for the development of adaptive immunity. PMID:27056670

  11. Cell culture media supplementation of bioflavonoids for the targeted reduction of acidic species charge variants on recombinant therapeutic proteins.

    PubMed

    Hossler, Patrick; Wang, Min; McDermott, Sean; Racicot, Christopher; Chemfe, Kofi; Zhang, Yun; Chumsae, Christopher; Manuilov, Anton

    2015-01-01

    Charge variants in recombinant proteins are an important series of protein modifications, whose potential role on protein stability, activity, immunogenicity, and pharmacokinetics continues to be studied. Monoclonal antibodies in particular have been shown to have a wide range of acidic species variants, including those associated with the addition of covalent modifications as well as the chemical degradation at specific peptide regions on the antibody. These variants play a significant role toward the overall heterogeneity of recombinant therapeutic proteins and are typically monitored during manufacturing to ensure they lie within proven acceptable ranges. In this work, it has been found that the supplementation of members of the bioflavonoid chemical family into mammalian cell culture media was effective toward the reduction of acidic species charge variants on recombinant monoclonal antibodies and dual variable domain immunoglobulins. The demonstrated reduction in acidic species through the use of bioflavonoids facilitates the manufacturing of a less heterogeneous product with potential improvements in antibody structure and function. PMID:25920009

  12. Biochemical Characterization of a Recombinant TRIM5α Protein That Restricts Human Immunodeficiency Virus Type 1 Replication▿ †

    PubMed Central

    Langelier, Charles R.; Sandrin, Virginie; Eckert, Debra M.; Christensen, Devin E.; Chandrasekaran, Viswanathan; Alam, Steven L.; Aiken, Christopher; Olsen, John C.; Kar, Alak Kanti; Sodroski, Joseph G.; Sundquist, Wesley I.

    2008-01-01

    The rhesus monkey intrinsic immunity factor TRIM5αrh recognizes incoming capsids from a variety of retroviruses, including human immunodeficiency virus type 1 (HIV-1) and equine infectious anemia virus (EIAV), and inhibits the accumulation of viral reverse transcripts. However, direct interactions between restricting TRIM5α proteins and retroviral capsids have not previously been demonstrated using pure recombinant proteins. To facilitate structural and mechanistic studies of retroviral restriction, we have developed methods for expressing and purifying an active chimeric TRIM5αrh protein containing the RING domain from the related human TRIM21 protein. This recombinant TRIM5-21R protein was expressed in SF-21 insect cells and purified through three chromatographic steps. Two distinct TRIM5-21R species were purified and shown to correspond to monomers and dimers, as analyzed by analytical ultracentrifugation. Chemically cross-linked recombinant TRIM5-21R dimers and mammalian-expressed TRIM5-21R and TRIM5α proteins exhibited similar sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobilities, indicating that mammalian TRIM5α proteins are predominantly dimeric. Purified TRIM5-21R had ubiquitin ligase activity and could autoubquitylate with different E2 ubiquitin conjugating enzymes in vitro. TRIM5-21R bound directly to synthetic capsids composed of recombinant HIV-1 CA-NC proteins and to authentic EIAV core particles. HIV-1 CA-NC assemblies bound dimeric TRIM5-21R better than either monomeric TRIM5-21R or TRIM5-21R constructs that lacked the SPRY domain or its V1 loop. Thus, our studies indicate that TRIM5α proteins are dimeric ubiquitin E3 ligases that recognize retroviral capsids through direct interactions mediated by the SPRY domain and demonstrate that these activities can be recapitulated in vitro using pure recombinant proteins. PMID:18799573

  13. 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

  14. Recombinant Sheep Pox Virus Proteins Elicit Neutralizing Antibodies.

    PubMed

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

    2016-01-01

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

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

    PubMed Central

    Chakrabarti, Sanjukta; Barrow, Colin J.; Kanwar, Rupinder K.; Ramana, Venkata; Kanwar, Jagat R.

    2016-01-01

    Clipping of recombinant proteins is a major issue in animal cell cultures. A recombinant Fc-fusion protein, VEGFR1(D1–D3)-Fc expressed in CHOK1SV GS-KO cells was observed to be undergoing clippings in lab scale cultures. Partial cleaving of expressed protein initiated early on in cell culture and was observed to increase over time in culture and also on storage. In this study, a few parameters were explored in a bid to inhibit clipping in the fusion protein The effects of culture temperature, duration of culture, the addition of an anti-clumping agent, ferric citrate and use of protease inhibitor cocktail on inhibition of proteolysis of the Fc fusion were studied. Lowering of culture temperature from 37 to 30 °C alone appears to be the best solution for reducing protein degradation from the quality, cost and regulatory points of view. The obtained Fc protein was characterized and found to be in its stable folded state, exhibiting a high affinity for its ligand and also biological and functional activities. PMID:27294920

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

    PubMed

    Chakrabarti, Sanjukta; Barrow, Colin J; Kanwar, Rupinder K; Ramana, Venkata; Kanwar, Jagat R

    2016-01-01

    Clipping of recombinant proteins is a major issue in animal cell cultures. A recombinant Fc-fusion protein, VEGFR1(D1-D3)-Fc expressed in CHOK1SV GS-KO cells was observed to be undergoing clippings in lab scale cultures. Partial cleaving of expressed protein initiated early on in cell culture and was observed to increase over time in culture and also on storage. In this study, a few parameters were explored in a bid to inhibit clipping in the fusion protein The effects of culture temperature, duration of culture, the addition of an anti-clumping agent, ferric citrate and use of protease inhibitor cocktail on inhibition of proteolysis of the Fc fusion were studied. Lowering of culture temperature from 37 to 30 °C alone appears to be the best solution for reducing protein degradation from the quality, cost and regulatory points of view. The obtained Fc protein was characterized and found to be in its stable folded state, exhibiting a high affinity for its ligand and also biological and functional activities. PMID:27294920

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

    PubMed Central

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

    2001-01-01

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

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

    PubMed

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

    2001-04-01

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

  19. 75 FR 42114 - Office of Biotechnology Activities; Recombinant DNA Research: Proposed Action Under the NIH...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-07-20

    ... HUMAN SERVICES National Institutes of Health Office of Biotechnology Activities; Recombinant DNA Research: Proposed Action Under the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH... transgenic rodents by recombinant DNA technology must be registered with the Institutional...

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

    PubMed

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

    2015-07-01

    MAP30 (Momordica Antiviral Protein 30 Kd), a single-stranded type-I ribosome inactivating protein, possesses versatile biological activities including anti-tumor abilities. However, the low efficiency penetrating into tumor cells hampers the tumoricidal effect of MAP30. This paper describes MAP30 fused with a human-derived cell penetrating peptide HBD which overcome the low uptake efficiency by tumor cells and exhibits higher anti-tumor bioactivity. MAP30 gene was cloned from the genomic DNA of Momordica charantia and the recombinant plasmid pET28b-MAP30-HBD was established and transferred into Escherichia coli BL21 (DE3). The recombinant MAP30-HBD protein (rMAP30-HBD) was expressed in a soluble form after being induced by 0.5mM IPTG for 14h at 15°C. The recombinant protein was purified to greater than 95% purity with Ni-NTA affinity chromatography. The rMAP30-HBD protein not only has topological inactivation and protein translation inhibition activity but also showed significant improvements in cytotoxic activity compared to that of the rMAP30 protein without HBD in the tested tumor cell lines, and induced higher apoptosis rates in HeLa cells analyzed by Annexin V-FITC with FACS. This paper demonstrated a new method for improving MAP30 protein anti-tumor activity and might have potential applications in cancer therapy area. PMID:25797209

  1. Miz-1 regulates translation of Trp53 via ribosomal protein L22 in cells undergoing V(D)J recombination

    PubMed Central

    Rashkovan, Marissa; Vadnais, Charles; Ross, Julie; Gigoux, Mathieu; Suh, Woong-Kyung; Gu, Wei; Kosan, Christian; Möröy, Tarik

    2014-01-01

    To be effective, the adaptive immune response requires a large repertoire of antigen receptors, which are generated through V(D)J recombination in lymphoid precursors. These precursors must be protected from DNA damage-induced cell death, however, because V(D)J recombination generates double-strand breaks and may activate p53. Here we show that the BTB/POZ domain protein Miz-1 restricts p53-dependent induction of apoptosis in both pro-B and DN3a pre-T cells that actively rearrange antigen receptor genes. Miz-1 exerts this function by directly activating the gene for ribosomal protein L22 (Rpl22), which binds to p53 mRNA and negatively regulates its translation. This mechanism limits p53 expression levels and thus contains its apoptosis-inducing functions in lymphocytes, precisely at differentiation stages in which V(D)J recombination occurs. PMID:25468973

  2. Molluscan mobile elements similar to the vertebrate recombination-activating genes

    PubMed Central

    Panchin, Yuri; Moroz, Leonid L.

    2009-01-01

    Animal genomes contain ~20,000 genes. Additionally millions of genes for antigen receptors are generated in cells of the immune system from the sets of separate gene segments by a mechanism known as the V(D)J somatic recombination. The components of the V(D)J recombination system, Recombination-Activating Gene proteins (RAG1 and RAG2) and recombination signal sequence (RSS), are thought to have “entered” the vertebrate genome as a hypothetical “RAG transposon”. Recently discovered mobile elements have terminal inverted repeats (TIRs) similar to RSS and may encode proteins with a different degree of similarity to RAG1. We describe a novel N-RAG-TP transposon identified from the sea slug Aplysia californica that encodes a protein similar to the N-terminal part of RAG1 in vertebrates. This refines the “RAG transposon” hypothesis and allows us to propose a scenario for V(D)J recombination machinery evolution from a relic transposon related to the existing mobile elements N-RAG-TP, Chapaev and Transib. PMID:18313399

  3. Directed Ig class switch recombination in activated murine B cells.

    PubMed Central

    Winter, E; Krawinkel, U; Radbruch, A

    1987-01-01

    Immunoglobulin class switch recombination occurs at frequencies of up to 10%/cell/generation in activated murine B-lymphocytes. We analysed cH gene rearrangements and switch recombinations from active and inactive IgH loci of B-cells activated in various ways and immortalized by cell fusion. Although about half of the IgM+ cells show rearrangement of c mu genes, the deletion of c mu is a rare event. Half of the IgG3+ and IgG1+ cells show rearrangement of c mu genes on the inactive IgH locus and the other half of the IgG+ cells have deleted c mu from both IgH loci by switch recombination. This recombination is directed to the same switch regions on both IgH loci in 60-80% of all cases. Interleukin 4 may play a critical role in programming murine B-lymphocytes for specific switch recombination. Images Fig. 1. Fig. 2. Fig. 6. PMID:3038529

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

    PubMed

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

    2015-01-01

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

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

    PubMed

    Kovalskaya, Natalia; Hammond, Rosemarie W

    2009-01-01

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

  6. A Recombinant Multiepitope Protein for Hepatitis B Diagnosis

    PubMed Central

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

    2013-01-01

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

  7. GTPase activity and biochemical characterization of a recombinant cotton fiber annexin

    SciTech Connect

    Shin, H.; Brown, R.M. Jr. . Dept. of Botany)

    1999-03-01

    A cDNA encoding annexin was isolated from a cotton (Gossypium hirsutum) fiber cDNA library. The cDNA was expressed in Escherichia coli, and the resultant recombinant protein was purified. The authors then investigated some biochemical properties of the recombinant annexin based on the current understanding of plant annexins. An add-back experiment was performed to study the effect of the recombinant annexin on [beta]-glucan synthase activity, but no effect was found. However, it was found that the recombinant annexin could display ATPase/GTPase activities. The recombinant annexin showed much higher GTPase than ATPase activity. Mg[sup 2+] was essential for these activities, whereas a high concentration of Ca[sup 2+] was inhibitory. A photolabeling assay showed that this annexin could bind GTP more specifically than ATP. The GTP-binding site on the annexin was mapped into the carboxyl-terminal fourth repeat of annexin from the photolabeling experiment using domain-deletion mutants of this annexin. Northern-blot analysis showed that the annexin gene was highly expressed in the elongation stages of cotton fiber differentiation, suggesting a role of this annexin in cell elongation.

  8. Effect of Chemical Chaperones in Improving the Solubility of Recombinant Proteins in Escherichia coli▿†

    PubMed Central

    Prasad, Shivcharan; Khadatare, Prashant B.; Roy, Ipsita

    2011-01-01

    The recovery of active proteins from inclusion bodies usually involves chaotrope-induced denaturation, followed by refolding of the unfolded protein. The efficiency of renaturation is low, leading to reduced yield of the final product. In this work, we report that recombinant proteins can be overexpressed in the soluble form in the host expression system by incorporating compatible solutes during protein expression. Green fluorescent protein (GFP), which was otherwise expressed as inclusion bodies, could be made to partition off into the soluble fraction when sorbitol and arginine, but not ethylene glycol, were present in the growth medium. Arginine and sorbitol increased the production of soluble protein, while ethylene glycol did not. Production of ATP increased in the presence of sorbitol and arginine, but not ethylene glycol. A control experiment with fructose addition indicated that protein solubilization was not due to a simple ATP increase. We have successfully reproduced these results with the N-terminal domain of HypF (HypF-N), a bacterial protein which forms inclusion bodies in Escherichia coli. Instead of forming inclusion bodies, HypF-N could be expressed as a soluble protein in the presence of sorbitol, arginine, and trehalose in the expression medium. PMID:21551288

  9. [Expression, purification of recombinant cationic peptide AIK in Escherichia coli and its antitumor activity].

    PubMed

    Fan, Fangfang; Sun, Huiying; Xu, Hui; Liu, Jiawei; Zhang, Haiyuan; Li, Yilan; Ning, Xuelian; Sun, Yue; Bai, Jing; Fu, Songbin; Zhou, Chunshui

    2015-12-01

    AIK is a novel cationic peptide with potential antitumor activity. In order to construct the AIK expression vector by Gateway technology, and establish an optimal expression and purification method for recombinant AIK, a set of primers containing AttB sites were designed and used to create the AttB-TEV-FLAG-AIR fusion gene by overlapping PCR. The resulting fusion gene was cloned into the donor vector pDONR223 by attB and attP mediated recombination (BP reaction), then, transferred into the destination vector pDESTl 5 by attL and attR mediated recombination (LR reaction). All the cloning was verified by both colony PCR and DNA sequencing. The BL21 F. coli transformed by the GST-AIR expression plasmid was used to express the GST-AIK fusion protein with IPTG induction and the induction conditions were optimized. GST-AIR fusion protein was purified by glutathione magnetic beads, followed by rTEV cleavage to remove GST tag and MTS assay to test the growth inhibition activity of the recombinant AIR on human leukemia HL-60 cells. We found that a high level of soluble expression of GST-AIK protein (more than 30% out of the total bacterial proteins) was achieved upon 0.1 mmol/L ITPG induction for 4 h at 37 °C in the transformed BL21 F. coli with starting OD₆₀₀ at 1.0. Through GST affinity purification and rTEV cleavage, the purity of the resulting recombinant AIK was greater than 95%. And the MTS assays on HL-60 cells confirmed that the recombinant AIK retains an antitumor activity at a level similar to the chemically synthesized AIK. Taken together, we have established a method for expression and purification of recombinant AIK with a potent activity against tumor cells, which will be beneficial for the large-scale production and application of recombinant AIK in the future. PMID:27093838

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

    PubMed Central

    Capaldi, Stefano

    2014-01-01

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

  11. Maximal stimulation of meiotic recombination by a yeast transcription factor requires the transcription activation domain and a DNA-binding domain.

    PubMed Central

    Kirkpatrick, D T; Fan, Q; Petes, T D

    1999-01-01

    The DNA sequences located upstream of the yeast HIS4 represent a very strong meiotic recombination hotspot. Although the activity of this hotspot requires the transcription activator Rap1p, the level of HIS4 transcription is not directly related to the level of recombination. We find that the recombination-stimulating activity of Rap1p requires the transcription activation domain of the protein. We show that a hybrid protein with the Gal4p DNA-binding domain and the Rap1p activation domain can stimulate recombination in a strain in which Gal4p-binding sites are inserted upstream of HIS4. In addition, we find recombination hotspot activity associated with the Gal4p DNA-binding sites that is independent of known transcription factors. We suggest that yeast cells have two types of recombination hotspots, alpha (transcription factor dependent) and beta (transcription factor independent). PMID:10224246

  12. Vaccinia virus recombinants expressing chimeric proteins of human immunodeficiency virus and gamma interferon are attenuated for nude mice.

    PubMed Central

    Giavedoni, L D; Jones, L; Gardner, M B; Gibson, H L; Ng, C T; Barr, P J; Yilma, T

    1992-01-01

    We have developed a method for attenuating vaccinia virus recombinants by expressing a fusion protein of a lymphokine and an immunogen. Chimeric genes were constructed that coded for gamma interferon (IFN-gamma) and structural proteins of the human immunodeficiency virus type 1 (HIV-1). In this study, we describe the biological and immunological properties of vaccinia virus recombinants expressing chimeric genes of murine or human IFN-gamma with glycoprotein gp120, gag, and a fragment of gp41. All fusion proteins retained the antigenic characteristics of both IFN-gamma and HIV as shown by immunoblot analysis. However, the antiviral activity of IFN-gamma could be demonstrated only for the IFN-gamma-gag fusion protein. In contrast, the attenuating activity of IFN-gamma for nude mice was retained by all of the recombinants, albeit at various rates. Unlike the antiviral activity, the attenuating activity of IFN-gamma was not species specific. Implications for the development of attenuated live recombinant vaccines for AIDS are discussed. Images PMID:1565633

  13. 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

  14. 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).

  15. Genetically encoded optical activation of DNA recombination in human cells.

    PubMed

    Luo, J; Arbely, E; Zhang, J; Chou, C; Uprety, R; Chin, J W; Deiters, A

    2016-06-30

    We developed two tightly regulated, light-activated Cre recombinase enzymes through site-specific incorporation of two genetically-encoded photocaged amino acids in human cells. Excellent optical off to on switching of DNA recombination was achieved. Furthermore, we demonstrated precise spatial control of Cre recombinase through patterned illumination. PMID:27277957

  16. Recombination activity of interfaces in multicrystalline silicon

    SciTech Connect

    Peshcherova, S. M.; Yakimov, E. B.; Nepomnyashchikh, A. I.; Pavlova, L. A.; Feklisova, O. V.

    2015-06-15

    The electrical activity of grain boundaries in multicrystalline silicon grown from metallurgical silicon by the Bridgman method is investigated by the method of electron-beam induced current. The main tendencies of atypical manifestation of the local electrical activity of Σ3(111) and Σ9(110) special boundaries are revealed. The structural features of the grain boundaries after selective etching and the impurity-distribution characteristics in multicrystalline silicon are determined by the methods of electron backscattering diffraction and electron-probe microanalysis.

  17. The RAG proteins and V(D)J recombination: complexes, ends, and transposition.

    PubMed

    Fugmann, S D; Lee, A I; Shockett, P E; Villey, I J; Schatz, D G

    2000-01-01

    V(D)J recombination proceeds through a series of protein:DNA complexes mediated in part by the RAG1 and RAG2 proteins. These proteins are responsible for sequence-specific DNA recognition and DNA cleavage, and they appear to perform multiple postcleavage roles in the reaction as well. Here we review the interaction of the RAG proteins with DNA, the chemistry of the cleavage reaction, and the higher order complexes in which these events take place. We also discuss postcleavage functions of the RAG proteins, including recent evidence indicating that they initiate the process of coding end processing by nicking hairpin DNA termini. Finally, we discuss the evolutionary and functional implications of the finding that RAG1 and RAG2 constitute a transposase, and we consider RAG protein biochemistry in the context of several bacterial transposition systems. This suggests a model of the RAG protein active site in which two divalent metal ions serve alternating and opposite roles as activators of attacking hydroxyl groups and stabilizers of oxyanion leaving groups. PMID:10837067

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

    PubMed

    Hollinger; Uludag; Winn

    1998-05-01

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

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

    PubMed

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

    1987-06-01

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

  20. A vector system for ABC transporter-mediated secretion and purification of recombinant proteins in Pseudomonas species.

    PubMed

    Ryu, Jaewook; Lee, Ukjin; Park, Jiye; Yoo, Do-Hyun; Ahn, Jung Hoon

    2015-03-01

    Pseudomonas fluorescens is an efficient platform for recombinant protein production. P. fluorescens has an ABC transporter secreting endogenous thermostable lipase (TliA) and protease, which can be exploited to transport recombinant proteins across the cell membrane. In this study, the expression vector pDART was constructed by inserting tliDEF, genes encoding the ABC transporter, along with the construct of the lipase ABC transporter recognition domain (LARD), into pDSK519, a widely used shuttle vector. When the gene for the target protein was inserted into the vector, the C-terminally fused LARD allowed it to be secreted through the ABC transporter into the extracellular medium. After secretion of the fused target protein, the LARD containing a hydrophobic C terminus enabled its purification through hydrophobic interaction chromatography (HIC) using a methyl-Sepharose column. Alkaline phosphatase (AP) and green fluorescent protein (GFP) were used to validate the expression, export, and purification of target proteins by the pDART system. Both proteins were secreted into the extracellular medium in P. fluorescens. In particular, AP was secreted in several Pseudomonas species with its enzymatic activity in extracellular media. Furthermore, purification of the target protein using HIC yielded some degree of AP and GFP purification, where AP was purified to almost a single product. The pDART system will provide greater convenience for the secretory production and purification of recombinant proteins in Gram-negative bacteria, such as Pseudomonas species. PMID:25548043

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

    PubMed

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

    2003-04-01

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

  2. Protection of mice against H. somni septicemia by vaccination with recombinant immunoglobulin binding protein subunits

    PubMed Central

    Geertsema, Roger S.; Worby, Carolyn; Kruger, Robert P.; Tagawa, Yuichi; Russo, Riccardo; Herdman, D. Scott; Lo, Kimby; Kimball, Richard A.; Dixon, Jack; Corbeil, Lynette B.

    2008-01-01

    Haemophilus somni causes bovine pneumonia as well as septicemia and its sequelae but mechanisms of virulence and protective immunity are poorly understood. Since surface immunoglobulin binding proteins are virulence factors, we addressed their role as protective antigens in a mouse model of H. somni septicemia. Immunoglobulin binding protein A (IbpA), has homology to Bordetella pertussis filamentous hemagglutinin and other large bacterial exoproteins. IbpA is a major surface antigen encoded by the ibpA gene with many domains that may be important in pathogenesis and immune protection. Three IbpA recombinant protein subunits, IbpA3, IbpA5 and IbpADR2 were chosen for study because of putative functional domains and motifs. These recombinant GST fusion subunit proteins were compared with GST (negative control), formalin-killed H. somni (commercial vaccine control), live H. somni (to induce convalescent immunity) and H. somni culture supernatant (containing IbpA shed from the bacterial surface). In vaccination/challenge studies, both live H. somni (convalescent immunity) and supernatant protected equally but formalin-killed H. somni and GST did not protect against septicemia. The DR2 and A3 subunits protected moderately well and induced antibody responses against supernatant antigen and the homologous subunit in ELISA but not against whole cell antigens. Supernatant immunization protected better than the IbpA subunit antigens and induced high antibody activity against both whole cells and supernatant antigens. The results indicate that culture supernatant antigens or perhaps recombinant IbpA subunits may be useful in H. somni vaccines. These studies also provide insight into the contribution of IbpA domains to pathogenesis of H. somni septicemia. PMID:18590787

  3. Protection of mice against H. somni septicemia by vaccination with recombinant immunoglobulin binding protein subunits.

    PubMed

    Geertsema, Roger S; Worby, Carolyn; Kruger, Robert P; Tagawa, Yuichi; Russo, Riccardo; Herdman, D Scott; Lo, Kimby; Kimball, Richard A; Dixon, Jack; Corbeil, Lynette B

    2008-08-18

    Histophilus somni causes bovine pneumonia as well as septicemia and its sequelae but mechanisms of virulence and protective immunity are poorly understood. Since surface immunoglobulin binding proteins are virulence factors, we addressed their role as protective antigens in a mouse model of H. somni septicemia. Immunoglobulin binding protein A (IbpA), has homology to Bordetella pertussis filamentous hemagglutinin and other large bacterial exoproteins. IbpA is a major surface antigen encoded by the ibpA gene with many domains that may be important in pathogenesis and immune protection. Three IbpA recombinant protein subunits, IbpA3, IbpA5 and IbpADR2 were chosen for study because of putative functional domains and motifs. These recombinant GST fusion subunit proteins were compared with GST (negative control), formalin-killed H. somni (commercial vaccine control), live H. somni (to induce convalescent immunity) and H. somni culture supernatant (containing IbpA shed from the bacterial surface). In vaccination/challenge studies, both live H. somni (convalescent immunity) and supernatant protected equally but formalin-killed H. somni and GST did not protect against septicemia. The DR2 and A3 subunits protected moderately well and induced antibody responses against supernatant antigen and the homologous subunit in ELISA but not against whole cell antigens. Supernatant immunization protected better than the IbpA subunit antigens and induced high antibody activity against both whole cells and supernatant antigens. The results indicate that culture supernatant antigens or perhaps recombinant IbpA subunits may be useful in H. somni vaccines. These studies also provide insight into the contribution of IbpA domains to pathogenesis of H. somni septicemia. PMID:18590787

  4. Rational Design of a Carrier Protein for the Production of Recombinant Toxic Peptides in Escherichia coli.

    PubMed

    Pane, Katia; Durante, Lorenzo; Pizzo, Elio; Varcamonti, Mario; Zanfardino, Anna; Sgambati, Valeria; Di Maro, Antimo; Carpentieri, Andrea; Izzo, Viviana; Di Donato, Alberto; Cafaro, Valeria; Notomista, Eugenio

    2016-01-01

    Commercial uses of bioactive peptides require low cost, effective methods for their production. We developed a new carrier protein for high yield production of recombinant peptides in Escherichia coli very well suited for the production of toxic peptides like antimicrobial peptides. GKY20, a short antimicrobial peptide derived from the C-terminus of human thrombin, was fused to the C-terminus of Onconase, a small ribonuclease (104 amino acids), which efficiently drove the peptide into inclusion bodies with very high expression levels (about 200-250 mg/L). After purification of the fusion protein by immobilized metal ion affinity chromatography, peptide was obtained by chemical cleavage in diluted acetic acid of an acid labile Asp-Pro sequence with more than 95% efficiency. To improve peptide purification, Onconase was mutated to eliminate all acid labile sequences thus reducing the release of unwanted peptides during the acid cleavage. Mutations were chosen to preserve the differential solubility of Onconase as function of pH, which allows its selective precipitation at neutral pH after the cleavage. The improved carrier allowed the production of 15-18 mg of recombinant peptide per liter of culture with 96-98% purity without the need of further chromatographic steps after the acid cleavage. The antimicrobial activity of the recombinant peptide, with an additional proline at the N-terminus, was tested on Gram-negative and Gram-positive strains and was found to be identical to that measured for synthetic GKY20. This finding suggests that N-terminal proline residue does not change the antimicrobial properties of recombinant (P)GKY20. The improved carrier, which does not contain cysteine and methionine residues, Asp-Pro and Asn-Gly sequences, is well suited for the production of peptides using any of the most popular chemical cleavage methods. PMID:26808536

  5. Rational Design of a Carrier Protein for the Production of Recombinant Toxic Peptides in Escherichia coli

    PubMed Central

    Pizzo, Elio; Varcamonti, Mario; Zanfardino, Anna; Sgambati, Valeria; Di Maro, Antimo; Carpentieri, Andrea; Izzo, Viviana; Di Donato, Alberto; Cafaro, Valeria; Notomista, Eugenio

    2016-01-01

    Commercial uses of bioactive peptides require low cost, effective methods for their production. We developed a new carrier protein for high yield production of recombinant peptides in Escherichia coli very well suited for the production of toxic peptides like antimicrobial peptides. GKY20, a short antimicrobial peptide derived from the C-terminus of human thrombin, was fused to the C-terminus of Onconase, a small ribonuclease (104 amino acids), which efficiently drove the peptide into inclusion bodies with very high expression levels (about 200–250 mg/L). After purification of the fusion protein by immobilized metal ion affinity chromatography, peptide was obtained by chemical cleavage in diluted acetic acid of an acid labile Asp-Pro sequence with more than 95% efficiency. To improve peptide purification, Onconase was mutated to eliminate all acid labile sequences thus reducing the release of unwanted peptides during the acid cleavage. Mutations were chosen to preserve the differential solubility of Onconase as function of pH, which allows its selective precipitation at neutral pH after the cleavage. The improved carrier allowed the production of 15–18 mg of recombinant peptide per liter of culture with 96–98% purity without the need of further chromatographic steps after the acid cleavage. The antimicrobial activity of the recombinant peptide, with an additional proline at the N-terminus, was tested on Gram-negative and Gram-positive strains and was found to be identical to that measured for synthetic GKY20. This finding suggests that N-terminal proline residue does not change the antimicrobial properties of recombinant (P)GKY20. The improved carrier, which does not contain cysteine and methionine residues, Asp-Pro and Asn-Gly sequences, is well suited for the production of peptides using any of the most popular chemical cleavage methods. PMID:26808536

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

    PubMed Central

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

    2015-01-01

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

  7. ATM increases activation-induced cytidine deaminase activity at downstream S regions during class-switch recombination.

    PubMed

    Khair, Lyne; Guikema, Jeroen E J; Linehan, Erin K; Ucher, Anna J; Leus, Niek G J; Ogilvie, Colin; Lou, Zhenkun; Schrader, Carol E; Stavnezer, Janet

    2014-05-15

    Activation-induced cytidine deaminase (AID) initiates Ab class-switch recombination (CSR) in activated B cells resulting in exchanging the IgH C region and improved Ab effector function. During CSR, AID instigates DNA double-strand break (DSB) formation in switch (S) regions located upstream of C region genes. DSBs are necessary for CSR, but improper regulation of DSBs can lead to chromosomal translocations that can result in B cell lymphoma. The protein kinase ataxia telangiectasia mutated (ATM) is an important proximal regulator of the DNA damage response (DDR), and translocations involving S regions are increased in its absence. ATM phosphorylates H2AX, which recruits other DNA damage response (DDR) proteins, including mediator of DNA damage checkpoint 1 (Mdc1) and p53 binding protein 1 (53BP1), to sites of DNA damage. As these DDR proteins all function to promote repair and recombination of DSBs during CSR, we examined whether mouse splenic B cells deficient in these proteins would show alterations in S region DSBs when undergoing CSR. We find that in atm(-/-) cells Sμ DSBs are increased, whereas DSBs in downstream Sγ regions are decreased. We also find that mutations in the unrearranged Sγ3 segment are reduced in atm(-/-) cells. Our data suggest that ATM increases AID targeting and activity at downstream acceptor S regions during CSR and that in atm(-/-) cells Sμ DSBs accumulate as they lack a recombination partner. PMID:24729610

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

    PubMed

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

    2016-01-01

    We previously described a procedure for the use of plant protease inhibitors as "companion" accessory proteins to prevent unwanted proteolysis of clinically useful recombinant proteins in leaf crude protein extracts (Benchabane et al. Methods Mol Biol 483:265-273, 2009). Here we describe the use of these inhibitors for the protection of recombinant proteins in planta, before their extraction from leaf tissues. A procedure is first described involving inhibitors co-expressed along-and co-migrating-with the protein of interest in host plant cells. An alternative, single transgene scheme is then described involving translational fusions of the recombinant protein and companion inhibitor. These approaches may allow for a significant improvement of protein steady-state levels in leaves, comparable to yield improvements observed with protease-deficient strains of less complex protein expression hosts such as E. coli or yeasts. PMID:26614285

  9. Effect of periplasmic expression of recombinant mouse interleukin-4 on hydrogen peroxide concentration and catalase activity in Escherichia coli.

    PubMed

    Mehdizadeh Aghdam, Elnaz; Mahmoudi Azar, Lena; Barzegari, Abolfazl; Karimi, Farrokh; Mesbahfar, Majid; Samadi, Naser; Hejazi, Mohammad Saeid

    2012-12-15

    Oxidative stress occurs as a result of imbalance between generation and detoxification of reactive oxygen species (ROS). This kind of stress was rarely discussed in connection with foreign protein production in Escherichia coli. Relation between cytoplasmic recombinant protein expression with H(2)O(2) concentration and catalase activity variation was already reported. The periplasmic space of E. coli has different oxidative environment in relative to cytoplasm and there are some benefits in periplasmic expression of recombinant proteins. In this study, hydrogen peroxide concentration and catalase activity following periplasmic expression of mouse IL-4 were measured in E. coli. After construction of pET2mIL4 plasmid, the expression of recombinant mouse interleukin-4 (mIL-4) was confirmed. Then, the H(2)O(2) concentration and catalase activity variation in the cells were studied in exponential and stationary phases at various ODs and were compared to those of wild type cells and empty vector transformed cells. It was revealed that empty vector introduction and periplasmic recombinant protein expression increased significantly the H(2)O(2) concentration of the cells. However, the H(2)O(2) concentration in mIL-4 expressing cells was significantly higher than its concentration in empty vector transformed cells, demonstrating more effects of recombinant mIL-4 expression on H(2)O(2) elevation. Likewise, although catalase activity was reduced in foreign DNA introduced cells, it was more lowered following expression of recombinant proteins. Correlation between H(2)O(2) concentration elevation and catalase activity reduction with cell growth depletion is also demonstrated. It was also found that recombinant protein expression results in cell size increase. PMID:23000065

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

    PubMed

    Fuhrmann, Markus

    2004-01-01

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

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

    PubMed

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

    2016-04-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-06-28

    Brucellosis is a zoonotic disease caused by Brucella, a genus of gram-negative bacteria. Cytokines have key roles in the activation of innate and acquired immunities. Despite several research attempts to reveal the immune responses, the mechanism of Brucella infection remains unclear. Therefore, immune responses were analyzed in mice immunized with nine recombinant proteins. Cytokine production profiles were analyzed in the RAW 264.7 cells and naive splenocytes after stimulation with three recombinant proteins, metal-dependent hydrolase (r0628), bacterioferritin (rBfr), and thiamine transporter substrate-binding protein (rTbpA). Immune responses were analyzed by ELISA and ELISpot assay after immunization with proteins in mice. The production levels of NO, TNF-α, and IL-6 were time-dependently increased after having been stimulated with proteins in the RAW 264.7 cells. In naive splenocytes, the production of IFN-γ and IL-2 was increased after stimulation with the proteins. It was concluded that two recombinant proteins, r0628 and rTbpA, showed strong immunogenicity that was induced with Th1-related cytokines IFN-γ, IL-2, and TNF-α more than Th2-related cytokines IL-6, IL-4, and IL-5 in vitro. Conversely, a humoral immune response was activated by increasing the number of antigen-secreting cells specifically. Furthermore, these could be candidate diagnosis antigens for better understanding of brucellosis. PMID:27012238

  14. High-Level Expression of Recombinant Bovine Lactoferrin in Pichia pastoris with Antimicrobial Activity.

    PubMed

    Iglesias-Figueroa, Blanca; Valdiviezo-Godina, Norberto; Siqueiros-Cendón, Tania; Sinagawa-García, Sugey; Arévalo-Gallegos, Sigifredo; Rascón-Cruz, Quintín

    2016-01-01

    In this study, bovine lactoferrin (bLf), an iron-binding glycoprotein considered an important nutraceutical protein because of its several properties, was expressed in Pichia pastoris KM71-H under AOX1 promoter control, using pJ902 as the recombinant plasmid. Dot blotting analysis revealed the expression of recombinant bovine lactoferrin (rbLf) in Pichia pastoris. After Bach fermentation and purification by molecular exclusion, we obtained an expression yield of 3.5 g/L of rbLf. rbLf and predominantly pepsin-digested rbLf (rbLfcin) demonstrated antibacterial activity against Escherichia coli (E. coli) BL21DE3, Staphylococcus aureus (S. aureus) FRI137, and, in a smaller percentage, Pseudomonas aeruginosa (Ps. Aeruginosa) ATCC 27833. The successful expression and characterization of functional rbLf expressed in Pichia pastoris opens a prospect for the development of natural antimicrobial agents produced recombinantly. PMID:27294912

  15. High-Level Expression of Recombinant Bovine Lactoferrin in Pichia pastoris with Antimicrobial Activity

    PubMed Central

    Iglesias-Figueroa, Blanca; Valdiviezo-Godina, Norberto; Siqueiros-Cendón, Tania; Sinagawa-García, Sugey; Arévalo-Gallegos, Sigifredo; Rascón-Cruz, Quintín

    2016-01-01

    In this study, bovine lactoferrin (bLf), an iron-binding glycoprotein considered an important nutraceutical protein because of its several properties, was expressed in Pichia pastoris KM71-H under AOX1 promoter control, using pJ902 as the recombinant plasmid. Dot blotting analysis revealed the expression of recombinant bovine lactoferrin (rbLf) in Pichia pastoris. After Bach fermentation and purification by molecular exclusion, we obtained an expression yield of 3.5 g/L of rbLf. rbLf and predominantly pepsin-digested rbLf (rbLfcin) demonstrated antibacterial activity against Escherichia coli (E. coli) BL21DE3, Staphylococcus aureus (S. aureus) FRI137, and, in a smaller percentage, Pseudomonas aeruginosa (Ps. Aeruginosa) ATCC 27833. The successful expression and characterization of functional rbLf expressed in Pichia pastoris opens a prospect for the development of natural antimicrobial agents produced recombinantly. PMID:27294912

  16. Role of recombination activating genes in the generation of antigen receptor diversity and beyond.

    PubMed

    Nishana, Mayilaadumveettil; Raghavan, Sathees C

    2012-12-01

    V(D)J recombination is the process by which antibody and T-cell receptor diversity is attained. During this process, antigen receptor gene segments are cleaved and rejoined by non-homologous DNA end joining for the generation of combinatorial diversity. The major players of the initial process of cleavage are the proteins known as RAG1 (recombination activating gene 1) and RAG2. In this review, we discuss the physiological function of RAGs as a sequence-specific nuclease and its pathological role as a structure-specific nuclease. The first part of the review discusses the basic mechanism of V(D)J recombination, and the last part focuses on how the RAG complex functions as a sequence-specific and structure-specific nuclease. It also deals with the off-target cleavage of RAGs and its implications in genomic instability. PMID:23039142

  17. Selective Blockade of Trypanosomatid Protein Synthesis by a Recombinant Antibody Anti-Trypanosoma cruzi P2β Protein

    PubMed Central

    Simonetti, Leandro; Duffy, Tomas; Longhi, Silvia A.; Gómez, Karina A.; Hoebeke, Johan; Levin, Mariano J.; Smulski, Cristian R.

    2012-01-01

    The ribosomal P proteins are located on the stalk of the ribosomal large subunit and play a critical role during the elongation step of protein synthesis. The single chain recombinant antibody C5 (scFv C5) directed against the C-terminal region of the Trypanosoma cruzi P2β protein (TcP2β) recognizes the conserved C-terminal end of all T. cruzi ribosomal P proteins. Although this region is highly conserved among different species, surface plasmon resonance analysis showed that the scFv C5 possesses very low affinity for the corresponding mammalian epitope, despite having only one single amino-acid change. Crystallographic analysis, in silico modelization and NMR assays support the analysis, increasing our understanding on the structural basis of epitope specificity. In vitro protein synthesis experiments showed that scFv C5 was able to specifically block translation by T. cruzi and Crithidia fasciculata ribosomes, but virtually had no effect on Rattus norvegicus ribosomes. Therefore, we used the scFv C5 coding sequence to make inducible intrabodies in Trypanosoma brucei. Transgenic parasites showed a strong decrease in their growth rate after induction. These results strengthen the importance of the P protein C terminal regions for ribosomal translation activity and suggest that trypanosomatid ribosomal P proteins could be a possible target for selective therapeutic agents that could be derived from structural analysis of the scFv C5 antibody paratope. PMID:22570698

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

    PubMed

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

    2015-12-10

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

  19. Expression, purification and characterization of the recombinant ribonuclease P protein component from Bacillus subtilis.

    PubMed Central

    Niranjanakumari, S; Kurz, J C; Fierke, C A

    1998-01-01

    Ribonuclease P is a ribonucleoprotein complex that catalyzes the essential 5' maturation of all precursor tRNA molecules. The protein component both alters the conformation of the RNA component and enhances the substrate affinity and specificity. To facilitate biochemical and biophysical studies, the protein component of Bacillus subtilis ribonuclease P (RNase P) was overproduced in Escherichia coli using the native amino acid sequence with the initial 20 codons optimized for expression in E.coli . A simple purification procedure using consecutive cation exchange chromatography steps in the presence and absence of urea was developed to purify large quantities of P protein without contaminating nucleic acids. The identity of the recombinant protein as a cofactor of RNase P was established by its ability to stimulate the activity of the RNA component in low ionic strength buffer in a 1:1 stoichiometry. Circular dichroism studies indicate that P protein is a combination of alpha-helix and beta-sheet secondary structures and is quite stable, with a T m of 67 degrees C. The described methods facilitated the large scale purification of homogeneous, RNA-free P protein required for high resolution crystallographic analyses and may be useful for the preparation of other RNA binding proteins. PMID:9628904

  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. PMID:26805756

  1. Involvement of a periplasmic protein kinase in DNA strand break repair and homologous recombination in Escherichia coli.

    PubMed

    Khairnar, Nivedita P; Kamble, Vidya A; Mangoli, Suhas H; Apte, Shree K; Misra, Hari S

    2007-07-01

    The involvement of signal transduction in the repair of radiation-induced damage to DNA has been known in eukaryotes but remains understudied in bacteria. This article for the first time demonstrates a role for the periplasmic lipoprotein (YfgL) with protein kinase activity transducing a signal for DNA strand break repair in Escherichia coli. Purified YfgL protein showed physical as well as functional interaction with pyrroloquinoline-quinone in solution and the protein kinase activity of YfgL was strongly stimulated in the presence of pyrroloquinoline-quinone. Transgenic E. coli cells producing Deinococcus radiodurans pyrroloquinoline-quinone synthase showed nearly four log cycle improvement in UVC dark survival and 10-fold increases in gamma radiation resistance as compared with untransformed cells. Pyrroloquinoline-quinone enhanced the UV resistance of E. coli through the YfgL protein and required the active recombination repair proteins. The yfgL mutant showed higher sensitivity to UVC, mitomycin C and gamma radiation as compared with wild-type cells and showed a strong impairment in homologous DNA recombination. The mutant expressing an active YfgL in trans recovered the lost phenotypes to nearly wild-type levels. The results strongly suggest that the periplasmic phosphoquinolipoprotein kinase YfgL plays an important role in radiation-induced DNA strand break repair and homologous recombination in E. coli. PMID:17630970

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

    PubMed

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

    2016-05-01

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

  3. Passive immunization by recombinant ferric enterobactin protein (FepA) from Escherichia coli O157

    PubMed Central

    Larrie-Bagha, Seyed Mehdi; Rasooli, Iraj; Mousavi-Gargari, Seyed Latif; Rasooli, Zohreh; Nazarian, Shahram

    2013-01-01

    Background and Objectives Enterohemorrhagic Escherichia coli (EHEC) O157:H7 has been recognized as a major food borne pathogen responsible for frequent hemorrhagic colitis and hemolytic uremic syndrome in humans. Cattle are important reservoirs of E. coli O157:H7, in which the organism colonizes the intestinal tract and is shed in the feces. Objective Vaccination of cattle has significant potential as a pre-harvest intervention strategy for E. coli O157:H7. The aim of this study was to evaluate active and passive immunization against E. coli O157:H7 using a recombinant protein. Materials and Methods The recombinant FepA protein induced by IPTG was purified by nickel affinity chromatography. Antibody titre was determined by ELISA in FepA immunized rabbits sera. Sera collected from vaccinated animals were used for bacterial challenge in passive immunization studies. Results The results demonstrate that passive immunization with serum raised against FepA protects rabbits from subsequent infection. Conclusion Significant recognition by the antibody of ferric enterobactin binding protein may lead to its application in the restriction of Enterobacteriaceae propagation. PMID:23825727

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

    SciTech Connect

    Gizatullina, Albina K.; Finkina, Ekaterina I.; Mineev, Konstantin S.; Melnikova, Daria N.; Bogdanov, Ivan V.; Shenkarev, Zakhar O.; Ovchinnikova, Tatiana V.

    2013-10-04

    Highlights: •Lipid transfer protein from lentil seeds (Lc-LTP2) was overexpressed in E. coli. •Antimicrobial activity and spatial structure of the recombinant Lc-LTP2 were examined. •Internal tunnel-like lipid-binding cavity occupies ∼7% of the total Lc-LTP2 volume. •Binding of DMPG lipid induces moderate rearrangements in the Lc-LTP2 structure. •Lc-LTP2/DMPG complex has limited lifetime and dissociates within tens of hours. -- Abstract: Lipid transfer protein, designated as Lc-LTP2, was isolated from seeds of the lentil Lens culinaris. The protein has molecular mass 9282.7 Da, consists of 93 amino acid residues including 8 cysteines forming 4 disulfide bonds. Lc-LTP2 and its stable isotope labeled analogues were overexpressed in Escherichia coli and purified. Antimicrobial activity of the recombinant protein was examined, and its spatial structure was studied by NMR spectroscopy. The polypeptide chain of Lc-LTP2 forms four α-helices (Cys4-Leu18, Pro26-Ala37, Thr42-Ala56, Thr64-Lys73) and a long C-terminal tail without regular secondary structure. Side chains of the hydrophobic residues form a relatively large internal tunnel-like lipid-binding cavity (van der Waals volume comes up to ∼600 Å{sup 3}). The side-chains of Arg45, Pro79, and Tyr80 are located near an assumed mouth of the cavity. Titration with dimyristoyl phosphatidylglycerol (DMPG) revealed formation of the Lc-LTP2/lipid non-covalent complex accompanied by rearrangements in the protein spatial structure and expansion of the internal cavity. The resultant Lc-LTP2/DMPG complex demonstrates limited lifetime and dissociates within tens of hours.

  5. 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

  6. DNA binding specificities of the long zinc-finger recombination protein PRDM9

    PubMed Central

    2013-01-01

    Background Meiotic recombination ensures proper segregation of homologous chromosomes and creates genetic variation. In many organisms, recombination occurs at limited sites, termed 'hotspots', whose positions in mammals are determined by PR domain member 9 (PRDM9), a long-array zinc-finger and chromatin-modifier protein. Determining the rules governing the DNA binding of PRDM9 is a major issue in understanding how it functions. Results Mouse PRDM9 protein variants bind to hotspot DNA sequences in a manner that is specific for both PRDM9 and DNA haplotypes, and that in vitro binding parallels its in vivo biological activity. Examining four hotspots, three activated by Prdm9Cst and one activated by Prdm9Dom2, we found that all binding sites required the full array of 11 or 12 contiguous fingers, depending on the allele, and that there was little sequence similarity between the binding sites of the three Prdm9Cst activated hotspots. The binding specificity of each position in the Hlx1 binding site, activated by Prdm9Cst, was tested by mutating each nucleotide to its three alternatives. The 31 positions along the binding site varied considerably in the ability of alternative bases to support binding, which also implicates a role for additional binding to the DNA phosphate backbone. Conclusions These results, which provide the first detailed mapping of PRDM9 binding to DNA and, to our knowledge, the most detailed analysis yet of DNA binding by a long zinc-finger array, make clear that the binding specificities of PRDM9, and possibly other long-array zinc-finger proteins, are unusually complex. PMID:23618393

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

    PubMed

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

    2016-01-01

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

  8. 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.

  9. Humoral immune response against human cytomegalovirus (HCMV)-specific proteins after HCMV infection in lung transplantation as detected with recombinant and naturally occurring proteins.

    PubMed Central

    van Zanten, J; Harmsen, M C; van der Giessen, M; van der Bij, W; Prop, J; de Leij, L; The, T H

    1995-01-01

    The humoral immune response to four intracellularly located cytomegalovirus (CMV) proteins was studied in 15 lung transplant recipients experiencing active CMV infections. Five patients had primary infections, and 10 had secondary infections. Antibodies of the immunoglobulin M (IgM) and IgG classes were measured in an enzyme-linked immunosorbent assay (ELISA) system in which procaryotically expressed recombinant proteins were used as a substrate and also in a monoclonal antibody-based capture ELISA which uses naturally occurring proteins as a substrate. The proteins investigated were the lower matrix protein pp65 (ppUL83), the major DNA-binding protein p52 (ppUL44), and the two immediate early proteins IE1 and IE2 (different splicing products of UL123). Higher levels of antibodies were found to pp65 and especially to p52 than to the immediate early antigens. Antibody levels detected in the recombinant protein-based ELISAs were generally lower than antibody responses detected with the matching antigen capture ELISA. Moreover, some patients appeared to have antibodies mainly to epitopes present on naturally occurring proteins. The antibody responses detected in both assays were related to the viral load during infection as assessed by the CMV antigenemia test, which is a quantitative marker for CMV load. It was found that although epitopes on naturally occurring proteins induce higher antibody responses and responses in more patients, antibodies directed to epitopes present on the recombinant proteins were inversely related to the viral load during a CMV infection. Therefore, antibodies to epitopes on the recombinant proteins might be more clinically relevant in this group of lung transplant recipients. PMID:7535179

  10. 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.

  11. 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.

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

    PubMed

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

    2011-12-01

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

  13. Transduction of Recombinant M3-p53-R12 Protein Enhances Human Leukemia Cell Apoptosis

    PubMed Central

    Lu, Tsung Chi; Zhao, Guan- Hao; Chen, Yao Yun; Chien, Chia-Ying; Huang, Chi-Hung; Lin, Kwang Hui; Chen, Shen Liang

    2016-01-01

    Tumor suppressor protein p53 plays important roles in initiating cell cycle arrest and promoting tumor cell apoptosis. Previous studies have shown that p53 is either mutated or defective in approximately 50% of human cancers; therefore restoring normal p53 activity in cancer cells might be an effective anticancer therapeutic approach. Herein, we designed a chimeric p53 protein flanked with the MyoD N-terminal transcriptional activation domain (amino acids 1-62, called M3) and a poly-arginine (R12) cell penetrating signal in its N-and C-termini respectively. This chimeric protein, M3-p53-R12, can be expressed in E. coli and purified using immobilized metal ion chromatography followed by serial refolding dialysis. The purified M3-p53-R12 protein retains DNA-binding activity and gains of cell penetrating ability. Using MTT assay, we demonstrated that M3-p53-R12 inhibited the growth of K562, Jurkat as well as HL-60 leukemia cells carrying mutant p53 genes. Results from FACS analysis also demonstrated that transduction of M3-p53-R12 protein induced cell cycle arrest of these leukemia cells. Of special note, M3-p53-R12 has no apoptotic effect on normal mesenchymal stem cells (MSC) and leukocytes, highlighting its differential effects on normal and tumor cells. To sum up, our results reveal that purified recombinant M3-p53-R12 protein has functions of suppressing the leukemia cell lines' proliferation and launching cell apoptosis, suggesting the feasibility of using M3-p53-R12 protein as an anticancer drug. In the future we will test whether this chimeric protein can preferentially trigger the death of malignant cancer cells without affecting normal cells in animals carrying endogenous or xenographic tumors. PMID:27390612

  14. Development of a highly efficient protein-secreting system in recombinant Lactobacillus casei.

    PubMed

    Kajikawa, Akinobu; Ichikawa, Eiko; Igimi, Shizunobu

    2010-02-01

    The available techniques for heterologous protein secretion in Lactobacillus strains are limited. The aim of the present study was to develop an efficient protein-secretion system using recombinant lactobacilli for various applications such as live delivery of biotherapeutics. For the construction of expression vectors, the Lactobacillus brevis slpA promoter, Lactobacillus casei prtP signal sequence, and mouse IL-10 sequences were used as a model system. Interestingly, the slpA promoter exhibited strong activity in L. casei contrary to previous observations. In order to stabilize replication of the plasmid in E. coli, a removable terminator sequence was built into the promoter region. For the improvement of secretion efficiency, a DTNSD oligopeptide was added to the cleavage site of signal peptidase. The resulting plasmids provided remarkably efficient IL-10 secretion. Accumulation of the protein in the culture supernatant varied widely according to the pH conditions. By analysis of the secreted protein, formation of homodimers and biological activity, IL-10 was confirmed to be functional. The presently constructed plasmids could be useful tools for heterologous protein-secretion in L. casei. PMID:20208444

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

    PubMed Central

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

    1988-01-01

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

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

    PubMed Central

    Grishchuk, A L; Kohli, J

    2003-01-01

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

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

    PubMed Central

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

    2010-01-01

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

  18. Surface Display of Recombinant Proteins on Bacillus subtilis Spores

    PubMed Central

    Isticato, Rachele; Cangiano, Giuseppina; Tran, Hoa T.; Ciabattini, Annalisa; Medaglini, Donata; Oggioni, Marco R.; De Felice, Maurilio; Pozzi, Gianni; Ricca, Ezio

    2001-01-01

    We developed a novel surface display system based on the use of bacterial spores. A protein of the Bacillus subtilis spore coat, CotB, was found to be located on the spore surface and used as fusion partner to express the 459-amino-acid C-terminal fragment of the tetanus toxin (TTFC). Western, dot blot and fluorescent-activated cell sorting analyses were used to monitor TTFC surface expression on purified spores. We estimated that more than 1.5 × 103 TTFC molecules were exposed on the surface of each spore and recognized by TTFC-specific antibodies. The efficient surface presentation of the heterologous protein, together with the simple purification procedure and the high stability and safety record of B. subtilis spores, makes this spore-based display system a potentially powerful approach for surface expression of bioactive molecules. PMID:11591673

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

    PubMed Central

    Almo, Steven C.; Love, James D.

    2014-01-01

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

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

    PubMed

    Almo, Steven C; Love, James D

    2014-06-01

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

  1. A recombinant triblock protein polymer with dispersant and binding properties for digital printing.

    PubMed

    Qi, Min; O'Brien, John P; Yang, Jianjun

    2008-01-01

    A structured triblock protein was designed to explore the potential of engineered peptides to function as high-performance ink dispersants and binders. The protein consists of three functional elements, including a pigment binding domain, a hydrophilic linker, and a printing surface binding domain. To construct such a chimeric protein, a carbon black binding peptide, FHENWPS, and a cellulose binding peptide, THKTSTQRLLAA, were identified from phage display libraries through biopanning, based on their strong and specific binding affinities to carbon black and cellulose. They were used as carbon black and cellulose binding domains, respectively, in a recombinant triblock protein. A linker sequence, PTPTPTPTPTPTPTPTPTPTPTP, was adapted from endoglucanase A of the bacterium Cellulomonas fimi, as a small, rigid, and hydrophilic interdomain linker. When incorporated into the triblock structure between the carbon black and cellulose binding sequences, the linker sufficiently isolates these two elements and allows dual binding activity. The structured triblock protein was shown to disperse carbon black particles and attach it to paper surfaces. Thus, the utility of structured proteins having useful dispersant and binding properties for digital printing inks was demonstrated. PMID:17972282

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

    PubMed Central

    de Marco, Ario

    2009-01-01

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

  3. Structural and Functional Characterization of Recombinant Isoforms of the Lentil Lipid Transfer Protein.

    PubMed

    Bogdanov, I V; Finkina, E I; Balandin, S V; Melnikova, D N; Stukacheva, E A; Ovchinnikova, T V

    2015-01-01

    The recombinant isoforms Lc-LTP1 and Lc-LTP3 of the lentil lipid transfer protein were overexpressed in E. coli cells. It was confirmed that both proteins are stabilized by four disulfide bonds and characterized by a high proportion of the α-helical structure. It was found that Lc-LTP1 and Lc-LTP3 possess antimicrobial activity and can bind fatty acids. Both isoforms have the ability to bind specific IgE from sera of patients with food allergies, which recognize similar epitopes of the major peach allergen Pru p 3. Both isoforms were shown to have immunological properties similar to those of other plant allergenic LTPs, but Lc-LTP3 displayed a less pronounced immunoreactivity. PMID:26483961

  4. Structural and Functional Characterization of Recombinant Isoforms of the Lentil Lipid Transfer Protein

    PubMed Central

    Bogdanov, I. V.; Finkina, E. I.; Balandin, S. V.; Melnikova, D. N.; Stukacheva, E. A.; Ovchinnikova, T. V.

    2015-01-01

    The recombinant isoforms Lc-LTP1 and Lc-LTP3 of the lentil lipid transfer protein were overexpressed in E. coli cells. It was confirmed that both proteins are stabilized by four disulfide bonds and characterized by a high proportion of the α-helical structure. It was found that Lc-LTP1 and Lc-LTP3 possess antimicrobial activity and can bind fatty acids. Both isoforms have the ability to bind specific IgE from sera of patients with food allergies, which recognize similar epitopes of the major peach allergen Pru p 3. Both isoforms were shown to have immunological properties similar to those of other plant allergenic LTPs, but Lc-LTP3 displayed a less pronounced immunoreactivity. PMID:26483961

  5. Anti-angiogenesis and anti-tumor activity of recombinant anginex

    SciTech Connect

    Brandwijk, Ricardo J.M.G.E.; Dings, Ruud P.M.; Linden, Edith van der; Mayo, Kevin H.; Thijssen, Victor L.J.L.; Griffioen, Arjan W. . E-mail: aw.griffioen@path.unimaas.nl

    2006-10-27

    Anginex, a synthetic 33-mer angiostatic peptide, specifically inhibits vascular endothelial cell proliferation and migration along with induction of apoptosis in endothelial cells. Here we report on the in vivo characterization of recombinant anginex and use of the artificial anginex gene for gene therapy approaches. Tumor growth of human MA148 ovarian carcinoma in athymic mice was inhibited by 80% when treated with recombinant anginex. Histological analysis of the tumors showed an approximate 2.5-fold reduction of microvessel density, suggesting that angiogenesis inhibition is the cause of the anti-tumor effect. Furthermore, there was a significant correlation between the gene expression patterns of 16 angiogenesis-related factors after treatment with both recombinant and synthetic anginex. To validate the applicability of the anginex gene for gene therapy, stable transfectants of murine B16F10 melanoma cells expressing recombinant anginex were made. Supernatants of these cells inhibited endothelial cell proliferation in vitro. Furthermore, after subcutaneous injection of these cells in C57BL/6 mice, an extensive delay in tumor growth was observed. These data show that the artificial anginex gene can be used to produce a recombinant protein with similar activity as its synthetic counterpart and that the gene can be applied in gene therapy approaches for cancer treatment.

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

    DOEpatents

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

    2016-08-09

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

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

    PubMed

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

    2016-06-01

    Fusion proteins have become essential for the expression and purification of recombinant proteins in Escherichia coli. The metal-binding protein CusF has shown several features that make it an attractive fusion protein and affinity tag: "Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF" (Cantu-Bustos et al., 2016 [1]). Here we present accompanying data from protein expression experiments; we tested different protein tags, temperatures, expression times, cellular compartments, and concentrations of inducer in order to obtain soluble protein and low formation of inclusion bodies. Additionally, we present data from the purification of the green fluorescent protein (GFP) tagged with CusF, using Ag(I) metal affinity chromatography. PMID:27014739

  8. 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

  9. Efficient expression and purification of recombinant therapeutic protein candidates, human midkine and pleiotrophin.

    PubMed

    Murasugi, Akira

    2013-01-01

    Midkine is a heparin-binding growth factor that promotes cell growth, survival, and migration. Externally added midkine prevents ventricular remodeling and improves long-term survival after myocardial infarction in the mouse. Preclinical testing of this protein is in progress. Externally added pleiotrophin, a member of the midkine protein family, promotes functional recovery after neural transplantation in rats. Thus, pleiotrophin is also a candidate therapeutic protein. Large amounts of these proteins were obtained by using the heterologous protein expression system of Pichia pastoris, and the recombinant P. pastoris clones were cultured in a controlled fermentor. Intracellular expression yielded about 300 mg/L recombinant human (rh)-midkine, which was extracted, renatured, and purified. From 1 L of the culture, 64 mg of rh-midkine was purified. Secretory expression induced by the midkine secretion signal resulted in about 100 mg of rhmidkine in 1 L of the culture supernatant, but over 70% of the rh-midkine had yeast-specific glycosylation. Three threonyl residues that are targets for glycosylation were substituted with alanyl residues, and nonglycosylated, active rh-midkine was obtained. In secretory expression using α-mating factor prepro-sequence, about 640 mg/L rh-midkine was obtained, but it was partially truncated. Therefore, a protease-deficient host was used, and about 360 mg/L intact rh-midkine was then obtained. The rh-midkine was recovered and purified, with 70% final yield. All purified rh-midkine, regardless of expression method, was able to promote mammalian cell proliferation. In secretory expression of rh-pleiotrophin using α- mating factor prepro-sequence, 260 mg/L rh-pleiotrophin could be secreted. The rh-pleiotrophin was recovered and efficiently purified with 72% final yield. PMID:24372230

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

    PubMed

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

    2015-08-01

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

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

    PubMed Central

    Baganz, Frank; Purton, Saul

    2015-01-01

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

  12. Native and recombinant Pg-AMP1 show different antibacterial activity spectrum but similar folding behavior.

    PubMed

    Porto, William F; Nolasco, Diego O; Franco, Octavio L

    2014-05-01

    Glycine-rich proteins (GRPs) derived from plants compose a family of proteins and peptides that share a glycine repeat domain and they can perform diverse functions. Two structural conformations have been proposed for GRPs: glycine loops arranged as a Velcro and an anti-parallel β-sheet with several β-strands. The antimicrobial peptide Pg-AMP1 is the only plant GRP with antibacterial activity reported so far and its structure remains unclear. Recently, its recombinant expression was reported, where the recombinant peptide had an additional methionine residue at the N-terminal and a histidine tag at the C-terminal (His6-tag). These changes seem to change the peptide's activity, generating a broader spectrum of antibacterial activity. In this report, through ab initio molecular modelling and molecular dynamics, it was observed that both native and recombinant peptide structures were composed of an N-terminal α-helix and a dynamic loop that represents two-thirds of the protein. In contrast to previous reports, it was observed that there is a tendency to adopt a globular fold instead of an extended one, which could be in both, glycine loops or anti-parallel β-sheet conformation. The recombinant peptide showed a slightly higher solvated potential energy compared to the native form, which could be related to the His6-tag exposition. In fact, the His6-tag could be mainly responsible for the broader spectrum of activity, but it does not seem to cause great structural changes. However, novel studies are needed for a better characterization of its pharmacological properties so that in the future novel drugs may be produced based on this peptide. PMID:24582624

  13. 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. PMID:27288064

  14. Elimination of truncated recombinant protein expressed in Escherichia coli by removing cryptic translation initiation site.

    PubMed

    Jennings, Matthew J; Barrios, Adam F; Tan, Song

    2016-05-01

    Undesirable truncated recombinant protein products pose a special expression and purification challenge because such products often share similar chromatographic properties as the desired full length protein. We describe here our observation of both full length and a truncated form of a yeast protein (Gcn5) expressed in Escherichia coli, and the reduction or elimination of the truncated form by mutating a cryptic Shine-Dalgarno or START codon within the Gcn5 coding region. Unsuccessful attempts to engineer in a cryptic translation initiation site into other recombinant proteins suggest that cryptic Shine-Dalgarno or START codon sequences are necessary but not sufficient for cryptic translation in E. coli. PMID:26739786

  15. Protease-Triggered, Integrin-Targeted Cellular Uptake of Recombinant Protein Micelles.

    PubMed

    Gao, Chen; Vargo, Kevin B; Hammer, Daniel A

    2016-09-01

    Targeting nanoparticles for drug delivery has great potential for improving efficacy and reducing side effects from systemic toxicity. New developments in the assembly of materials afford the opportunity to expose cryptic targeting domains in tissue-specific microenvironments in which certain proteases are expressed. Here, recombinant proteins are designed to combine the responsiveness to environmental proteases with specific targeting. Materials made recombinantly allow complete control over amino acid sequence, in which each molecule is identically functionalized. Previously, oleosin, a naturally occurring plant protein that acts as a surfactant, has been engineered to self-assemble into spherical micelles-a useful structure for drug delivery. To make oleosins that are locally activated to bind receptors, oleosin is genetically modified to incorporate the integrin-binding motif RGDS just behind a domain cleavable by thrombin. The resulting modified oleosin self-assembles into spherical micelles in aqueous environments, with the RGDS motif protected by the thrombin-cleavable domain. Upon the addition of thrombin, the RGDS is exposed and the binding of the spherical micelles to breast cancer cells is increased fourfold. PMID:27284959

  16. Balancing the Expression and Production of a Heterodimeric Protein: Recombinant Agkisacutacin as a Novel Antithrombotic Drug Candidate.

    PubMed

    Guo, Yugang; Wu, Jing; Jia, Hao; Chen, Wei; Shao, Changsheng; Zhao, Lei; Ma, Jiajia; Li, Rui; Zhong, Yongjun; Fang, Fang; Wang, Dong; Sun, Jie; Qian, Fang; Dai, Xiangrong; Zhang, Guohui; Tian, Zhigang; Xiaoyi Li, Benjamin; Xiao, Weihua

    2015-01-01

    Agkisacucetin extracted from the venom of Agkistrodon acutus has been demonstrated to be a promising antithrombotic drug candidate in clinical studies due to its function as a novel platelet membrane glycoprotein (GP) Ib inhibitor. Agkisacucetin is a heterodimeric protein composed of α- and β-subunits with seven disulphide bonds. Both subunits form inactive homodimeric products, which cause difficulties for recombinant production. In this study, Agkisacucetin α- and β-subunits were inserted sequentially into the chromosome of Pichia pastoris at the mutant histidinol dehydrogenase gene and ribosomal DNA repeat sites, respectively. By optimizing the gene copies and productivity of each subunit by drug screening, we successfully obtained a recombinant strain with balanced expression of the two subunits. Using this strain, a yield greater than 100 mg/L recombinant Agkisacucetin in fed-batch fermentation was reached. The recombinant Agkisacucetin possessed extremely similar binding affinity to recombinant GPIb and human platelets in in vitro assays, and its ristocetin-induced platelet aggregation activity ex vivo was identical to that of the extracted native Agkisacucetin, demonstrating that the yeast-derived Agkisacucetin could be an effective alternative to native Agkisacucetin. Moreover, this study provides an effective strategy for balancing the expression and production of heterodimeric proteins in P. pastoris. PMID:26144864

  17. Quality Control of Widely Used Therapeutic Recombinant Proteins by a Novel Real-Time PCR Approach

    PubMed Central

    Mamnoon, Babak; Naserpour Farivar, Taghi; Kamyab, Ahmad Reza; Ilghari, Dariush; Khamesipour, Ali; Karimi Arzenani, Mohsen

    2016-01-01

    Background: Existence of bacterial host-cell DNA contamination in biopharmaceutical products is a potential risk factor for patients receiving these drugs. Hence, the quantity of contamination must be controlled under the regulatory standards. Although different methods such as hybridization assays have been employed to determine DNA impurities, these methods are labor intensive and rather expensive. In this study, a rapid real-time PCR test was served as a method of choice to quantify the E. coli host- cell DNA contamination in widely used recombinant streptokinase (rSK), and alpha interferon (IFN-α) preparations. Methods: A specific primer pair was designed to amplify a sequence inside the E. coli 16S rRNA gene. Serial dilutions of DNA extracted from E. coli host cells, along with DNA extracted from Active Pharmaceutical Ingredients of rSK, and IFN-α samples were subjected to an optimized real-time PCR assay based on SYBR Green chemistry. Results: The test enabled us to detect a small quantity of genomic DNA contamination as low as 0.0002 pg in recombinant protein-based drugs. For the first time, this study showed that DNA contamination in rSK and IFN-α preparation manufactured in Pasteur Institute of Iran is much lower than the safety limit suggested by the US FDA. Conclusion: Real-time PCR is a reliable test for rapid detection of host-cell DNA contamination, which is a major impurity of therapeutic recombinant proteins to keep manufacturers’ minds on refining drugs, and provides consumers with safer biopharmaceuticals. PMID:26047906

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

    PubMed

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

    2012-12-01

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

  19. Lifecycle management for recombinant protein production using mammalian cell culture technology.

    PubMed

    Moran, Enda; Gammell, Patrick

    2014-01-01

    Product lifecycle management refers to the oversight process and activities carried out to fully realize the commercial potential and value of a product in the marketplace. It is typical for many changes to be introduced to the production processes and testing methods for biopharmaceutical drugs over their lifetime in the commercial marketplace. Technology lifecycle management, as discussed here, refers to the management of the different phases or generations of processes and methods used to make and test the active biopharmaceutical ingredient or drug product, and the adoption of different devices used to present the drug product to patients. The factors to consider when making changes to a commercial biopharmaceutical manufacturing process as part of a technology lifecycle management program are discussed. A case study outlines one approach taken in bringing forward a major process change to a cell culture process for the production of a therapeutic recombinant protein. PMID:24196316

  20. Natural poly-histidine affinity tag for purification of recombinant proteins on cobalt(II)-carboxymethylaspartate crosslinked agarose.

    PubMed

    Chaga, G; Bochkariov, D E; Jokhadze, G G; Hopp, J; Nelson, P

    1999-12-24

    A natural 19-amino-acid poly-histidine affinity tag was cloned at the N-terminus of three recombinant proteins. The vectors containing the DNA of the fusion proteins were used for transformation of Escherichia coli DH5alpha cells. Each protein was expressed, extracted and purified in one chromatographic step. The purification procedure for each protein can be accomplished in less than 1 h. A new type of immobilized metal ion affinity chromatography adsorbent--Co2+-carboxymethylaspartate agarose Superflow--was utilized at linear flow-rates as high as 5 cm/min. The final preparation of each protein is with purity greater than 95% as ascertained by sodium dodecyl sulfate-electrophoresis. Recovery for each purified protein was higher than 77% of the initial loaded amount as judged by biological activity. The operational capacity of Co2+-carboxymethylaspartate agarose for each protein was determined. PMID:10669292

  1. Highly efficient recombinant production and purification of streptococcal cysteine protease streptopain with increased enzymatic activity.

    PubMed

    Lane, Michael D; Seelig, Burckhard

    2016-05-01

    Streptococcus pyogenes produces the cysteine protease streptopain (SpeB) as a critical virulence factor for pathogenesis. Despite having first been described seventy years ago, this protease still holds mysteries which are being investigated today. Streptopain can cleave a wide range of human proteins, including immunoglobulins, the complement activation system, chemokines, and structural proteins. Due to the broad activity of streptopain, it has been challenging to elucidate the functional results of its action and precise mechanisms for its contribution to S. pyogenes pathogenesis. To better study streptopain, several expression and purification schemes have been developed. These methods originally involved isolation from S. pyogenes culture but were more recently expanded to include recombinant Escherichia coli expression systems. While substantially easier to implement, the latter recombinant approach can prove challenging to reproduce, often resulting in mostly insoluble protein and poor purification yields. After extensive optimization of a wide range of expression and purification conditions, we applied the autoinduction method of protein expression and developed a two-step column purification scheme that reliably produces large amounts of purified soluble and highly active streptopain. This method reproducibly yielded 3 mg of streptopain from 50 mL of expression culture at >95% purity, with an activity of 5306 ± 315 U/mg, and no remaining affinity tags or artifacts from recombinant expression. This improved method therefore enables the facile production of the important virulence factor streptopain at higher yields, with no purification scars that might bias functional studies, and with an 8.1-fold increased enzymatic activity compared to previously described procedures. PMID:26773742

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

    PubMed Central

    Vidi, Pierre-Alexandre; Kessler, Felix; Bréhélin, Claire

    2007-01-01

    Background The potential of transgenic plants for cost-effective production of pharmaceutical molecules is now becoming apparent. Plants have the advantage over established fermentation systems (bacterial, yeast or animal cell cultures) to circumvent the risk of pathogen contamination, to be amenable to large scaling up and to necessitate only established farming procedures. Chloroplasts have proven a useful cellular compartment for protein accumulation owing to their large size and number, as well as the possibility for organellar transformation. They therefore represent the targeting destination of choice for recombinant proteins in leaf crops such as tobacco. Extraction and purification of recombinant proteins from leaf material contribute to a large extent to the production costs. Developing new strategies facilitating these processes is therefore necessary. Results Here, we evaluated plastoglobule lipoprotein particles as a new subchloroplastic destination for recombinant proteins. The yellow fluorescent protein as a trackable cargo was targeted to plastoglobules when fused to plastoglobulin 34 (PGL34) as the carrier. Similar to adipocyte differentiation related protein (ADRP) in animal cells, most of the protein sequence of PGL34 was necessary for targeting to lipid bodies. The recombinant protein was efficiently enriched in plastoglobules isolated by simple flotation centrifugation. The viability of plants overproducing the recombinant protein was not affected, indicating that plastoglobule targeting did not significantly impair photosynthesis or sugar metabolism. Conclusion Our data identify plastoglobules as a new targeting destination for recombinant protein in leaf crops. The wide-spread presence of plastoglobules and plastoglobulins in crop species promises applications comparable to those of transgenic oilbody-oleosin technology in molecular farming. PMID:17214877

  3. Novel osmotically induced antifungal chitinases and bacterial expression of an active recombinant isoform.

    PubMed Central

    Yun, D J; D'Urzo, M P; Abad, L; Takeda, S; Salzman, R; Chen, Z; Lee, H; Hasegawa, P M; Bressan, R A

    1996-01-01

    NaCl (428 mM)-adapted tobacco (Nicotiana tabacum L. var Wisconsin 38) cells accumulate and secrete several antifungal chitinases. The predominant protein secreted to the culture medium was a 29-kD peptide that, based on internal amino acid sequence, was determined to be a class II acidic chitinase with similarity to PR-Q. The four predominant chitinases (T1, T2, T3, and T4) that accumulated intracellularly in 428 mM NaCl-adapted cells were purified. Based on N-terminal sequence analyses, two of these were identified as class I chitinase isoforms, one similar to the N. tomentosiformis (H. Shinshi, J.M. Neuhaus, J. Ryals, F. Meins [1990] Plant Mol Biol 14:357-368) protein (T1) and the other homologous to the N. sylvestris (Y. Fukuda, M. Ohme, H. Shinshi [1991] Plant Mol Biol 16:1-10) protein (T2). The other two proteins (T3 and T4) were determined to be novel chitinases that have sequence similarity with class I chitinases, but each lacks a chitin-binding domain. All four chitinases inhibited Fusarium oxysporum f. sp. lycopersici and Trichoderma longibrachiatum hyphal growth in vitro, although the isoforms containing a chitin-binding domain were somewhat more active. Conditions were established for the successful expression of soluble and active bacterial recombinant T2. Expression of soluble recombinant T2 was achieved when isopropyl beta-D-thiogalactopyranoside induction occurred at 18 degrees C but not at 25 or 37 degrees C. The purified recombinant protein exhibited antifungal activity comparable to a class I chitinase purified from NaCl-adapted tobacco cells. PMID:8756502

  4. Use of a 24-kilodalton Trypanosoma cruzi recombinant protein to monitor cure of human Chagas' disease.

    PubMed Central

    Krautz, G M; Galvão, L M; Cançado, J R; Guevara-Espinoza, A; Ouaissi, A; Krettli, A U

    1995-01-01

    A 24-kDa recombinant protein from Trypanosoma cruzi (rTc24) was evaluated by enzyme-linked immunosorbent assay (ELISA) and Western blot (immunoblot) tests to identify treated chagasic patients considered parasitologically cured on the basis of persistently negative tests of hemocultures and lytic antibodies. Some of these patients were termed dissociated because their sera, although negative by the complement-mediated lysis test, were positive by conventional serology. The negative lysis test indicates the absence of active infection after specific treatment, but this assay requires live and infectious parasites and cannot be used easily in a laboratory routine. Here we tested rTc24 by ELISA and Western blotting as an alternative for the complement-mediated lysis test. For the group of patients with active infection despite the treatment (uncured patients), all the sera tested recognized rTc24 in both tests. For the dissociated patients, approximately 80% of the sera did not react with rTc24 in the ELISA or in Western blots, in agreement with the negative complement-mediated lysis tests. Thus, the 24-kDa T. cruzi recombinant antigen, when used for initial trials to evaluate cure of chagasic patients submitted to specific treatment, will allow the identification of most, but not all, cases. PMID:7559953

  5. 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. PMID:23857606

  6. An Acidic Thermostable Recombinant Aspergillus nidulans Endoglucanase Is Active towards Distinct Agriculture Residues

    PubMed Central

    Tavares, Eveline Queiroz de Pinho; Rubini, Marciano Regis; Mello-de-Sousa, Thiago Machado; Duarte, Gilvan Caetano; de Faria, Fabrícia Paula; Ferreira Filho, Edivaldo Ximenes; Kyaw, Cynthia Maria; Silva-Pereira, Ildinete; Poças-Fonseca, Marcio Jose

    2013-01-01

    Aspergillus nidulans is poorly exploited as a source of enzymes for lignocellulosic residues degradation for biotechnological purposes. This work describes the A. nidulans Endoglucanase A heterologous expression in Pichia pastoris, the purification and biochemical characterization of the recombinant enzyme. Active recombinant endoglucanase A (rEG A) was efficiently secreted as a 35 kDa protein which was purified through a two-step chromatography procedure. The highest enzyme activity was detected at 50°C/pH 4. rEG A retained 100% of activity when incubated at 45 and 55°C for 72 h. Purified rEG A kinetic parameters towards CMC were determined as Km = 27.5 ± 4.33 mg/mL, Vmax = 1.185 ± 0.11 mmol/min, and 55.8 IU (international units)/mg specific activity. Recombinant P. pastoris supernatant presented hydrolytic activity towards lignocellulosic residues such as banana stalk, sugarcane bagasse, soybean residues, and corn straw. These data indicate that rEG A is suitable for plant biomass conversion into products of commercial importance, such as second-generation fuel ethanol. PMID:23936633

  7. Protein-protein complex structure predictions by multimeric threading and template recombination

    PubMed Central

    Mukherjee, Srayanta; Zhang, Yang

    2011-01-01

    Summary The number of protein-protein complex structures is nearly 6-times smaller than that of tertiary structures in PDB which limits the power of homology-based approaches to complex structure modeling. We present a new threading-recombination approach, COTH, to boost the protein complex structure library by combining tertiary structure templates with complex alignments. The query sequences are first aligned to complex templates using a modified dynamic programming algorithm, guided by ab initio binding-site predictions. The monomer alignments are then shifted to the multimeric template framework by structural alignments. COTH was tested on 500 non-homologous dimeric proteins, which can successfully detect correct templates for half of the cases after homologous templates are excluded, which significantly outperforms conventional homology modeling algorithms. It also shows a higher accuracy in interface modeling than rigid-body docking of unbound structures from ZDOCK although with lower coverage. These data demonstrate new avenues to model complex structures from non-homologous templates. PMID:21742262

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

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    2014-09-01

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

  10. Hyper-Enhanced Production of Foreign Recombinant Protein by Fusion with the Partial Polyhedrin of Nucleopolyhedrovirus

    PubMed Central

    Bae, Sung Min; Kim, Hee Jung; Lee, Jun Beom; Choi, Jae Bang; Shin, Tae Young; Koo, Hyun Na; Choi, Jae Young; Lee, Kwang Sik; Je, Yeon Ho; Jin, Byung Rae; Yoo, Sung Sik; Woo, Soo Dong

    2013-01-01

    To enhance the production efficiency of foreign protein in baculovirus expression systems, the effects of polyhedrin fragments were investigated by fusion expressing them with the enhanced green fluorescent protein (EGFP). Recombinant viruses were generated to express EGFP fused with polyhedrin fragments based on the previously reported minimal region for self-assembly and the KRKK nuclear localization signal (NLS). Fusion expressions with polyhedrin amino acids 19 to 110 and 32 to 110 lead to localization of recombinant protein into the nucleus and mediate its assembly. The marked increase of EGFP by these fusion expressions was confirmed through protein and fluorescence intensity analyses. The importance of nuclear localization for enhanced production was shown by the mutation of the NLS within the fused polyhedrin fragment. In addition, when the polyhedrin fragment fused with EGFP was not localized in the nucleus, some fragments increased the production of protein. Among these fragments, some degradation of only the fused polyhedrin was observed in the fusion of amino acids 19 to 85 and 32 to 85. The fusion of amino acids 32 to 85 may be more useful for the enhanced and intact production of recombinant protein. The production of E2 protein, which is a major antigen of classical swine fever virus, was dramatically increased by fusion expression with polyhedrin amino acids 19 to 110, and its preliminary immunogenicity was verified using experimental guinea pigs. This study suggests a new option for higher expression of useful foreign recombinant protein by using the partial polyhedrin in baculovirus. PMID:23593321

  11. 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.

  12. LC-MS and MS/MS in the analysis of recombinant proteins

    NASA Astrophysics Data System (ADS)

    Coulot, M.; Domon, B.; Grossenbacher, H.; Guenat, C.; Maerki, W.; Müller, D. R.; Richter, W. J.

    1993-03-01

    Applicability and performance of electrospray ionization mass spectrometry (ESIMS) is demonstrated for protein analysis. ESIMS is applied in conjunction with on-line HPLC (LC-ESlMS) and direct tandem mass spectrometry (positive and negative ion mode ESlMS/MS) to the structural characterization of a recombinant protein (r-hirudin variant 1) and a congener phosphorylated at threonine 45 (RP-1).

  13. Recombinant Paracoccin Reproduces the Biological Properties of the Native Protein and Induces Protective Th1 Immunity against Paracoccidioides brasiliensis Infection

    PubMed Central

    Alegre, Ana Claudia Paiva; Oliveira, Aline Ferreira; Dos Reis Almeida, Fausto Bruno; Roque-Barreira, Maria Cristina; Hanna, Ebert Seixas

    2014-01-01

    Background Paracoccin is a dual-function protein of the yeast Paracoccidioides brasiliensis that has lectin properties and N-acetylglucosaminidase activities. Proteomic analysis of a paracoccin preparation from P. brasiliensis revealed that the sequence matched that of the hypothetical protein encoded by PADG-3347 of isolate Pb-18, with a polypeptide sequence similar to the family 18 endochitinases. These endochitinases are multi-functional proteins, with distinct lectin and enzymatic domains. Methodology/principal findings The multi-exon assembly and the largest exon of the predicted ORF (PADG-3347), was cloned and expressed in Escherichia coli cells, and the features of the recombinant proteins were compared to those of the native paracoccin. The multi-exon protein was also used for protection assays in a mouse model of paracoccidioidomycosis. Conclusions/Significance Our results showed that the recombinant protein reproduced the biological properties described for the native protein—including binding to laminin in a manner that is dependent on carbohydrate recognition—showed N-acetylglucosaminidase activity, and stimulated murine peritoneal macrophages to produce high levels of TNF-α and nitric oxide. Considering the immunomodulatory potential of glycan-binding proteins, we also investigated whether prophylactic administration of recombinant paracoccin affected the course of experimental paracoccidioidomycosis in mice. In comparison to animals injected with vehicle (controls), mice treated with recombinant paracoccin displayed lower pulmonary fungal burdens and reduced pulmonary granulomas. These protective effects were associated with augmented pulmonary levels of IL-12 and IFN-γ. We also observed that injection of paracoccin three days before challenge was the most efficient administration protocol, as the induced Th1 immunity was balanced by high levels of pulmonary IL-10, which may prevent the tissue damage caused by exacerbated inflammation. The

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

    PubMed

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

    2015-11-01

    Recombinant protein overexpression, an important biotechnological process, is ruled by complex biological rules which are mostly unknown, is in need of an intelligent algorithm so as to avoid resource-intensive lab-based trial and error experiments in order to determine the expression level of the recombinant protein. The purpose of this study is to propose a predictive model to estimate the level of recombinant protein overexpression for the first time in the literature using a machine learning approach based on the sequence, expression vector, and expression host. The expression host was confined to Escherichia coli which is the most popular bacterial host to overexpress recombinant proteins. To provide a handle to the problem, the overexpression level was categorized as low, medium and high. A set of features which were likely to affect the overexpression level was generated based on the known facts (e.g. gene length) and knowledge gathered from related literature. Then, a representative sub-set of features generated in the previous objective was determined using feature selection techniques. Finally a predictive model was developed using random forest classifier which was able to adequately classify the multi-class imbalanced small dataset constructed. The result showed that the predictive model provided a promising accuracy of 80% on average, in estimating the overexpression level of a recombinant protein. PMID:26476414

  15. Increasing recombinant protein production in Escherichia coli K12 through metabolic engineering.

    PubMed

    Waegeman, Hendrik; De Lausnay, Stijn; Beauprez, Joeri; Maertens, Jo; De Mey, Marjan; Soetaert, Wim

    2013-01-25

    Escherichia coli strains are widely used as host for the production of recombinant proteins. Compared to E. coli K12, E. coli BL21 (DE3) has several biotechnological advantages, such as a lower acetate yield and a higher biomass yield, which have a beneficial effect on protein production. In a previous study (BMC Microbiol. 2011, 11:70) we have altered the metabolic fluxes of a K12 strain (i.e. E. coli MG1655) by deleting the regulators ArcA and IclR in such a way that the biomass yield is remarkably increased, while the acetate production is decreased to a similar value as for BL21 (DE3). In this study we show that the increased biomass yield beneficially influences recombinant protein production as a higher GFP yield was observed for the double knockout strain compared to its wild type. However, at higher cell densities (>2 g L(-1) CDW), the GFP concentration decreases again, due to the activity of proteases which obstructs the application of the strain in high cell density cultivations. By further deleting the genes lon and ompT, which encode for proteases, this degradation could be reduced. Consequently, higher GFP yields were observed in the quadruple knockout strain as opposed to the double knockout strain and the MG1655 wild type and its yield approximates the GFP yield of E. coli BL21 (DE3), that is, 27±5 mg g(CDW)(-1) vs. 30±5 mg g(CDW)(-1), respectively. PMID:22115732

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

    PubMed Central

    Kaur, Jasvir; Reinhardt, Dieter P.

    2012-01-01

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

  17. Purified and Recombinant Hemopexin: Protease Activity and Effect on Neutrophil Chemotaxis

    PubMed Central

    Lin, Tian; Liu, Jialin; Huang, Feng; van Engelen, Tjitske SR; Thundivalappil, Sujatha R; Riley, Frank E; Super, Michael; Watters, Alexander L; Smith, Ann; Brinkman, Nathan; Ingber, Donald E; Warren, H Shaw

    2016-01-01

    Infusion of the heme-binding protein hemopexin has been proposed as a novel approach to decrease heme-induced inflammation in settings of red blood cell breakdown, but questions have been raised as to possible side effects related to protease activity and inhibition of chemotaxis. We evaluated protease activity and effects on chemotaxis of purified plasma hemopexin obtained from multiple sources as well as a novel recombinant fusion protein Fc-hemopexin. Amidolytic assay was performed to measure the protease activity of several plasma-derived hemopexin and recombinant Fc-hemopexin. Hemopexin was added to the human monocyte culture in the presence of lipopolysaccharides (LPS), and also injected into mice intravenously (i.v.) 30 min before inducing neutrophil migration via intraperitoneal (i.p.) injection of thioglycolate. Control groups received the same amount of albumin. Protease activity varied widely between hemopexins. Recombinant Fc-hemopexin bound heme, inhibited the synergy of heme with LPS on tumor necrosis factor (TNF) production from monocytes, and had minor but detectable protease activity. There was no effect of any hemopexin preparation on chemotaxis, and purified hemopexin did not alter the migration of neutrophils into the peritoneal cavity of mice. Heme and LPS synergistically induced the release of LTB4 from human monocytes, and hemopexin blocked this release, as well as chemotaxis of neutrophils in response to activated monocyte supernatants. These results suggest that hemopexin does not directly affect chemotaxis through protease activity, but may decrease heme-driven chemotaxis and secondary inflammation by attenuating the induction of chemoattractants from monocytes. This property could be beneficial in some settings to control potentially damaging inflammation induced by heme. PMID:26772775

  18. 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.

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

    PubMed Central

    2013-01-01

    Seeds must maintain a constant level of nitrogen in order to germinate. When recombinant proteins are produced while endogenous seed protein expression is suppressed, the production levels of the foreign proteins increase to compensate for the decreased synthesis of endogenous proteins. Thus, exchanging the production of endogenous seed proteins for that of foreign proteins is a promising approach to increase the yield of foreign recombinant proteins. Providing a space for the deposition of recombinant protein in the intracellular compartment is critical, at this would lessen any competition in this region between the endogenous seed proteins and the introduced foreign protein. The production yields of several recombinant proteins have been greatly increased by this strategy. PMID:23563599

  20. Molecular characterization of the thi3 gene involved in thiamine biosynthesis in Zea mays: cDNA sequence and enzymatic and structural properties of the recombinant bifunctional protein with 4-amino-5-hydroxymethyl-2-methylpyrimidine (phosphate) kinase and thiamine monophosphate synthase activities.

    PubMed

    Rapala-Kozik, Maria; Olczak, Mariusz; Ostrowska, Katarzyna; Starosta, Agata; Kozik, Andrzej

    2007-12-01

    A thiamine biosynthesis gene, thi3, from maize Zea mays has been identified through cloning and sequencing of cDNA and heterologous overexpression of the encoded protein, THI3, in Escherichia coli. The recombinant THI3 protein was purified to homogeneity and shown to possess two essentially different enzymatic activities of HMP(-P) [4-amino-5-hydroxymethyl-2-methylpyrimidine (phosphate)] kinase and TMP (thiamine monophosphate) synthase. Both activities were characterized in terms of basic kinetic constants, with interesting findings that TMP synthase is uncompetitively inhibited by excess of one of the substrates [HMP-PP (HMP diphosphate)] and ATP. A bioinformatic analysis of the THI3 sequence suggested that these activities were located in two distinct, N-terminal kinase and C-terminal synthase, domains. Models of the overall folds of THI3 domains and the arrangements of active centre residues were obtained with the SWISS-MODEL protein modelling server, on the basis of the known three-dimensional structures of Salmonella enterica serotype Typhimurium HMP(-P) kinase and Bacillus subtilis TMP synthase. The essential roles of Gln98 and Met134 residues for HMP kinase activity and of Ser444 for TMP synthase activity were experimentally confirmed by site-directed mutagenesis. PMID:17696876

  1. Identification and use of zinc finger transcription factors that increase production of recombinant proteins in yeast and mammalian cells.

    PubMed

    Park, Kyung-Soon; Seol, Wongi; Yang, Hyo-Young; Lee, Seong-Il; Kim, Sung Keun; Kwon, Ryuk Jun; Kim, Eui-Joong; Roh, Young-Hoon; Seong, Baik Lin; Kim, Jin-Soo

    2005-01-01

    Randomized ZFP-TF libraries could induce a specific phenotype without detailed knowledge about the phenotype of interest because, theoretically, the libraries could modulate any gene in the target organism. We have developed a novel method for enhancing the efficiency of recombinant protein production in mammalian and microbial cells using combinatorial libraries of zinc finger protein transcription factors. To this end, we constructed tens of thousands of zinc finger proteins (ZFPs) with distinct DNA-binding specificities and fused these ZFPs to either a transcriptional activation or repression domain to make transcriptional activators or repressors, respectively. Expression vectors that encode these artificial transcription factors were delivered into Saccharomyces cerevisiae or HEK 293 cells along with reporter plasmids that code for human growth hormone (hGH) or SEAP (secreted alkaline phosphatase) (for yeast or HEK, respectively). Expression of the reporter genes was driven by either the cytomegalovirus (CMV) or SV40 virus promoters. After transfection, we screened the cells for increased synthesis of the reporter proteins. From these cells, we then isolated several ZFP-transcription factors (ZFP-TFs) that significantly increased hGH or SEAP synthesis and subjected these regulatory proteins to further characterization. Our results show that randomized ZFP-TF libraries are useful tools for improving the yield of heterologous recombinant protein both in yeast and mammalian cells. PMID:15932240

  2. Generation of Recombinant Modified Vaccinia Virus Ankara Encoding VP2, NS1, and VP7 Proteins of Bluetongue Virus.

    PubMed

    Marín-López, Alejandro; Ortego, Javier

    2016-01-01

    Modified Vaccinia Virus Ankara (MVA) is employed widely as an experimental vaccine vector for its lack of replication in mammalian cells and high expression level of foreign/heterologous genes. Recombinant MVAs (rMVAs) are used as platforms for protein production as well as vectors to generate vaccines against a high number of infectious diseases and other pathologies. The portrait of the virus combines desirable elements such as high-level biological safety, the ability to activate appropriate innate immune mediators upon vaccination, and the capacity to deliver substantial amounts of heterologous antigens. Recombinant MVAs encoding proteins of bluetongue virus (BTV), an Orbivirus that infects domestic and wild ruminants transmitted by biting midges of the Culicoides species, are excellent vaccine candidates against this virus. In this chapter we describe the methods for the generation of rMVAs encoding VP2, NS1, and VP7 proteins of bluetongue virus as a model example for orbiviruses. The protocols included cover the cloning of VP2, NS1, and VP7 BTV-4 genes in a transfer plasmid, the construction of recombinant MVAs, the titration of virus working stocks and the protein expression analysis by immunofluorescence and radiolabeling of rMVA infected cells as well as virus purification. PMID:26458834

  3. 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. PMID:26883952

  4. 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

  5. 76 FR 44339 - Office of Biotechnology Activities; Recombinant DNA Research: Action Under the NIH Guidelines for...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-07-25

    ... HUMAN SERVICES National Institutes of Health Office of Biotechnology Activities; Recombinant DNA Research: Action Under the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines... attenuated strains of bacteria and viruses that are frequently used in recombinant DNA research. OBA is...

  6. 75 FR 69687 - Office of Biotechnology Activities Recombinant DNA Research: Proposed Actions Under the NIH...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-11-15

    ... HUMAN SERVICES National Institutes of Health Office of Biotechnology Activities Recombinant DNA Research: Proposed Actions Under the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines... the NIH Recombinant DNA Advisory Committee (RAC) and specifically approved by the NIH Director as...

  7. Expression and purification of recombinant antibody formats and antibody fusion proteins.

    PubMed

    Siegemund, Martin; Richter, Fabian; Seifert, Oliver; Unverdorben, Felix; Kontermann, Roland E

    2014-01-01

    In the laboratory-scale production of antibody fragments or antibody fusion proteins, it is often difficult to keep track on the most suitable affinity tags for protein purification from either prokaryotic or eukaryotic host systems. Here, we describe how such recombinant proteins derived from Escherichia coli lysates as well as HEK293 cell culture supernatants are purified by IMAC and by different affinity chromatography methods based on fusions to FLAG-tag, Strep-tag, and Fc domains. PMID:24515473

  8. Enzymatic Properties of Populus α- and β-NAD-ME Recombinant Proteins

    PubMed Central

    Liu, Jinwen; Yu, Qiguo; Elsheery, Nabil I.; Cheng, Yuxiang

    2013-01-01

    Plant mitochondrial NAD-malic enzyme (NAD-ME), which is composed of α- and β-subunits in many species, participates in many plant biosynthetic pathways and in plant respiratory metabolism. However, little is known about the properties of woody plant NAD-MEs. In this study, we analyzed four NAD-ME genes (PtNAD-ME1 through PtNAD-ME4) in the genome of Populus trichocarpa. PtNAD-ME1 and -2 encode putative α-subunits, while PtNAD-ME3 and -4 encode putative β-subunits. The Populus NAD-MEs were expressed in Escherichia coli cells as GST-tagged fusion proteins. Each recombinant GST-PtNAD-ME protein was purified to near homogeneity by glutathione-Sepharose 4B affinity chromatography. Milligram quantities of each native protein were obtained from 1 L bacterial cultures after cleavage of the GST tag. Analysis of the enzymatic properties of these proteins in vitro indicated that α-NAD-MEs are more active than β-NAD-MEs and that α- and β-NAD-MEs presented different kinetic properties (Vmax, kcat and kcat/Km). The effect of different amounts of metabolites on the activities of Populus α- and β-NAD-MEs was assessed in vitro. While none of the metabolites evaluated in our assays activated Populus NAD-ME, oxalacetate and citrate inhibited all α- and β-NAD-MEs and glucose-6-P and fructose inhibited only the α-NAD-MEs. PMID:23797660

  9. Recombinant Expression, Biophysical Characterization, and Cardiolipin-Induced Changes of Two Caenorhabditis elegans Cytochrome c Proteins

    PubMed Central

    Vincelli, Amber J.; Pottinger, Danielle S.; Zhong, Fangfang; Hanske, Jonas; Rolland, Stéphane G.; Conradt, Barbara; Pletneva, Ekaterina V.

    2013-01-01

    Cytochrome c (cyt c) is one of the most widely studied biomolecules, but not much is known about this protein from nematodes. Recombinant expression of C. elegans CYC-2.1 and CYC-2.2 allowed for detailed characterization of their structural features, redox properties, stabilities, and interactions with cardiolipin (CL)-containing liposomes. Using a variety of spectroscopic tools, we show that CYC-2.1 and CYC-2.2 adopt a globular α-helical fold with His/Met heme ligation. The longer CYC-2.2 has a lower thermodynamic stability than CYC-2.1 and lacks His residues to misligate to the heme in the protein’s denatured state. Both C. elegans proteins bind to CL-containing liposomes and these interactions promote the proteins’ peroxidase activity but to a much greater degree for CYC-2.2. Dye-to-heme distance distributions from time-resolved FRET in bimane-labeled CYC-2.1 and CYC-2.2 revealed similar populations of extended and compact conformers for CL-bound proteins, suggesting that their distinct peroxidase activities in the presence of CL arise from differences in the local heme environments for the two polypeptide ensembles. Without inhibition from His misligation, a less stable and more prone to unfolding CYC-2.2 allows for better access of substrates to the heme and thus exhibits higher peroxidase activity. Similar features of the conformational ensembles of CYC-2.1 and CYC-2.2 to those of mammalian cyt c suggest that C. elegans proteins, particularly the former, could serve as useful models for examining the mechanism of cyt c-CL interactions in live organisms. PMID:23282202

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

    PubMed

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

    2013-10-01

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

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

    PubMed Central

    2011-01-01

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2015-07-01

    The ability of a new class of metal binding tags to facilitate the purification of recombinant proteins, exemplified by the tagged glutathione S-transferase and human growth hormone, from Escherichia coli fermentation broths and lysates has been further investigated. These histidine-containing tags exhibit high affinity for borderline metal ions chelated to the immobilised ligand, 1,4,7-triazacyclononane (tacn). The use of this tag-tacn immobilised metal ion affinity chromatography (IMAC) system engenders high selectivity with regard to host cell protein removal and permits facile tag removal from the E. coli-expressed recombinant protein. In particular, these tags were specifically designed to enable their efficient removal by the dipeptidyl aminopeptidase 1 (DAP-1), thus capturing the advantages of high substrate specificity and rates of cleavage. MALDI-TOF MS analysis of the cleaved products from the DAP-1 digestion of the recombinant N-terminally tagged proteins confirmed the complete removal of the tag within 4-12 h under mild experimental conditions. Overall, this study demonstrates that the use of tags specifically designed to target tacn-based IMAC resins offers a comprehensive and flexible approach for the purification of E. coli-expressed recombinant proteins, where complete removal of the tag is an essential prerequisite for subsequent application of the purified native proteins in studies aimed at delineating the molecular and cellular basis of specific biological processes. PMID:25727088

  14. Properties of the recombinant TNF-binding proteins from variola, monkeypox, and cowpox viruses are different.

    PubMed

    Gileva, Irina P; Nepomnyashchikh, Tatiana S; Antonets, Denis V; Lebedev, Leonid R; Kochneva, Galina V; Grazhdantseva, Antonina V; Shchelkunov, Sergei N

    2006-11-01

    Tumor necrosis factor (TNF), a potent proinflammatory and antiviral cytokine, is a critical extracellular immune regulator targeted by poxviruses through the activity of virus-encoded family of TNF-binding proteins (CrmB, CrmC, CrmD, and CrmE). The only TNF-binding protein from variola virus (VARV), the causative agent of smallpox, infecting exclusively humans, is CrmB. Here we have aligned the amino acid sequences of CrmB proteins from 10 VARV, 14 cowpox virus (CPXV), and 22 monkeypox virus (MPXV) strains. Sequence analyses demonstrated a high homology of these proteins. The regions homologous to cd00185 domain of the TNF receptor family, determining the specificity of ligand-receptor binding, were found in the sequences of CrmB proteins. In addition, a comparative analysis of the C-terminal SECRET domain sequences of CrmB proteins was performed. The differences in the amino acid sequences of these domains characteristic of each particular orthopoxvirus species were detected. It was assumed that the species-specific distinctions between the CrmB proteins might underlie the differences in these physicochemical and biological properties. The individual recombinant proteins VARV-CrmB, MPXV-CrmB, and CPXV-CrmB were synthesized in a baculovirus expression system in insect cells and isolated. Purified VARV-CrmB was detectable as a dimer with a molecular weight of 90 kDa, while MPXV- and CPXV-CrmBs, as monomers when fractioned by non-reducing SDS-PAGE. The CrmB proteins of VARV, MPXV, and CPXV differed in the efficiencies of inhibition of the cytotoxic effects of human, mouse, or rabbit TNFs in L929 mouse fibroblast cell line. Testing of CrmBs in the experimental model of LPS-induced shock using SPF BALB/c mice detected a pronounced protective effect of VARV-CrmB. Thus, our data demonstrated the difference in anti-TNF activities of VARV-, MPXV-, and CPXV-CrmBs and efficiency of VARV-CrmB rather than CPXV- or MPXV-CrmBs against LPS-induced mortality in mice. PMID:17070121

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

    PubMed

    Wakasa, Yuhya; Takaiwa, Fumio

    2016-01-01

    Rice seeds are an ideal production platform for high-value recombinant proteins in terms of economy, scalability, safety, and stability. Strategies for the expression of large amounts of recombinant proteins in rice seeds have been established in the past decade and transgenic rice seeds that accumulate recombinant products such as bioactive peptides and proteins, which promote the health and quality of life of humans, have been generated in many laboratories worldwide. One of the most important advantages is the potential for direct oral delivery of transgenic rice seeds without the need for recombinant protein purification (downstream processing), which has been attributed to the high expression levels of recombinant products. Transgenic rice will be beneficial as a delivery system for pharmaceuticals and nutraceuticals in the future. This chapter introduces the strategy for producing recombinant protein in the edible part (endosperm) of the rice grain and describes methods for the analysis of transgenic rice seeds in detail. PMID:26614293

  16. Recombinant Human Bone Morphogenetic Protein-2 in Development and Progression of Oral Squamous Cell Carcinoma.

    PubMed

    Zaid, Khaled Waleed; Chantiri, Mansour; Bassit, Ghassan

    2016-01-01

    Bone morphogenetic proteins (BMPs), belonging to the transforming growth factor-β superfamily, regulate many cellular activities including cell migration, differentiation, adhesion, proliferation and apoptosis. Use of recombinant human bone morphogenic protein?2 (rhBMP?2) in oral and maxillofacial surgery has seen a tremendous increase. Due to its role in many cellular pathways, the influence of this protein on carcinogenesis in different organs has been intensively studied over the past decade. BMPs also have been detected to have a role in the development and progression of many tumors, particularly disease-specific bone metastasis. In oral squamous cell carcinoma - the tumor type accounting for more than 90% of head and neck malignancies- aberrations of both BMP expression and associated signaling pathways have a certain relation with the development and progression of the disease by regulating a range of biological functions in the altered cells. In the current review, we discuss the influence of BMPs -especially rhBMP-2- in the development and progression of oral squamous cell carcinoma. PMID:27039814

  17. Study on the immune response to recombinant Hsp70 protein from Megalobrama amblycephala.

    PubMed

    Chen, Nan; Wan, Xiao-Ling; Huang, Chun-Xiao; Wang, Wei-Min; Liu, Hong; Wang, Huan-Ling

    2014-11-01

    The expression of heat shock protein 70 (Hsp70) is induced in response to many factors including high temperature, infection, metal pollutants and toxic chemicals. In this study, Megalobrama amblycephala HSP70 promoter was cloned, and characteristic heat shock elements (HSEs) were identified in the promoter region. The recombinant M. amblycephala Hsp70 protein (rMaHsp70) was expressed and purified from Escherichia coli BL21 (DE3). To evaluate in vivo immune response of rMaHsp70, we administered intraperitoneal (IP) injection, and demonstrated that rMaHsp70 stimulated M. amblycephala immune activity by inducing the expression of HSP70, HIF-1α, HSC70, CXCR4b, TNF-α and IL-1β mRNAs in liver, headkidney, spleen and gill, as well as SOD, glutathione, lysozyme and interferon alpha proteins in serum and liver. The effect of rMaHsp70 as adjuvant against Aeromonas hydrophila was assessed by injecting a mixed vaccine of rMaHsp70 and A. hydrophila (A. hydrophila/Hsp70) into M. amblycephala, and the relative percent survival (RPS) in the A. hydrophila/Hsp70 group was 75% compared to 50% in the A. hydrophila/PBS group. Furthermore, rMaHsp70 also promoted the proliferation and suppressed apoptosis in M. amblycephala fin cells (MAF) in a dose-dependent manner. Taken together, these results suggest that rMaHsp70 can induce organic immune response and improve environmental tolerance. PMID:25113416

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

    PubMed Central

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

    2014-01-01

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

  19. Albinism-Causing Mutations in Recombinant Human Tyrosinase Alter Intrinsic Enzymatic Activity

    PubMed Central

    Dolinska, Monika B.; Kovaleva, Elena; Backlund, Peter; Wingfield, Paul T.; Brooks, Brian P.; Sergeev, Yuri V.

    2014-01-01

    Background Tyrosinase (TYR) catalyzes the rate-limiting, first step in melanin production and its gene (TYR) is mutated in many cases of oculocutaneous albinism (OCA1), an autosomal recessive cause of childhood blindness. Patients with reduced TYR activity are classified as OCA1B; some OCA1B mutations are temperature-sensitive. Therapeutic research for OCA1 has been hampered, in part, by the absence of purified, active, recombinant wild-type and mutant human enzymes. Methodology/Principal Findings The intra-melanosomal domain of human tyrosinase (residues 19–469) and two OCA1B related temperature-sensitive mutants, R422Q and R422W were expressed in insect cells and produced in T. ni larvae. The short trans-membrane fragment was deleted to avoid potential protein insolubility, while preserving all other functional features of the enzymes. Purified tyrosinase was obtained with a yield of >1 mg per 10 g of larval biomass. The protein was a monomeric glycoenzyme with maximum enzyme activity at 37°C and neutral pH. The two purified mutants when compared to the wild-type protein were less active and temperature sensitive. These differences are associated with conformational perturbations in secondary structure. Conclusions/Significance The intramelanosomal domains of recombinant wild-type and mutant human tyrosinases are soluble monomeric glycoproteins with activities which mirror their in vivo function. This advance allows for the structure – function analyses of different mutant TYR proteins and correlation with their corresponding human phenotypes; it also provides an important tool to discover drugs that may improve tyrosinase activity and treat OCA1. PMID:24392141

  20. Three faces of recombination activating gene 1 (RAG1) mutations.

    PubMed

    Patiroglu, Turkan; Akar, Himmet Haluk; Van Der Burg, Mirjam

    2015-12-01

    Severe combined immune deficiency (SCID) is a group of genetic disorder associated with development of T- and/or B-lymphocytes. Recombination-activating genes (RAG1/2) play a critical role on VDJ recombination process that leads to the production of a broad T-cell receptor (TCR) and B-cell receptor (BCR) repertoire in the development of T and B cells. RAG1/2 genes mutations result in various forms of primary immunodeficiency, ranging from classic SCID to Omenn syndrome (OS) to atypical SCID with such as granuloma formation and autoimmunity. Herein, we reported 4 patients with RAG1 deficiency: classic SCID was seen in two patients who presented with recurrent pneumonia and chronic diarrhoea, and failure to thrive. OS was observed in one patient who presented with chronic diarrhoea, skin rash, recurrent lower respiratory infections, and atypical SCID was seen in one patient who presented with Pyoderma gangrenosum (PG) and had novel RAG1 mutation. PMID:26689875

  1. 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

  2. Inhibition of homologous recombination by the PCNA-interacting protein PARI.

    PubMed

    Moldovan, George-Lucian; Dejsuphong, Donniphat; Petalcorin, Mark I R; Hofmann, Kay; Takeda, Shunichi; Boulton, Simon J; D'Andrea, Alan D

    2012-01-13

    Inappropriate homologous recombination (HR) causes genomic instability and cancer. In yeast, the UvrD family helicase Srs2 is recruited to sites of DNA replication by SUMO-modified PCNA, where it acts to restrict HR by disassembling toxic RAD51 nucleofilaments. How human cells control recombination at replication forks is unknown. Here, we report that the protein PARI, containing a UvrD-like helicase domain, is a PCNA-interacting partner required for preservation of genome stability in human and DT40 chicken cells. Using cell-based and biochemical assays, we show that PARI restricts unscheduled recombination by interfering with the formation of RAD51-DNA HR structures. Finally, we show that PARI knockdown suppresses the genomic instability of Fanconi Anemia/BRCA pathway-deficient cells. Thus, we propose that PARI is a long sought-after factor that suppresses inappropriate recombination events at mammalian replication forks. PMID:22153967

  3. Expression of recombinant protein using Corynebacterium Glutamicum: progress, challenges and applications.

    PubMed

    Liu, Xiuxia; Yang, Yankun; Zhang, Wei; Sun, Yang; Peng, Feng; Jeffrey, Laura; Harvey, Linda; McNeil, Brian; Bai, Zhonghu

    2016-08-01

    Corynebacterium glutamicum (C. glutamicum) is a highly promising alternative prokaryotic host for recombinant protein expression, as it possesses several significant advantages over Escherichia coli (E. coli), the currently leading bacterial protein expression system. During the past decades, several experimental techniques and vector components for genetic manipulation of C. glutamicum have been developed and validated, including strong promoters for tightly regulating target gene expression, various types of plasmid vectors, protein secretion systems and methods of genetically modifying the host strain genome to improve protein production potential. This review critically discusses current progress in establishing C. glutamicum as a host for recombinant protein expression, and examines, in depth, some successful case studies of actual application of this expression system. The established "expression tool box" for developing novel constructs based on C. glutamicum as a host are also evaluated. Finally, the existing issues and solutions in process development with C. glutamicum as a host are specifically addressed. PMID:25714007

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

    PubMed

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

    1997-12-01

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

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

    PubMed

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

    2015-04-10

    Human glycoproteins, expressed in hamster cell lines, show similar glycosylation patterns to naturally occurring human molecules except for a minute difference in the linkage of terminal sialic acid: both cell types lack α2,6-galactosyl-sialyltransferase, abundantly expressed in human hepatocytes and responsible for the α2,6-sialylation of circulating glycoproteins. This minute difference, which is currently not known to have any physiological relevance, was the basis for the selective measurement of recombinant glycoproteins in the presence of their endogenous counterparts. The assay is based on using the lectin Sambucus nigra agglutinin (SNA), selectively binding to α2,6-sialylated N-glycans. Using von Willebrand factor (VWF), factor IX (FIX), and factor VIIa (FVIIa), it was demonstrated that (i) the plasma-derived proteins, but not the corresponding recombinant proteins, specifically bind to SNA and (ii) this binding can be used to deplete the plasma-derived proteins. The feasibility of this approach was confirmed in spike-recovery studies for all three recombinant coagulation proteins in human plasma and for recombinant VWF (rVWF) in macaque plasma. Analysis of plasma samples from macaques after administration of recombinant and a plasma-derived VWF demonstrated the suitability and robustness of this approach. Data showed that rVWF could be selectively measured without changing the ELISAs and furthermore revealed the limitations of baseline adjustment using a single measurement of the predose concentration only. The SNA gel-based depletion procedure can easily be integrated in existing procedures as a specific sample pre-treatment step. While ELISA-based methods were used to measure the recombinant coagulation proteins in the supernatants obtained by depletion, this procedure is applicable for all biochemical analyses. PMID:25703236

  6. Effect of codon-optimized E. coli signal peptides on recombinant Bacillus stearothermophilus maltogenic amylase periplasmic localization, yield and activity.

    PubMed

    Samant, Shalaka; Gupta, Gunja; Karthikeyan, Subbulakshmi; Haq, Saiful F; Nair, Ayyappan; Sambasivam, Ganesh; Sukumaran, Sunilkumar

    2014-09-01

    Recombinant proteins can be targeted to the Escherichia coli periplasm by fusing them to signal peptides. The popular pET vectors facilitate fusion of target proteins to the PelB signal. A systematic comparison of the PelB signal with native E. coli signal peptides for recombinant protein expression and periplasmic localization is not reported. We chose the Bacillus stearothermophilus maltogenic amylase (MA), an industrial enzyme widely used in the baking and brewing industry, as a model protein and analyzed the competence of seven, codon-optimized, E. coli signal sequences to translocate MA to the E. coli periplasm compared to PelB. MA fusions to three of the signals facilitated enhanced periplasmic localization of MA compared to the PelB fusion. Interestingly, these three fusions showed greatly improved MA yields and between 18- and 50-fold improved amylase activities compared to the PelB fusion. Previously, non-optimal codon usage in native E. coli signal peptide sequences has been reported to be important for protein stability and activity. Our results suggest that E. coli signal peptides with optimal codon usage could also be beneficial for heterologous protein secretion to the periplasm. Moreover, such fusions could even enhance activity rather than diminish it. This effect, to our knowledge has not been previously documented. In addition, the seven vector platform reported here could also be used as a screen to identify the best signal peptide partner for other recombinant targets of interest. PMID:25038884

  7. 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. PMID:21898365

  8. Properties and mutation studies of a bacteriophage-derived chimeric recombinant staphylolytic protein P128

    PubMed Central

    Saravanan, Sanjeev Rajagopalan; Paul, Vivek Daniel; George, Shilpa; Sundarrajan, Sudarson; Kumar, Nirmal; Hebbur, Madhavi; Kumar, Naveen; Veena, Ananda; Maheshwari, Uma; Appaiah, Chemira Biddappa; Chidambaran, Muralidharan; Bhat, Anuradha Gopal; Hariharan, Sukumar; Padmanabhan, Sriram

    2013-01-01

    P128 is a chimeric anti-staphylococcal protein having a catalytic domain from a Staphylococcus bacteriophage K tail associated structural protein and a cell wall targeting domain from the Staphylococcus bacteriocin-lysostaphin. In this study, we disclose additional properties of P128 and compared the same with lysostaphin. While lysostaphin was found to get inactivated by heat and was inactive on its parent strain S. simulans biovar staphylolyticus, P128 was thermostable and was lytic towards S. simulans biovar staphylolyticus demonstrating a difference in their mechanism of action. Selected mutation studies of the catalytic domain of P128 showed that arginine and cysteine, at 40th and 76th positions respectively, are critical for the staphylolytic activity of P128, although these amino acids are not conserved residues. In comparison to native P128, only the R40S mutant (P301) was catalytically active on zymogram gel and had a similar secondary structure, as assessed by circular dichroism analysis and in silico modeling with similar cell binding properties. Mutation of the arginine residue at 40th position of the P128 molecule caused dramatic reduction in the Vmax (∆OD600 [mg/min]) value (nearly 270 fold) and the recombinant lysostaphin also showed lesser Vmax value (nearly 1.5 fold) in comparison to the unmodified P128 protein. The kinetic parameters such as apparent Km (Km APP) and apparent Kcat (KcatAPP) of the native P128 protein also showed significant differences in comparison to the values observed for P301 and lysostaphin. PMID:24251076

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

    PubMed Central

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

    2002-01-01

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

  10. 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. PMID:15939754

  11. 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

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

    PubMed

    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

  13. Leaf proteome rebalancing in Nicotiana benthamiana for upstream enrichment of a transiently expressed recombinant protein.

    PubMed

    Robert, Stéphanie; Goulet, Marie-Claire; D'Aoust, Marc-André; Sainsbury, Frank; Michaud, Dominique

    2015-10-01

    A key factor influencing the yield of biopharmaceuticals in plants is the ratio of recombinant to host proteins in crude extracts. Postextraction procedures have been devised to enrich recombinant proteins before purification. Here, we assessed the potential of methyl jasmonate (MeJA) as a generic trigger of recombinant protein enrichment in Nicotiana benthamiana leaves before harvesting. Previous studies have reported a significant rebalancing of the leaf proteome via the jasmonate signalling pathway, associated with ribulose 1,5-bisphosphate carboxylase oxygenase (RuBisCO) depletion and the up-regulation of stress-related proteins. As expected, leaf proteome alterations were observed 7 days post-MeJA treatment, associated with lowered RuBisCO pools and the induction of stress-inducible proteins such as protease inhibitors, thionins and chitinases. Leaf infiltration with the Agrobacterium tumefaciens bacterial vector 24 h post-MeJA treatment induced a strong accumulation of pathogenesis-related proteins after 6 days, along with a near-complete reversal of MeJA-mediated stress protein up-regulation. RuBisCO pools were partly restored upon infiltration, but most of the depletion effect observed in noninfiltrated plants was maintained over six more days, to give crude protein samples with 50% less RuBisCO than untreated tissue. These changes were associated with net levels reaching 425 μg/g leaf tissue for the blood-typing monoclonal antibody C5-1 expressed in MeJA-treated leaves, compared to less than 200 μg/g in untreated leaves. Our data confirm overall the ability of MeJA to trigger RuBisCO depletion and recombinant protein enrichment in N. benthamiana leaves, estimated here for C5-1 at more than 2-fold relative to host proteins. PMID:26286859

  14. 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. PMID:26859695

  15. High-yield bacterial expression and structural characterization of recombinant human insulin-like growth factor binding protein-2

    PubMed Central

    Swain, Monalisa; Slomiany, Mark G.; Rosenzweig, Steven A.; Atreya, Hanudatta S.

    2010-01-01

    The diverse biological activities of the insulin-like growth factors (IGF-1 and IGF-2) are mediated by the IGF-1 receptor (IGF-IR). These actions are modulated by a family of six IGF-binding proteins (IGFBP-1–6; 22–31 kDa) that via high affinity binding to the IGFs (KD ~ 300–700 pM) both protect the IGFs in the circulation and attenuate IGF action by blocking their receptor access. In recent years, IGFBPs have been implicated in a variety of cancers. However, the structural basis of their interaction with IGFs and/or other proteins is not completely understood. A critical challenge in the structural characterization of full-length IGFBPs has been the difficulty in expressing these proteins at levels suitable for NMR/X-ray crystallography analysis. Here we describe the high-yield expression of full-length recombinant human IGFBP-2 (rhIGFBP-2) in E. coli. Using a single step purification protocol, rhIGFBP-2 was obtained with >95% purity and structurally characterized using NMR spectroscopy. The protein was found to exist as a monomer at the high concentrations required for structural studies and to exist in a single conformation exhibiting a unique intra-molecular disulfide-bonding pattern. The protein retained full biologic activity. This study represents the first high-yield expression of wild-type recombinant human IGFBP-2 in E. coli and first structural characterization of a full-length IGFBP. PMID:20541521

  16. 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

  17. Cleavage of recombinant proteins at poly-His sequences by Co(II) and Cu(II)

    PubMed Central

    Andberg, Martina; Jäntti, Jussi; Heilimo, Sara; Pihkala, Päivi; Paananen, Arja; Koskinen, Ari M.P.; Söderlund, Hans; Linder, Markus B.

    2007-01-01

    Improved ways to cleave peptide chains at engineered sites easily and specifically would form useful tools for biochemical research. Uses of such methods include the activation or inactivation of enzymes or the removal of tags for enhancement of recombinant protein expression or tags used for purification of recombinant proteins. In this work we show by gel electrophoresis and mass spectroscopy that salts of Co(II) and Cu(II) can be used to cleave fusion proteins specifically at sites where sequences of His residues have been introduced by protein engineering. The His residues could be either consecutive or spaced with other amino acids in between. The cleavage reaction required the presence of low concentrations of ascorbate and in the case of Cu(II) also hydrogen peroxide. The amount of metal ions required for cleavage was very low; in the case of Cu(II) only one to two molar equivalents of Cu(II) to protein was required. In the case of Co(II), 10 molar equivalents gave optimal cleavage. The reaction occurred within minutes, at a wide pH range, and efficiently at temperatures ranging from 0°C to 70°C. The work described here can also have implications for understanding protein stability in vitro and in vivo. PMID:17600148

  18. Rapid screening for the robust expression of recombinant proteins in algal plastids.

    PubMed

    Barrera, Daniel; Gimpel, Javier; Mayfield, Stephen

    2014-01-01

    Chlamydomonas reinhardtii has many advantages as a photosynthetic model organism. One of these is facile, targeted chloroplast transformation by particle bombardment. Functional recombinant proteins can be expressed to significant levels in this system, potentially outperforming higher plants in speed of scaling, cost, and space requirements. Several strategies and regulatory regions can be used for achieving transgene expression. Here we present two of those strategies: one makes use of the psbD promoter for expressing moderate levels of the recombinant protein in a photosynthetic background. The other strategy is based on the strong psbA promoter for obtaining high yields of the recombinant product in a non-photosynthetic strain. We herein describe the vectors, transformation procedures, and screening methods associated with these two strategies. PMID:24599869

  19. 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. PMID:26458807

  20. 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

  1. 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. PMID:24153406

  2. 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. PMID:27105777

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

    PubMed

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

    2016-05-01

    Pichia pastoris is used for commercial production of human therapeutic proteins, and genome-scale models of P. pastoris metabolism have been generated in the past to study the metabolism and associated protein production by this yeast. A major challenge with clinical usage of recombinant proteins produced by P. pastoris is the difference in N-glycosylation of proteins produced by humans and this yeast. However, through metabolic engineering, a P. pastoris strain capable of producing humanized N-glycosylated proteins was constructed. The current genome-scale models of P. pastoris do not address native nor humanized N-glycosylation, and we therefore developed ihGlycopastoris, an extension to the iLC915 model with both native and humanized N-glycosylation for recombinant protein production, but also an estimation of N-glycosylation of P. pastoris native proteins. This new model gives a better prediction of protein yield, demonstrates the effect of the different types of N-glycosylation of protein yield, and can be used to predict potential targets for strain improvement. The model represents a step towards a more complete description of protein production in P. pastoris, which is required for using these models to understand and optimize protein production processes. Biotechnol. Bioeng. 2016;113: 961-969. © 2015 Wiley Periodicals, Inc. PMID:26480251

  4. Recombinant FAdV-4 fiber-2 protein protects chickens against hepatitis-hydropericardium syndrome (HHS).

    PubMed

    Schachner, Anna; Marek, Ana; Jaskulska, Barbara; Bilic, Ivana; Hess, Michael

    2014-02-19

    Virulent fowl adenovirus (FAdV) serotype 4 strains are the etiological agents of hepatitis-hydropericardium syndrome (HHS), a highly infectious disease in chickens with severe economic impact. In the present study, three different FAdV-4 derived capsid proteins, fiber-1, fiber-2, and hexon loop-1, were expressed in a baculovirus system and tested for their capacity to induce protection in chickens. Purified recombinant proteins were administered to day-old specific pathogen-free (SPF) chickens allocated in three separate groups and challenged with virulent FAdV-4 at 21 days of life. Two additional groups served as controls, a challenge control group with mock-vaccinated but infected birds and a negative control group with PBS injection substituting both vaccination and challenge. The fiber-2 vaccinated group displayed high resistance against the adverse effects of the challenge with only one dead bird out of 28, as compared to the challenge control group where the infection caused 78% mortality. A moderate protective effect resulting in 38% mortality was observed for fiber-1, whereas the hexon loop-1 vaccinated group was not effectively protected as manifested by 73% mortality. While a fiber-2 specific ELISA showed a gradual antibody increase after immunization of birds with the homologous protein, a commercial ELISA did not detect vaccination-induced antibodies in any of the groups but displayed a difference in challenge virus-directed response in protected and non-protected birds. Although immunoblotting confirmed the presence of specific antibodies in all vaccinated groups, the anti-protein sera did not exhibit neutralizing activity. Fecal excretion of challenge virus DNA was detected with a real-time PCR in the majority of tested birds until termination of the study independent of protection, indicating the prevention of clinical symptoms, but not infection, by vaccination. In conclusion, recombinant fiber-2 was identified as a protective immunogen and is

  5. Solution Structure and DNA-binding Properties of the Winged Helix Domain of the Meiotic Recombination HOP2 Protein*

    PubMed Central

    Moktan, Hem; Guiraldelli, Michel F.; Eyster, Craig A.; Zhao, Weixing; Lee, Chih-Ying; Mather, Timothy; Camerini-Otero, R. Daniel; Sung, Patrick; Zhou, Donghua H.; Pezza, Roberto J.

    2014-01-01

    The HOP2 protein is required for efficient double-strand break repair which ensures the proper synapsis of homologous chromosomes and normal meiotic progression. We previously showed that in vitro HOP2 shows two distinctive activities: when it is incorporated into a HOP2-MND1 heterodimer, it stimulates DMC1 and RAD51 recombination activities, and the purified HOP2 alone is proficient in promoting strand invasion. The structural and biochemical basis of HOP2 action in recombination are poorly understood; therefore, they are the focus of this work. Herein, we present the solution structure of the amino-terminal portion of mouse HOP2, which contains a typical winged helix DNA-binding domain. Together with NMR spectral changes in the presence of double-stranded DNA, protein docking on DNA, and mutation analysis to identify the amino acids involved in DNA coordination, our results on the three-dimensional structure of HOP2 provide key information on the fundamental structural and biochemical requirements directing the interaction of HOP2 with DNA. These results, in combination with mutational experiments showing the role of a coiled-coil structural feature involved in HOP2 self-association, allow us to explain important aspects of the function of HOP2 in recombination. PMID:24711446

  6. Production and characterization of ZFP36L1 antiserum against recombinant protein from Escherichia coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tristetraprolin (TTP/ZFP36) family proteins are anti-inflammatory. They bind and destabilize some AU-rich element-containing mRNAs such as tumor necrosis factor mRNA. In this study, recombinant ZFP36L1/TIS11B (a TTP homologue) was over-expressed in E. coli, purified, and used for polyclonal antibody...

  7. Antigenic Profiles of Recombinant Proteins from Mycobacterium avium subsp paratuberculosis in Sheep with Johne's Disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Methods to improve the ELISA test to detect Mycobacterium avium subsp paratuberculosis have been explored over several years. Previously, selected recombinant proteins of M. avium subspecies paratuberculosis were found to be immunogenic in cattle with Johne’s disease. In the present study, antibo...

  8. A proposed feeding strategy for the overproduction of recombinant proteins in Escherichia coli.

    PubMed

    Babaeipour, Valiollah; Shojaosadati, Seyed Abbas; Khalilzadeh, Rasoul; Maghsoudi, Nader; Tabandeh, Fatemeh

    2008-02-01

    Different feeding strategies for the production of human interferon-gamma using an isopropyl beta-D-thiogalactoside-inducible expression system in recombinant Escherichia coli BL21(DE3) (plasmid pET3a-ifngamma) were studied. Four fed-batch modes were designed to compare the effect of mu (specific growth rate) on recombinant-protein production, substrate consumption, by-product formation and plasmid stability during pre- and post-chemical induction in high-cell-density cultures of E. coli. It was found that Y(p/s), the product/substrate yield of interferon-gamma was significantly affected by mu throughout the process, but product/biomass yield (Y(p/x)) was influenced by mu at the pre-induction stage. By applying an efficient feeding strategy, in which the mu was maintained at the maximum attainable level, recombinant protein was accumulated up to a level of 60% of the total cell protein and its productivity was increased significantly. In this case, the overall productivities of biomass and recombinant protein were 6.36 g l(-1) h(-1) and 2.1 g l(-1) h(-1) respectively, in comparison with 1.91 g l(-1) h(-1) and 0.16 g l(-1) h(-1) during exponential feeding, in which the specific growth rate was kept constant throughout the entire process. PMID:17630954

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2012-05-01

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

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

    PubMed Central

    Haon, Mireille; Grisel, Sacha; Navarro, David; Gruet, Antoine; Berrin, Jean-Guy; Bignon, Christophe

    2015-01-01

    Filamentous fungi are the predominant source of lignocellulolytic enzymes used in industry for the transformation of plant biomass into high-value molecules and biofuels. The rapidity with which new fungal genomic and post-genomic data are being produced is vastly outpacing functional studies. This underscores the critical need for developing platforms dedicated to the recombinant expression of enzymes lacking confident functional annotation, a prerequisite to their functional and structural study. In the last decade, the yeast Pichia pastoris has become increasingly popular as a host for the production of fungal biomass-degrading enzymes, and particularly carbohydrate-active enzymes (CAZymes). This study aimed at setting-up a platform to easily and quickly screen the extracellular expression of biomass-degrading enzymes in P. pastoris. We first used three fungal glycoside hydrolases (GHs) that we previously expressed using the protocol devised by Invitrogen to try different modifications of the original protocol. Considering the gain in time and convenience provided by the new protocol, we used it as basis to set-up the facility and produce a suite of fungal CAZymes (GHs, carbohydrate esterases and auxiliary activity enzyme families) out of which more than 70% were successfully expressed. The platform tasks range from gene cloning to automated protein purifications and activity tests, and is open to the CAZyme users’ community. PMID:26441929

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

    PubMed

    Haon, Mireille; Grisel, Sacha; Navarro, David; Gruet, Antoine; Berrin, Jean-Guy; Bignon, Christophe

    2015-01-01

    Filamentous fungi are the predominant source of lignocellulolytic enzymes used in industry for the transformation of plant biomass into high-value molecules and biofuels. The rapidity with which new fungal genomic and post-genomic data are being produced is vastly outpacing functional studies. This underscores the critical need for developing platforms dedicated to the recombinant expression of enzymes lacking confident functional annotation, a prerequisite to their functional and structural study. In the last decade, the yeast Pichia pastoris has become increasingly popular as a host for the production of fungal biomass-degrading enzymes, and particularly carbohydrate-active enzymes (CAZymes). This study aimed at setting-up a platform to easily and quickly screen the extracellular expression of biomass-degrading enzymes in P. pastoris. We first used three fungal glycoside hydrolases (GHs) that we previously expressed using the protocol devised by Invitrogen to try different modifications of the original protocol. Considering the gain in time and convenience provided by the new protocol, we used it as basis to set-up the facility and produce a suite of fungal CAZymes (GHs, carbohydrate esterases and auxiliary activity enzyme families) out of which more than 70% were successfully expressed. The platform tasks range from gene cloning to automated protein purifications and activity tests, and is open to the CAZyme users' community. PMID:26441929

  13. Beyond the cytoplasm of Escherichia coli: localizing recombinant proteins where you want them.

    PubMed

    Boock, Jason T; Waraho-Zhmayev, Dujduan; Mizrachi, Dario; DeLisa, Matthew P

    2015-01-01

    Recombinant protein expression in Escherichia coli represents a cornerstone of the biotechnology enterprise. While cytoplasmic expression in this host has received the most attention, achieving substantial yields of correctly folded proteins in this compartment can sometimes be met with difficulties. These issues can often be overcome by targeting protein expression to extracytoplasmic compartments (e.g., membrane, periplasm) or to the culture medium. This chapter discusses various strategies for exporting proteins out of the cytoplasm as well as tools for monitoring and optimizing these different export mechanisms. PMID:25447860

  14. UvrD controls the access of recombination proteins to blocked replication forks.

    PubMed

    Lestini, Roxane; Michel, Bénédicte

    2007-08-22

    Blocked replication forks often need to be processed by recombination proteins prior to replication restart. In Escherichia coli, the UvrD repair helicase was recently shown to act at inactivated replication forks, where it counteracts a deleterious action of RecA. Using two mutants affected for different subunits of the polymerase III holoenzyme (Pol IIIh), we show here that the anti-RecA action of UvrD at blocked forks reflects two different activities of this enzyme. A defective UvrD mutant is able to antagonize RecA in cells affected for the Pol IIIh catalytic subunit DnaE. In this mutant, RecA action at blocked forks specifically requires the protein RarA (MgsA). We propose that UvrD prevents RecA binding, possibly by counteracting RarA. In contrast, at forks affected for the Pol IIIh clamp (DnaN), RarA is not required for RecA binding and the ATPase function of UvrD is essential to counteract RecA, supporting the idea that UvrD removes RecA from DNA. UvrD action on RecA is conserved in evolution as it can be performed in E. coli by the UvrD homologue from Bacillus subtilis, PcrA. PMID:17641684

  15. UvrD controls the access of recombination proteins to blocked replication forks

    PubMed Central

    Lestini, Roxane; Michel, Bénédicte

    2007-01-01

    Blocked replication forks often need to be processed by recombination proteins prior to replication restart. In Escherichia coli, the UvrD repair helicase was recently shown to act at inactivated replication forks, where it counteracts a deleterious action of RecA. Using two mutants affected for different subunits of the polymerase III holoenzyme (Pol IIIh), we show here that the anti-RecA action of UvrD at blocked forks reflects two different activities of this enzyme. A defective UvrD mutant is able to antagonize RecA in cells affected for the Pol IIIh catalytic subunit DnaE. In this mutant, RecA action at blocked forks specifically requires the protein RarA (MgsA). We propose that UvrD prevents RecA binding, possibly by counteracting RarA. In contrast, at forks affected for the Pol IIIh clamp (DnaN), RarA is not required for RecA binding and the ATPase function of UvrD is essential to counteract RecA, supporting the idea that UvrD removes RecA from DNA. UvrD action on RecA is conserved in evolution as it can be performed in E. coli by the UvrD homologue from Bacillus subtilis, PcrA. PMID:17641684

  16. ERCC4 (XPF) encodes a human nucleotide excision repair protein with eukaryotic recombination homologs.

    PubMed

    Brookman, K W; Lamerdin, J E; Thelen, M P; Hwang, M; Reardon, J T; Sancar, A; Zhou, Z Q; Walter, C A; Parris, C N; Thompson, L H

    1996-11-01

    ERCC4 is an essential human gene in the nucleotide excision repair (NER) pathway, which is responsible for removing UV-C photoproducts and bulky adducts from DNA. Among the NER genes, ERCC4 and ERCC1 are also uniquely involved in removing DNA interstrand cross-linking damage. The ERCC1-ERCC4 heterodimer, like the homologous Rad10-Rad1 complex, was recently found to possess an endonucleolytic activity that incises on the 5' side of damage. The ERCC4 gene, assigned to chromosome 16p13.1-p13.2, was previously isolated by using a chromosome 16 cosmid library. It corrects the defect in Chinese hamster ovary (CHO) mutants of NER complementation group 4 and is implicated in complementation group F of the human disorder xeroderma pigmentosum. We describe the ERCC4 gene structure and functional cDNA sequence encoding a 916-amino-acid protein (104 kDa), which has substantial homology with the eukaryotic DNA repair and recombination proteins MEI-9 (Drosophila melanogaster), Rad16 (Schizosaccharomyces pombe), and Rad1 (Saccharomyces cerevisiae). ERCC4 cDNA efficiently corrected mutants in rodent NER complementation groups 4 and 11, showing the equivalence of these groups, and ERCC4 protein levels were reduced in mutants of both groups. In cells of an XP-F patient, the ERCC4 protein level was reduced to less than 5%, consistent with XPF being the ERCC4 gene. The considerable identity (40%) between ERCC4 and MEI-9 suggests a possible involvement of ERCC4 in meiosis. In baboon tissues, ERCC4 was expressed weakly and was not significantly higher in testis than in nonmeiotic tissues. PMID:8887684

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

    PubMed

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

    2011-07-01

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

  18. 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

  19. Intratracheal Recombinant Surfactant Protein D Prevents Endotoxin Shock in the Newborn Preterm Lamb

    PubMed Central

    Ikegami, Machiko; Carter, Karen; Bishop, Kimberly; Yadav, Annuradha; Masterjohn, Elizabeth; Brondyk, William; Scheule, Ronald K.; Whitsett, Jeffrey A.

    2006-01-01

    Rationale: The susceptibility of neonates to pulmonary and systemic infection has been associated with the immaturity of both lung structure and the immune system. Surfactant protein (SP) D is a member of the collectin family of innate immune molecules that plays an important role in innate host defense of the lung. Objectives: We tested whether treatment with recombinant human SP-D influenced the response of the lung and systemic circulation to intratracheally administered Escherichia coli lipopolysaccharides. Methods: After intratracheal lipopolysaccharide instillation, preterm newborn lambs were treated with surfactant and ventilated for 5 h. Measurement: Survival rate, physiologic lung function, lung and systemic inflammation, and endotoxin level in plasma were evaluated. Main Results: In control lambs, intratracheal lipopolysaccharides caused septic shock and death associated with increased endotoxin in plasma. In contrast, all lambs treated with recombinant human SP-D were physiologically stable and survived. Leakage of lipopolysaccharides from the lungs to the systemic circulation was prevented by intratracheal recombinant human SP-D. Recombinant human SP-D prevented systemic inflammation and decreased the expression of IL-1β, IL-8, and IL-6 in the spleen and liver. Likewise, recombinant human SP-D decreased IL-1β and IL-6 in the lung and IL-8 in the plasma. Recombinant human SP-D did not alter pulmonary mechanics following endotoxin exposure. Recombinant human SP-D was readily detected in the lung 5 h after intratracheal instillation. Conclusions: Intratracheal recombinant human SP-D prevented shock caused by endotoxin released from the lung during ventilation in the premature newborn. PMID:16556693

  20. 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

  1. 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

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

    PubMed

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

    2016-09-01

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

  3. 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

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

    PubMed

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

    2016-04-27

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

  5. 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

  6. 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. PMID:26435147

  7. Discovery of an Active RAG Transposon Illuminates the Origins of V(D)J Recombination.

    PubMed

    Huang, Shengfeng; Tao, Xin; Yuan, Shaochun; Zhang, Yuhang; Li, Peiyi; Beilinson, Helen A; Zhang, Ya; Yu, Wenjuan; Pontarotti, Pierre; Escriva, Hector; Le Petillon, Yann; Liu, Xiaolong; Chen, Shangwu; Schatz, David G; Xu, Anlong

    2016-06-30

    Co-option of RAG1 and RAG2 for antigen receptor gene assembly by V(D)J recombination was a crucial event in the evolution of jawed vertebrate adaptive immunity. RAG1/2 are proposed to have arisen from a transposable element, but definitive evidence for this is lacking. Here, we report the discovery of ProtoRAG, a DNA transposon family from lancelets, the most basal extant chordates. A typical ProtoRAG is flanked by 5-bp target site duplications and a pair of terminal inverted repeats (TIRs) resembling V(D)J recombination signal sequences. Between the TIRs reside tail-to-tail-oriented, intron-containing RAG1-like and RAG2-like genes. We demonstrate that ProtoRAG was recently active in the lancelet germline and that the lancelet RAG1/2-like proteins can mediate TIR-dependent transposon excision, host DNA recombination, transposition, and low-efficiency TIR rejoining using reaction mechanisms similar to those used by vertebrate RAGs. We propose that ProtoRAG represents a molecular "living fossil" of the long-sought RAG transposon. PMID:27293192

  8. Activation of Coagulation by Administration of Recombinant Factor VIIa Elicits Interleukin 6 (IL-6) and IL-8 Release in Healthy Human Subjects

    PubMed Central

    de Jonge, Evert; Friederich, Philip W.; Vlasuk, George P.; Rote, William E.; Vroom, Margaretha B.; Levi, Marcel; van der Poll, Tom

    2003-01-01

    The activation of coagulation has been shown to contribute to proinflammatory responses in animal and in vitro experiments. Here we report that the activation of coagulation in healthy human subjects by the administration of recombinant factor VIIa also elicits a small but significant increase in the concentrations of interleukin 6 (IL-6) and IL-8 in plasma. This increase was absent when the subjects were pretreated with recombinant nematode anticoagulant protein c2, the inhibitor of tissue factor-factor VIIa. PMID:12738659

  9. Producing Recombinant mTEX101; a Murine Testis Specific Protein

    PubMed Central

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

    2009-01-01

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

  10. 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. PMID:27076136

  11. RIG-I ligand enhances the immunogenicity of recombinant H7HA protein.

    PubMed

    Cao, Weiping; Liepkalns, Justine S; Kamal, Ram P; Reber, Adrian J; Kim, Jin Hyang; Hofstetter, Amelia R; Amoah, Samuel; Stevens, James; Ranjan, Priya; Gangappa, Shivaprakash; York, Ian A; Sambhara, Suryaprakash

    2016-01-01

    Avian H7N9 influenza virus infection with fatal outcomes continues to pose a pandemic threat and highly immunogenic vaccines are urgently needed. In this report we show that baculovirus-derived recombinant H7 hemagglutinin protein, when delivered with RIG-I ligand, induced enhanced antibody and T cell responses and conferred protection against lethal challenge with a homologous H7N9 virus. These findings indicate the potential utility of RIG-I ligands as vaccine adjuvants to increase the immunogenicity of recombinant H7 hemagglutinin. PMID:27106062

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

    PubMed

    Gottschamel, Johanna; Lössl, Andreas

    2016-01-01

    Plastid transformation for the expression of recombinant proteins and entire enzymatic pathways has become a promising tool for plant biotechnology in the past decade. Several improvements of the technology have turned plant plastids into robust and dependable expression platforms for multiple high value compounds. In this chapter, we describe our current methodology based on Gateway(®) recombinant cloning, which we have adapted for plastid transformation. We describe the steps required for cloning, biolistic transformation, identification, and regeneration of transplastomic plant lines and Western blot analysis. PMID:26614278

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

    PubMed

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

    2013-11-01

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

  14. Canine Antibodies against Salivary Recombinant Proteins of Phlebotomus perniciosus: A Longitudinal Study in an Endemic Focus of Canine Leishmaniasis

    PubMed Central

    Kostalova, Tatiana; Lestinova, Tereza; Sumova, Petra; Vlkova, Michaela; Rohousova, Iva; Berriatua, Eduardo; Oliva, Gaetano; Fiorentino, Eleonora; Scalone, Aldo; Gramiccia, Marina; Gradoni, Luigi; Volf, Petr

    2015-01-01

    Background Phlebotomine sand flies are vectors of Leishmania parasites. During blood feeding, sand flies deposit into the host skin immunogenic salivary proteins which elicit specific antibody responses. These anti-saliva antibodies enable an estimate of the host exposure to sand flies and, in leishmaniasis endemic areas, also the risk for Leishmania infections. However, the use of whole salivary gland homogenates as antigen has several limitations, and therefore, recombinant salivary proteins have been tested to replace them in antibody detection assays. In this study, we have used for the first time sand fly salivary recombinant proteins in a longitudinal field study on dogs. Methodology/Principal Findings Sera from dogs naturally exposed to P. perniciosus bites over two consecutive transmission seasons in a site endemic for canine leishmaniasis (CanL) were tested at different time points by ELISA for the antibodies recognizing whole saliva, single salivary 43 kDa yellow-related recombinant protein (rSP03B), and a combination of two salivary recombinant proteins, 43 kDa yellow-related protein and 35.5 kDa apyrase (rSP01). Dogs were also tested for Leishmania infantum positivity by serology, culture, and PCR and the infection status was evaluated prospectively. We found a significant association between active CanL infection and the amount of anti-P. perniciosus saliva antibodies. Importantly, we detected a high correlation between IgG antibodies recognizing rSP03B protein and the whole salivary antigen. The kinetics of antibody response showed for both a whole saliva and rSP03B a similar pattern that was clearly related to the seasonal abundance of P. perniciosus. Conclusions These results suggest that P. perniciosus rSP03B protein is a valid alternative to whole saliva and could be used in large-scale serological studies. This novel method could be a practical and economically-sound tool to detect the host exposure to sand fly bites in CanL endemic areas. PMID

  15. RAC-tagging: Recombineering And Cas9-assisted targeting for protein tagging and conditional analyses.

    PubMed

    Baker, Oliver; Gupta, Ashish; Obst, Mandy; Zhang, Youming; Anastassiadis, Konstantinos; Fu, Jun; Stewart, A Francis

    2016-01-01

    A fluent method for gene targeting to establish protein tagged and ligand inducible conditional loss-of-function alleles is described. We couple new recombineering applications for one-step cloning of gRNA oligonucleotides and rapid generation of short-arm (~1 kb) targeting constructs with the power of Cas9-assisted targeting to establish protein tagged alleles in embryonic stem cells at high efficiency. RAC (Recombineering And Cas9)-tagging with Venus, BirM, APEX2 and the auxin degron is facilitated by a recombineering-ready plasmid series that permits the reuse of gene-specific reagents to insert different tags. Here we focus on protein tagging with the auxin degron because it is a ligand-regulated loss-of-function strategy that is rapid and reversible. Furthermore it includes the additional challenge of biallelic targeting. Despite high frequencies of monoallelic RAC-targeting, we found that simultaneous biallelic targeting benefits from long-arm (>4 kb) targeting constructs. Consequently an updated recombineering pipeline for fluent generation of long arm targeting constructs is also presented. PMID:27216209

  16. RAC-tagging: Recombineering And Cas9-assisted targeting for protein tagging and conditional analyses

    PubMed Central

    Baker, Oliver; Gupta, Ashish; Obst, Mandy; Zhang, Youming; Anastassiadis, Konstantinos; Fu, Jun; Stewart, A. Francis

    2016-01-01

    A fluent method for gene targeting to establish protein tagged and ligand inducible conditional loss-of-function alleles is described. We couple new recombineering applications for one-step cloning of gRNA oligonucleotides and rapid generation of short-arm (~1 kb) targeting constructs with the power of Cas9-assisted targeting to establish protein tagged alleles in embryonic stem cells at high efficiency. RAC (Recombineering And Cas9)-tagging with Venus, BirM, APEX2 and the auxin degron is facilitated by a recombineering-ready plasmid series that permits the reuse of gene-specific reagents to insert different tags. Here we focus on protein tagging with the auxin degron because it is a ligand-regulated loss-of-function strategy that is rapid and reversible. Furthermore it includes the additional challenge of biallelic targeting. Despite high frequencies of monoallelic RAC-targeting, we found that simultaneous biallelic targeting benefits from long-arm (>4 kb) targeting constructs. Consequently an updated recombineering pipeline for fluent generation of long arm targeting constructs is also presented. PMID:27216209

  17. Gene A protein cleavage of recombinant plasmids containing the phi X174 replication origin.

    PubMed Central

    Fluit, A C; Baas, P D; Van Boom, J H; Veeneman, G H; Jansz, H S

    1984-01-01

    Synthetic oligonucleotides, DNA ligase and DNA polymerase were used to construct double-stranded DNA fragments homologous to the first 25, 27 or 30 b.p. of the origin of replication of bacteriophage phi X174 (nucleotides 4299-4328 of the phi X174 DNA sequence). The double-stranded DNA fragments were cloned into the unique SmaI or HindIII restriction sites in the kanamycin-resistance gene of pACYC177 (AmpR, KmR). Recombinant plasmids were picked up by colony hybridization. DNA sequencing showed that not only recombinant plasmids with the expected insert were formed, but also recombinant plasmids with a shorter insert. Recombinant plasmids with an insert homologous to the first 24, 25, 26, 27, 28 or all 30 b.p. of the phi X174 origin region were thus obtained. Supercoiled plasmids containing a sequence homologous to the first 27, 28 or 30 b.p. of the phi X174 origin region are nicked by the phi X174 gene A protein. However, the other supercoiled plasmids are not nicked by the phi X174 gene A protein. These results show that the first 27 b.p. of the phi X174 origin region are sufficient as well as required for the initiation step in phi X174 RF DNA replication, i.e. the cleavage by gene A protein. Images PMID:6236428

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

    PubMed Central

    Chapple, Susan DJ; Crofts, Anna M; Shadbolt, S Paul; McCafferty, John; Dyson, Michael R

    2006-01-01

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

  19. Tissue Plasminogen Activator Neurotoxicity is Neutralized by Recombinant ADAMTS 13

    PubMed Central

    Fan, Mengchen; Xu, Haochen; Wang, Lixiang; Luo, Haiyu; Zhu, Ximin; Cai, Ping; Wei, Lixiang; Lu, Lu; Cao, Yongliang; Ye, Rong; Fan, Wenying; Zhao, Bing-Qiao

    2016-01-01

    Tissue plasminogen activator (tPA) is an effective treatment for ischemic stroke, but its neurotoxicity is a significant problem. Here we tested the hypothesis that recombinant ADAMTS 13 (rADAMTS 13) would reduce tPA neurotoxicity in a mouse model of stroke. We show that treatment with rADAMTS 13 in combination with tPA significantly reduced infarct volume compared with mice treated with tPA alone 48 hours after stroke. The combination treatment significantly improved neurological deficits compared with mice treated with tPA or vehicle alone. These neuroprotective effects were associated with significant reductions in fibrin deposits in ischemic vessels and less severe cell death in ischemic brain. The effect of rADAMTS13 on tPA neurotoxicity was mimicked by the N-methyl-D-aspartate (NMDA) receptor antagonist M-801, and was abolished by injection of NMDA. Moreover, rADAMTS 13 prevents the neurotoxicity effect of tPA, by blocking its interaction with the NMDA receptor NR2B and the attendant phosphorylation of NR2B and activation of ERK1/2. Finally, the NR2B-specific NMDA receptor antagonist ifenprodil abolished tPA neurotoxicity and rADAMTS 13 treatment had no further beneficial effect. Our data suggest that the combination of rADAMTS 13 and tPA may provide a novel treatment of ischemic stroke by diminishing the neurotoxic effects of exogenous tPA. PMID:27181025

  20. Tissue Plasminogen Activator Neurotoxicity is Neutralized by Recombinant ADAMTS 13.

    PubMed

    Fan, Mengchen; Xu, Haochen; Wang, Lixiang; Luo, Haiyu; Zhu, Ximin; Cai, Ping; Wei, Lixiang; Lu, Lu; Cao, Yongliang; Ye, Rong; Fan, Wenying; Zhao, Bing-Qiao

    2016-01-01

    Tissue plasminogen activator (tPA) is an effective treatment for ischemic stroke, but its neurotoxicity is a significant problem. Here we tested the hypothesis that recombinant ADAMTS 13 (rADAMTS 13) would reduce tPA neurotoxicity in a mouse model of stroke. We show that treatment with rADAMTS 13 in combination with tPA significantly reduced infarct volume compared with mice treated with tPA alone 48 hours after stroke. The combination treatment significantly improved neurological deficits compared with mice treated with tPA or vehicle alone. These neuroprotective effects were associated with significant reductions in fibrin deposits in ischemic vessels and less severe cell death in ischemic brain. The effect of rADAMTS13 on tPA neurotoxicity was mimicked by the N-methyl-D-aspartate (NMDA) receptor antagonist M-801, and was abolished by injection of NMDA. Moreover, rADAMTS 13 prevents the neurotoxicity effect of tPA, by blocking its interaction with the NMDA receptor NR2B and the attendant phosphorylation of NR2B and activation of ERK1/2. Finally, the NR2B-specific NMDA receptor antagonist ifenprodil abolished tPA neurotoxicity and rADAMTS 13 treatment had no further beneficial effect. Our data suggest that the combination of rADAMTS 13 and tPA may provide a novel treatment of ischemic stroke by diminishing the neurotoxic effects of exogenous tPA. PMID:27181025

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

    PubMed

    Petit, Marie-Agnès; Ehrlich, Dusko

    2002-06-17

    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

  2. Tankyrases Promote Homologous Recombination and Check Point Activation in Response to DSBs.

    PubMed

    Nagy, Zita; Kalousi, Alkmini; Furst, Audrey; Koch, Marc; Fischer, Benoit; Soutoglou, Evi

    2016-02-01

    DNA lesions are sensed by a network of proteins that trigger the DNA damage response (DDR), a signaling cascade that acts to delay cell cycle progression and initiate DNA repair. The Mediator of DNA damage Checkpoint protein 1 (MDC1) is essential for spreading of the DDR signaling on chromatin surrounding Double Strand Breaks (DSBs) by acting as a scaffold for PI3K kinases and for ubiquitin ligases. MDC1 also plays a role both in Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR) repair pathways. Here we identify two novel binding partners of MDC1, the poly (ADP-ribose) Polymerases (PARPs) TNKS1 and 2. We find that TNKSs are recruited to DNA lesions by MDC1 and regulate DNA end resection and BRCA1A complex stabilization at lesions leading to efficient DSB repair by HR and proper checkpoint activation. PMID:26845027

  3. Tankyrases Promote Homologous Recombination and Check Point Activation in Response to DSBs

    PubMed Central

    Furst, Audrey; Koch, Marc; Fischer, Benoit; Soutoglou, Evi

    2016-01-01

    DNA lesions are sensed by a network of proteins that trigger the DNA damage response (DDR), a signaling cascade that acts to delay cell cycle progression and initiate DNA repair. The Mediator of DNA damage Checkpoint protein 1 (MDC1) is essential for spreading of the DDR signaling on chromatin surrounding Double Strand Breaks (DSBs) by acting as a scaffold for PI3K kinases and for ubiquitin ligases. MDC1 also plays a role both in Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR) repair pathways. Here we identify two novel binding partners of MDC1, the poly (ADP-ribose) Polymerases (PARPs) TNKS1 and 2. We find that TNKSs are recruited to DNA lesions by MDC1 and regulate DNA end resection and BRCA1A complex stabilization at lesions leading to efficient DSB repair by HR and proper checkpoint activation. PMID:26845027

  4. 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.

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

    SciTech Connect

    Kashyap, Sanjay; Woehl, Taylor; Valverde-Tercedor, Carmen; Sanchez-Quesada, Miguel; Lopez, Concepcion Jimenez; Prozorov, Tanya

    2014-03-07

    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 formation of biotemplated inorganic nanoparticles. The self-organization of MamC is compared to the behavior of another acidic recombinant iron-binding protein, Mms6.

  6. Autonomous induction of recombinant proteins by minimally rewiring native quorum sensing regulon of E. coli.

    PubMed

    Tsao, Chen-Yu; Hooshangi, Sara; Wu, Hsuan-Chen; Valdes, James J; Bentley, William E

    2010-05-01

    Quorum sensing (QS) enables an individual bacterium's metabolic state to be communicated to and ultimately control the phenotype of an emerging population. Harnessing the hierarchical nature of this signal transduction process may enable the exploitation of individual cell characteristics to direct or "program" entire populations of cells. We re-engineered the native QS regulon so that individual cell signals (autoinducers) are used to guide high level expression of recombinant proteins in E. coli populations. Specifically, the autoinducer-2 (AI-2) QS signal initiates and guides the overexpression of green fluorescent protein (GFP), chloramphenicol acetyl transferase (CAT) and beta-galactosidase (LacZ). The new process requires no supervision or input (e.g., sampling for optical density measurement, inducer addition, or medium exchange) and represents a low-cost, high-yield platform for recombinant protein production. Moreover, rewiring a native signal transduction circuit exemplifies an emerging class of metabolic engineering approaches that target regulatory functions. PMID:20060924

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

    DOE PAGESBeta

    Kashyap, Sanjay; Woehl, Taylor; Valverde-Tercedor, Carmen; Sánchez-Quesada, Miguel; Jiménez López, Concepción; Prozorov, Tanya

    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

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

    PubMed

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

    2014-05-01

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

  9. Identification of Essential Genetic Baculoviral Elements for Recombinant Protein Expression by Transactivation in Sf21 Insect Cells

    PubMed Central

    Chen, Fang-Fang; Yen, Zen-Zen; Lindemann, Nils; Meyer, Steffen; Spehr, Johannes; van den Heuvel, Joop

    2016-01-01

    The Baculovirus Expression Vector System (BEVS) is widely used to produce high amounts of recombinant proteins. Nevertheless, generating recombinant baculovirus in high quality is rather time-consuming and labor-intensive. Alternatively, virus-free expression in insect cells did not achieve similar expression levels for most proteins so far. The transactivation method is a promising approach for protein expression in Sf21 cells. It combines advantages of BEVS and plasmid-based expression by activating strong virus-dependent promoters on a transfected plasmid by baculoviral coinfection. Here, we identified expression elements required for transactivation. Therefore, we designed several vectors comprising different viral promoters or promoter combinations and tested them for eGFP expression using the automated BioLector microcultivation system. Remarkably, only the combination of the very late promoter p10 together with the homologous region 5 (hr5) could boost expression during transactivation. Other elements, like p10 alone or the late viral promoter polH, did not respond to transactivation. A new combination of hr5 and p10 with the strongest immediate early OpMNPV viral promoter OpIE2 improved the yield of eGFP by ~25% in comparison to the previous applied hr5-IE1-p10 expression cassette. Furthermore, we observed a strong influence of the transcription termination sequence and vector backbone on the level of expression. Finally, the expression levels for transactivation, BEVS and solely plasmid-based expression were compared for the marker protein eGFP, underlining the potential of transactivation for fast recombinant protein expression in Sf21 cells. In conclusion, essential elements for transactivation could be identified. The optimal elements were applied to generate an improved vector applicable in virus-free plasmid-based expression, transactivation and BEVS. PMID:26934632

  10. 76 FR 3150 - Office of Biotechnology Activities; Recombinant DNA Research: Action Under the NIH Guidelines for...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-01-19

    ...). On July 20, 2010 the NIH Office of Biotechnology Activities (OBA) published a proposed action (75 FR... HUMAN SERVICES National Institutes of Health Office of Biotechnology Activities; Recombinant DNA Research: Action Under the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH...

  11. 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

  12. The recombinant LIC10508 is a plasma fibronectin, plasminogen, fibrinogen and C4BP-binding protein of Leptospira interrogans.

    PubMed

    Siqueira, Gabriela H; Teixeira, Aline F; Fernandes, Luis G; de Souza, Gisele O; Kirchgatter, Karin; Romero, Eliete C; Vasconcellos, Silvio A; Vieira, Monica L; Nascimento, Ana Lucia T O

    2016-03-01

    Leptospirosis is a zoonosis caused by pathogenic Leptospira spp. In this study, we report that the recombinant proteins LIC10507, LIC10508 and LIC10509 are recognized by confirmed leptospirosis serum samples at both phases of the disease. The recombinant rLIC10508 and rLIC10507 are plasminogen (PLG)-binding proteins, capable of generating plasmin in the presence of a PLG activator. The proteins bind to PLG in a dose-dependent and saturable manner, fulfilling host-ligand interaction. Furthermore, rLIC10508 interacts with fibrinogen (Fg), plasma fibronectin and C4b binding protein (C4BP). The binding of rLIC10508 to Fg decreases the fibrin clotting in a thrombin-catalyzed reaction. The incubation with 4 μM of protein promoted 40% inhibition upon clotting formation. C4BP bound to rLIC10508 retained its cofactor activity for factor I promoting the cleavage of C4b protein, which may reduce the membrane attack complex formation. Although these proteins have high amino acid sequence similarity, rLIC10508 is the most talented of the three, a behavior that might be explained by its unique putative 3D structure, whereas structures of rLIC10507 and rLIC10509 are very similar. Plasmin generation (rLIC10507 and rLIC10508), together with decreasing fibrin clot formation (rLIC10508) and impairment of the complement system (rLIC10508) may help the bacteria to overcome host defense, facilitating the infection process. PMID:26657108

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

    PubMed

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

    2016-08-01

    Napin and napin-like proteins belong to the 2S albumin seed storage family of proteins and have been shown to display a variety of biological activities. However, due to a high degree of polymorphism, purification of a single napin or napin-like protein exhibiting biological activity is extremely difficult. In the present study, we have produced the napin-like protein of Momordica charantia using the methylotrophic Pichia pastoris expression system. The recombinant napin-like protein (rMcnapin) secreted in the extracellular culture supernatant was enriched by ammonium sulfate precipitation, and purified using size exclusion chromatography at a yield of ∼290 mg/L of culture. Secondary structure analysis of the purified rMcnapin revealed it to be predominantly α-helical with minimal β strand content. CD spectroscopic and fluorescence spectroscopic analyses revealed the rMcnapin to be stable at a wide range of temperatures and pH. The rMcnapin exhibited antifungal activity against Trichoderma viride with an IC50 of ∼3.7 μg/ml and trypsin inhibitor activity with an IC50 of 4.2 μM. Thus, large amounts of homogenous preparations of the biologically active rMcnapin could be obtained at shake flask level, which is otherwise difficult from its natural source. PMID:27020281

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

    PubMed

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

    2016-01-01

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

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

    PubMed

    Nevalainen, Helena; Peterson, Robyn

    2014-01-01

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

  16. 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. PMID:26116677

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

    PubMed Central

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

    2015-01-01

    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. PMID:26116677

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

    PubMed

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

    2008-06-01

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

  19. Substrate oscillations boost recombinant protein release from Escherichia coli.

    PubMed

    Jazini, Mohammadhadi; Herwig, Christoph

    2014-05-01

    Intracellular production of recombinant proteins in prokaryotes necessitates subsequent disruption of cells for protein recovery. Since the cell disruption and subsequent purification steps largely contribute to the total production cost, scalable tools for protein release into the extracellular space is of utmost importance. Although there are several ways for enhancing protein release, changing culture conditions is rather a simple and scalable approach compared to, for example, molecular cell design. This contribution aimed at quantitatively studying process technological means to boost protein release of a periplasmatic recombinant protein (alkaline phosphatase) from E. coli. Quantitative analysis of protein in independent bioreactor runs could demonstrate that a defined oscillatory feeding profile was found to improve protein release, about 60 %, compared to the conventional constant feeding rate. The process technology included an oscillatory post-induction feed profile with the frequency of 4 min. The feed rate was oscillated triangularly between a maximum (1.3-fold of the maximum feed rate achieved at the end of the fed-batch phase) and a minimum (45 % of the maximum). The significant improvement indicates the potential to maximize the production rate, while this oscillatory feed profile can be easily scaled to industrial processes. Moreover, quantitative analysis of the primary metabolism revealed that the carbon dioxide yield can be used to identify the preferred feeding profile. This approach is therefore in line with the initiative of process analytical technology for science-based process understanding in process development and process control strategies. PMID:24114459

  20. Seroprevalence and specificity of human responses to the Plasmodium falciparum rhoptry protein Rhop-3 determined by using a C-terminal recombinant protein.

    PubMed Central

    Yang, J C; Blanton, R E; King, C L; Fujioka, H; Aikawa, M; Sam-Yellowe, T Y

    1996-01-01

    Rhoptry proteins participate in invasion of erythrocytes by malaria parasites. Antibodies to some of these proteins can inhibit invasion and partially protect monkeys from disease. To examine human serological responses to the 110-kDa component (Rhop-3) of the high-molecular-weight rhoptry protein complex, two cDNA clones corresponding to Rhop-3 were identified by immunologic screening. A recombinant protein representing the C-terminal one-third of the Rhop-3 was used to assess the seroprevalence to this protein in geographically isolated populations with different patterns of malaria transmission. The immunoglobulin G (IgG) positivity rate for the recombinant Rhop-3 in an enzyme-linked immunosorbent assay was 30% in an area of Papua New Guinea where malaria is holoendemic. In Kenya, the prevalence rates were 43 and 36%, respectively, in an area of hyperendemicity and an area of seasonal transmission. By contrast, rates of IgG seroprevalence to an extract of Gambian strain of Plasmodium falciparum were 48, 90, and 97% respectively, in these populations. In these areas, the pattern of antibody recognition of Rhop-3 is more similar (1.7-fold maximum difference) than the parasite extract (5-fold difference). The difference in seroresponses may represent antigenic polymorphism in different parasite strains, while their similarity for the Rhop-3 fragment may represent conservation of this protein. Recombinant- and parasite extract-specific IgG was not found in individuals infected only with Plasmodium vivax. Cross-reactivity was seen in the IgM assay. In Mombasa (Kenya), maternal and cord Rhop-3-specific IgG activities were similar. Fetal antigen-specific IgM reactivity was generally undetectable for all antigens. PMID:8751903

  1. Immune responses to a recombinant Rv0057-Rv1352 fusion protein of Mycobacterium tuberculosis.

    PubMed

    Yang, Yourong; Feng, Jindong; Zhang, Junxian; Zhao, Weiguo; Liu, Yu; Liang, Yan; Bai, Xuejuan; Wang, Lan; Wu, Xueqiong

    2015-01-01

    The identification and characterization of antigens of Mycobacterium tuberculosis help in understanding the mechanisms of protective immunity and in improving diagnostic methods for TB. Rv0057 and Rv1352 proteins are new T-cell antigens, found to play roles in TB infection. In this study, a recombinant fusion protein Rv0057-Rv1352 was made and analyzed for its immunological characteristics and potential utility. It showed good immunoreactivity with serum from TB patients by western blotting. The antibody levels against Rv0057-Rv1352 were significantly higher in sera from 69 TB patients than in sera from 60 patients with non-TB respiratory diseases (P<0.001). The sensitivities of a diagnostic ELISA test based on detecting Rv0057-Rv1352 antibody (60.3%) or 38 kDa-16 kDa antibody (58.8%) were comparable to commercial rapid test B (75.4%), and significantly higher (p<0.001) than bacteriological methods (15.9%), rapid test A (20.3%), or rapid test C (29.0%). The specificities of Rv0057-Rv1352 (93.3%) or 38 kDa-16 kDa antibody tests (93.3%) were equivalent to the rapid tests A (93.3%) and C (86.7%), and significantly higher than rapid test B (63.3%, p<0.001). When 38 kDa-16 kDa was used together with Rv0057-Rv1352, the test sensitivity reached 85.5%, and its specificity remained high (86.7%). The test was as sensitive with bacterium-positive TB patients as with bacterium-negative. In an ELISPOT assay for cellular immunity, Rv0057-Rv1352 stimulated T lymphocytes to produce fewer spots secreting IFN-γ than CFP10-ESAT6 fusion protein did (p>0.05). These results suggest that Rv0057-Rv1352 has potential for the serodiagnosis of active pulmonary TB. PMID:25696009

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

    PubMed Central

    Finkenwirth, Friedrich; Kirsch, Franziska

    2013-01-01

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

  3. Separating proteins with activated carbon.

    PubMed

    Stone, Matthew T; Kozlov, Mikhail

    2014-07-15

    Activated carbon is applied to separate proteins based on differences in their size and effective charge. Three guidelines are suggested for the efficient separation of proteins with activated carbon. (1) Activated carbon can be used to efficiently remove smaller proteinaceous impurities from larger proteins. (2) Smaller proteinaceous impurities are most efficiently removed at a solution pH close to the impurity's isoelectric point, where they have a minimal effective charge. (3) The most efficient recovery of a small protein from activated carbon occurs at a solution pH further away from the protein's isoelectric point, where it is strongly charged. Studies measuring the binding capacities of individual polymers and proteins were used to develop these three guidelines, and they were then applied to the separation of several different protein mixtures. The ability of activated carbon to separate proteins was demonstrated to be broadly applicable with three different types of activated carbon by both static treatment and by flowing through a packed column of activated carbon. PMID:24898563

  4. 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

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

    PubMed

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

    2015-12-01

    According to the protein-only hypothesis, infectious mammalian prions, which exist as distinct strains with discrete biological properties, consist of multichain assemblies of misfolded cellular prion protein (PrP). A critical test would be to produce prion strains synthetically from defined components. Crucially, high-titre 'synthetic' prions could then be used to determine the structural basis of infectivity and strain diversity at the atomic level. While there have been multiple reports of production of prions from bacterially expressed recombinant PrP using various methods, systematic production of high-titre material in a form suitable for structural analysis remains a key goal. Here, we report a novel high-throughput strategy for exploring a matrix of conditions, additives and potential cofactors that might generate high-titre prions from recombinant mouse PrP, with screening for infectivity using a sensitive automated cell-based bioassay. Overall, approximately 20,000 unique conditions were examined. While some resulted in apparently infected cell cultures, this was transient and not reproducible. We also adapted published methods that reported production of synthetic prions from recombinant hamster PrP, but again did not find evidence of significant infectious titre when using recombinant mouse PrP as substrate. Collectively, our findings are consistent with the formation of prion infectivity from recombinant mouse PrP being a rare stochastic event and we conclude that systematic generation of prions from recombinant PrP may only become possible once the detailed structure of authentic ex vivo prions is solved. PMID:26631378

  6. Protein extraction from activated sludge.

    PubMed

    Denecke, M

    2006-01-01

    Two methods for the separation of protein originating from activated sludge were compared. In one method, the total protein was isolated out of the activated sludge (crude extract). These samples included all dissolved proteins originating from the bacterial cells and biofilm made up of extracellular polymeric substances (EPS). Every time polyacrylamide gel electrophoresis (PAGE) was done, the protein bands from samples of crude extract were covered by polymeric substances including carbohydrates, uronic acids or humic compounds. Using the immunoblot technique it was possible to demonstrate the presence of the heat shock protein HSP70 in crude extracts of activated sludge. The comparison of protein fingerprints required that clear and distinct bands appear on the PAGE analysis. To this end, a procedure to separates bacterial cells from the EPS was developed. Bacterial cells were separated by incubation with EDTA and subsequent filtration. The isolated cells were directly incubated in a sample buffer. PMID:16898150

  7. [Construction of recombinant adenoviral vector expressing genes of the conservative influenza proteins M2 and nucleoprotein].

    PubMed

    Esmagambetov, I B; Sedova, E S; Shcherbinin, D N; Lysenko, A A; Garas, M N; Shmarov, M M; Logunov, D Iu

    2014-01-01

    Influenza is a highly contagious and one of the most massive infection diseases. General epidemiological significance has a strain, which belongs to subtype A. A high degree of genetic variety leads to the permanent changes in the antigenic structure of the influenza virus. Therefore, the current influenza vaccines require periodic updating of the composition of strains. Presently, it is important to develop a universal vaccine that can protect against different strains of influenza A virus at the same time and is based on the conserved antigens of the influenza virus. The recombinant adenovirus vectors expressing genes of conserved viral antigenes may be a promising candidate vaccine against influenza A. Using the method of the homologous recombination, we developed in this study recombinant adenovirus of fifth serotype that expresses genes of the ion channel M2 and nucleoprotein NP of the influenza virus A. Genes of the consensus protein M2 and NP of human influenza A virus were included into the structure of the viral genome. The expression of the antigens M2 and NP using recombinant adenovirus vector was detected by a Western blot assay. The immunogenicity of the developed recombinant adenovirus vector was demonstrated by the intranasal immunization of laboratory mice. PMID:25080815

  8. Antigenicity of Recombinant Maltose Binding Protein-Mycobacterium avium subsp. paratuberculosis Fusion Proteins with and without Factor Xa Cleaving

    PubMed Central

    Begg, Douglas J.; Purdie, Auriol C.; Bannantine, John P.; Whittington, Richard J.

    2013-01-01

    Mycobacterium avium subsp. paratuberculosis causes Johne's disease (JD) in ruminants. Proteomic studies have shown that M. avium subsp. paratuberculosis expresses certain proteins when exposed to in vitro physiological stress conditions similar to the conditions experienced within a host during natural infection. Such proteins are hypothesized to be expressed in vivo, are recognized by the host immune system, and may be of potential use in the diagnosis of JD. In this study, 50 recombinant maltose binding protein (MBP)-M. avium subsp. paratuberculosis fusion proteins were evaluated using serum samples from sheep infected with M. avium subsp. paratuberculosis, and 29 (58%) were found to be antigenic. Among 50 fusion proteins, 10 were evaluated in MBP fusion and factor Xa-cleaved forms. A total of 31 proteins (62%) were found to be antigenic in either MBP fusion or factor Xa-cleaved forms. Antigenicity after cleavage and removal of the MBP tag was marginally enhanced. PMID:24132604

  9. A Butter Aroma Recombinate Activates Human Class-I Odorant Receptors.

    PubMed

    Geithe, Christiane; Andersen, Gaby; Malki, Agne; Krautwurst, Dietmar

    2015-11-01

    With ∼400 olfactory G protein-coupled receptors (GPCR), humans sensitively perceive ∼230 key aroma compounds as best natural agonists of ∼10000 food volatiles. An understanding of odorant coding, thus, critically depends on the knowledge about interactions of key food aroma chemicals and their mixtures with their cognate receptors. Genetically designed test cell systems enable the screening, deorphaning, and characterization of single odorant receptors (OR). This study shows for the food aroma-specific and quantitative butter aroma recombinate, and its single components, specific in vitro class-I OR activity patterns, as well as the activation of selected OR in a concentration-dependent manner. Recently, chemosensory receptors, especially class-I OR, were demonstrated to be expressed on blood leukocytes, which may encounter foodborne aroma compounds postprandially. This study shows that butter aroma recombinate induced chemotaxis of isolated human neutrophils in a defined gradient, and in a concentration-dependent and pertussis toxin-sensitive manner, suggesting at least a GPCR-mediated activation of blood leukocytes by key food odorants. PMID:26451762

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

    PubMed Central

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

    2013-01-01

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

  11. Recombinant activated factor VII in post partum haemorrhage

    PubMed Central

    Magon, Navneet; Babu, K. M.; Kapur, Krishan; Chopra, Sanjiv; Joneja, Gurdarshan Singh

    2013-01-01

    Post-partum haemorrhage (PPH) is a life-threatening obstetric complication and the leading cause of maternal death. Any bleeding that results in or could result in haemodynamic instability, if untreated, must be considered as PPH. There is no controversy about the need for prevention and treatment of PPH. The keystone of management of PPH entails first, non-invasive and nonsurgical methods and then invasive and surgical methods. However, mortality remains high. Therefore, new advancements in the treatment are most crucial. One such advancement has been the use of recombinant activated factor VII (rFVIIa) in PPH. First used 12 years back in PPH, this universal haemostatic agent has been effectively used in controlling PPH. The best available indicator of rFVIIa efficacy is the arrest of haemorrhage, which is judged by visual evidence and haemodynamic stabilization. It also reduces costs of therapy and the use of blood components in massive PPH. In cases of intractable PPH with no other obvious indications for hysterectomy, administration of rFVIIa should be considered before surgery. We share our experience in a series of cases of PPH, successfully managed using rFVIIa. PMID:24403703

  12. 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

  13. 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

  14. 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.

  15. Active, soluble recombinant melittin purified by extracting insoluble lysate of Escherichia coli without denaturation

    PubMed Central

    Buhrman, Jason S.; Cook, Laura C.; Rayahin, Jamie E.; Federle, Michael J.; Gemeinhart, Richard A.

    2013-01-01

    Cell lytic peptides are a class of drugs that can be used to selectively kill invading organisms or diseased cells. Several of these peptides have been identified as potential therapeutics. Herein, we report a novel process for purifying recombinant melittin, a cell lytic peptide that inserts into the membranes of cells causing cell lysis, from Escherichia coli. The process involves surfactant and low pH to solubilize melittin fusion proteins from the insoluble fraction of bacterial lysates. We are able to significantly improve purity of the final product and confirm the activity of the peptide. The process yields recombinant melittin that is effective when used to treat U-87 MG glioma cells and inhibits growth of the Gram-positive pathogenic bacterium Streptococcus pyogenes. We demonstrate a method of repeated extraction of the insoluble protein fraction with mild detergent at a low pH that is able to generate a yield of pure, soluble melittin of approximately 0.5 to 1 mg/L of E. coli culture. PMID:23926061

  16. 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

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

    PubMed

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

    2014-09-01

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

  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. PMID:26742322

  19. Comparison of ADH3 promoter with commonly used promoters for recombinant protein production in Pichia pastoris.

    PubMed

    Karaoglan, Mert; Karaoglan, Fidan Erden; Inan, Mehmet

    2016-05-01

    Recombinant protein production under the control of the PADH3 was compared with Pichia pastoris PAOX1 and PGAP. The single-copy-clones expressing Aspergillus niger xylanase (XylB) gene with the three different promoters were tested in shake flask and 5 L fed-batch fermentation processes. Recombinant protein production with PADH3, PAOX1 and PGAP were initiated by addition of ethanol, methanol and glucose, respectively in the culture medium. The fermentation process was carried out for 72 h at 30 °C, pH 5 and 30% dissolved oxygen. Extracellular protein production yield for PADH3 (3725 U/mL) was higher than for PAOX1 (2095 U/mL) and PGAP (580 U/mL) at fermentor scale under the conditions tested. These results show that the PADH3 promoter is a promising tool for large scale production of recombinant proteins and can be an alternative to the PAOX1 and PGAP. PMID:26835836

  20. Immunotherapeutic potential of recombinant ESAT-6 protein in mouse model of experimental tuberculosis.

    PubMed

    Mir, Shabir Ahmad; Verma, Indu; Sharma, Sadhna

    2014-01-01

    Recent understanding of the pathogenesis of tuberculosis allows the possible application of immunotherapy for the treatment of tuberculosis. Therapies that would upregulate the host antimycobacterial immune response and/or attenuate T-cell suppressive and macrophage-deactivating cytokines may prove to be useful in the treatment of tuberculosis. ESAT6, 6-kDa early secreted antigenic target, is a potent protective antigen and is considered as major target for long-lived memory cells. In the present study the immunotherapeutic potential of ESAT-6 has been evaluated in mouse model of experimental tuberculosis. In the present study the ESAT-6 protein was cloned in Escherichia coli using pET23a(+) plasmid and purified by Ni(2+)-NTA chromatography. Further, the immunotherapeutic potential of the recombinant ESAT-6 (in terms of CFU enumeration in the target organs and histopathological analysis of lungs) was evaluated against experimental tuberculosis. The recombinant ESAT-6 with C-terminal histidine-tag and free N-terminus mimics the natural form of ESAT-6 has been successfully cloned and purified. The recombinant ESAT-6 protein adjuvanted with dimethyl dioctadecylammonium bromide (DDA) moderately reduced the bacterial load in the target organs of infected mice. Further, the formulation (ESAT-6-DDA) was able to act synergistically when given in combination with antituberculosis drugs. This recombinant ESAT-6 showed good immunotherapeutic potential against experimental tuberculosis and can be used as an adjunct to the conventional antituberculosis chemotherapy. PMID:24345702

  1. 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. PMID:26965414

  2. 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

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

    PubMed

    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

  4. Saccharomyces forkhead protein Fkh1 regulates donor preference during mating-type switching through the recombination enhancer

    PubMed Central

    Sun, Kaiming; Coïc, Eric; Zhou, Zhiqi; Durrens, Pascal; Haber, James E.

    2002-01-01

    Saccharomyces mating-type switching results from replacement by gene conversion of the MAT locus with sequences copied from one of two unexpressed donor loci, HML or HMR. MATa cells recombine with HMLα ∼90% of the time, whereas MATα cells choose HMRa 80%–90% of the time. HML preference in MATa is controlled by the cis-acting recombination enhancer (RE) that regulates recombination along the entire left arm of chromosome III. Comparison of RE sequences between S. cerevisiae, S. carlsbergensis, and S. bayanus defines four highly conserved regions (A, B, C, and D) within a 270-bp minimum RE. An adjacent E region enhances RE activity. Multimers of region A, D, or E are sufficient to promote selective use of HML. Regions A, D, and E each bind in vivo the transcription activator forkhead proteins Fkh1p and Fkh2p and their associated Ndd1p, although there are no adjacent open reading frames (ORFs). Deletion of FKH1 significantly reduces MATa's use of HML, as does mutation of the Fkh1/Fkh2-binding sites in a multimer of region A. We conclude that Fkh1p regulates MATa donor preference through direct interaction with RE. PMID:12183363

  5. Saccharomyces forkhead protein Fkh1 regulates donor preference during mating-type switching through the recombination enhancer.

    PubMed

    Sun, Kaiming; Coïc, Eric; Zhou, Zhiqi; Durrens, Pascal; Haber, James E

    2002-08-15

    Saccharomyces mating-type switching results from replacement by gene conversion of the MAT locus with sequences copied from one of two unexpressed donor loci, HML or HMR. MATa cells recombine with HMLalpha approximately 90% of the time, whereas MATalpha cells choose HMRa 80%-90% of the time. HML preference in MATa is controlled by the cis-acting recombination enhancer (RE) that regulates recombination along the entire left arm of chromosome III. Comparison of RE sequences between S. cerevisiae, S. carlsbergensis, and S. bayanus defines four highly conserved regions (A, B, C, and D) within a 270-bp minimum RE. An adjacent E region enhances RE activity. Multimers of region A, D, or E are sufficient to promote selective use of HML. Regions A, D, and E each bind in vivo the transcription activator forkhead proteins Fkh1p and Fkh2p and their associated Ndd1p, although there are no adjacent open reading frames (ORFs). Deletion of FKH1 significantly reduces MATa's use of HML, as does mutation of the Fkh1/Fkh2-binding sites in a multimer of region A. We conclude that Fkh1p regulates MATa donor preference through direct interaction with RE. PMID:12183363

  6. Construction of a Synthetically Engineered nirB Promoter for Expression of Recombinant Protein in Escherichia coli

    PubMed Central

    Nasr, Reza; Akbari Eidgahi, Mohammad Reza

    2014-01-01

    Background: Anaerobic-inducible promoters are alternatives of chemical-inducible promoters for expression of recombinant proteins especially in conditions where chemical induction is not possible or anaerobic conditions are preferable. The nirB promoter is the promoter of the first gene of nir operon in Escherichia coli, which encodes NADH-dependent nitrite reductase. This promoter is naturally induced under anaerobic conditions and upregulated by nitrite and nitrate. Objectives: The current study was carried out to construct a synthetic nirB promoter that does not respond to chemical inducers (nitrite or nitrate), but instead responds to anaerobic induction. For this purpose, a new plasmid was constructed (pFSnirB78-23LTB), which contains a synthetic nirB promoter. The activity of this plasmid was evaluated in E. coli under both aerobic and anaerobic conditions and in response to chemical inducers, nitrite and nitrate. Materials and Methods: A synthetic nirB promoter was firstly cloned into a pKK223 derivative plasmid and then the heat labile toxin B subunit gene (LTB) of entrotoxigenic E. coli was cloned under the control of this promoter. The inducibility of this plasmid in E. coli was measured under anaerobic conditions in the presence or absence of nitrite or nitrate by ganglioside GM1 ELISA. Results: Our data showed that this promoter is strongly induced under anaerobic conditions while it showed much lower activity (11%) under aerobic conditions. In contrast to the native promoter, this promoter was not induced by chemical inducers, nitrite or nitrate. Conclusions: This study showed that the recombinant protein produced under the control of synthetic nirB promoter has critical characteristics such as pentamer formation, receptor recognition ability and conservation of antigenic epitopes. In addition, the data showed anaerobiosis and chemical inducers had no adverse effects on recombinant proteins. Based on the results, this synthetic promoter is suitable for

  7. Myelostimulatory activity of recombinant human interleukin-2 in mice

    SciTech Connect

    Talmadge, J.E.; Schneider, M.; Keller, J.; Ruscetti, F.; Longo, D.; Pennington, R.; Bowersox, O.; Tribble, H.

    1989-05-01

    In a series of studies designed to extend our understanding of interleukin-2 (IL-2) and to study the effect of biologic response modifiers on bone marrow, we observed that administering recombinant human (rH) IL-2 to normal mice resulted in an increase in the frequency of colony-forming units-culture (CFU-C) in bone marrow. In addition, rH IL-2 was able to accelerate host recovery from cyclophosphamide (CTX)- or radiation-induced bone marrow depression and peripheral blood leukopenia. Not only can rH IL-2 accelerate, in a dose-dependent manner, the return of bone marrow, peripheral blood cellularity, and CFU-C frequency to normal levels following cytoreduction by CTX or irradiation, but it also significantly increases CFU-C frequency to greater than normal levels. Furthermore, rH IL-2 can significantly prolong survival of animals receiving a lethal dose of irradiation or CTX. Thus, multiple mechanisms are responsible for the synergistic therapeutic activity associated with rH IL-2 and CTX. rH IL-2 does not act only as an immunomodulatory agent in the presence or absence of suppressor T cells, but also accelerates host recovery from cytoreductive agents, resulting in decreased leukopenia and perhaps resistances to secondary infection. Thus, rH IL-2 plus chemotherapy may increase therapeutic activity against neoplastic disease, not only by adding immune stimulation to the direct antitumor effect of the drug but also by allowing delivery of higher, more effective doses of chemotherapy.

  8. Recombinant Bactericidal/Permeability-Increasing Protein rBPI21 Protects against Pneumococcal Disease▿

    PubMed Central

    Srivastava, Amit; Casey, Heather; Johnson, Nathaniel; Levy, Ofer; Malley, Richard

    2007-01-01

    Bactericidal/permeability-increasing (BPI) protein has been shown to play an important role in innate immunity to gram-negative bacteria, by direct microbicidal as well as endotoxin-neutralizing action. Here we examined potential interactions between a recombinant 21-kDa bioactive fragment of BPI, rBPI21, and the gram-positive pathogen Streptococcus pneumoniae. rBPI21 bound to pneumococci and pneumolysin (Ply) in a direct and specific fashion. We observed an enhanced inflammatory response in mouse macrophages when rBPI21 was combined with killed pneumococci or supernatant from overnight growth of pneumococci. In addition, rBPI21 augmented the proapoptotic activity of Ply+ (but not Ply−) pneumococci in TLR4-defective murine macrophages (known to be defective also in their apoptotic response to pneumolysin) in a tumor necrosis factor alpha-dependent manner. rBPI21 also enhanced the association of pneumococci with murine macrophages. In a model of invasive pneumococcal disease in TLR4-defective mice, the intranasal administration of rBPI21 following intranasal inoculation of Ply+ pneumococci both enhanced upper respiratory tract cell apoptosis and prolonged survival. We have thus discovered a novel interaction between pneumococcus and rBPI21, a potent antimicrobial peptide previously considered to target only gram-negative bacteria. PMID:17101667

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

    PubMed Central

    Randall, Richard E.; Elliott, Richard M.

    2015-01-01

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

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

    PubMed Central

    Bonander, Nicklas; Darby, Richard AJ; Grgic, Ljuban; Bora, Nagamani; Wen, Jikai; Brogna, Saverio; Poyner, David R; O'Neill, Michael AA; Bill, Roslyn M

    2009-01-01

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

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

    PubMed Central

    Crabb, J. W.; Carlson, A.; Chen, Y.; Goldflam, S.; Intres, R.; West, K. A.; Hulmes, J. D.; Kapron, J. T.; Luck, L. A.; Horwitz, J.; Bok, D.

    1998-01-01

    Cellular retinaldehyde-binding protein (CRALBP) is abundant in the retinal pigment epithelium (RPE) and Müller cells of the retina where it is thought to function in retinoid metabolism and visual pigment regeneration. The protein carries 11-cis-retinal and/or 11-cis-retinol as endogenous ligands in the RPE and retina and mutations in human CRALBP that destroy retinoid binding functionality have been linked to autosomal recessive retinitis pigmentosa. CRALBP is also present in brain without endogenous retinoids, suggesting other ligands and physiological roles exist for the protein. Human recombinant cellular retinaldehyde-binding protein (rCRALBP) has been over expressed as non-fusion and fusion proteins in Escherichia coli from pET3a and pET19b vectors, respectively. The recombinant proteins typically constitute 15-20% of the soluble bacterial lysate protein and after purification, yield about 3-8 mg per liter of bacterial culture. Liquid chromatography electrospray mass spectrometry, amino acid analysis, and Edman degradation were used to demonstrate that rCRALBP exhibits the correct primary structure and mass. Circular dichroism, retinoid HPLC, UV-visible absorption spectroscopy, and solution state 19F-NMR were used to characterize the secondary structure and retinoid binding properties of rCRALBP. Human rCRALBP appears virtually identical to bovine retinal CRALBP in terms of secondary structure, thermal stability, and stereoselective retinoid-binding properties. Ligand-dependent conformational changes appear to influence a newly detected difference in the bathochromic shift exhibited by bovine and human CRALBP when complexed with 9-cis-retinal. These recombinant preparations provide valid models for human CRALBP structure-function studies. PMID:9541407

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

    NASA Astrophysics Data System (ADS)

    Wang, Xiaoping; Lin, Huanping; Wang, Qiaoxia

    Purposes: To construct a recombinant vaccine alpha-fetoprotein (AFP)-heat shock protein (HSP70) complex, and study its ability to induce specific CTL response and its protective effect against AFP-producing tumor. Material/Methods: A recombinant vaccine was constructed by conjugating mouse alpha-fetoprotein to heat shock protein 70. By way of intracutaneous injection, mice were primed and boosted with recombinant vaccine mAFP/HSP70, whereas single mAFP or HSP70 injection as controls. The ELISPOT and ELISA were used to measure the frequency of cells producing the cytokine IFN-γ in splenocytes and the level of anti-AFP antibody of serum from immunized mice respectively. In vivo tumor challenge were carried out to assess the immune effect of the recombinant vaccine. Results: By recombinant mAFP/HSP70 vaccine immunization, the results of ELISPOT and ELISA showed that the number of splenic cells producing IFN-γ and the level of anti-AFP antibody of serum were significantly higher in mAFP/HSP70 group than those in mAFP and HSP70 groups (108.50±11.70 IFN-γ spots/106 cells vs 41.60±10.40 IFN-γ spots/106 cells, 7.32±3.14 IFN-γ spots/106 cells, P<0.01; 156.32±10.42 μg/mL vs 66.52±7.35 μg/mL, 5.73±2.89 μg/mL, P<0.01). The tumor volume in mAFP/HSP70 group was significantly smaller than that in mAFP and HSP70 groups (42.44±7.14 mm3 vs 392.23±12.46 mm3, 838.63±13.84 mm3, P<0.01). Conclusions: The study further confirmed the function of heat shock protein 70's immune adjuvant. Sequential immunization with recombinant mAFP/HSP70 vaccine could generate effective antitumor immunity on AFP-producing tumor. The recombined mAFP/HSP70 vaccine may be suitable for serving as an immunotherapy for hepatocellular carcinoma.

  13. Extracellular expression and efficient purification of a functional recombinant Volvariella volvacea immunomodulatory protein (FIP-vvo) using Pichia pastoris system.

    PubMed

    Sun, Xilin; Huang, Wei; Xiao, Sijia; Liang, Chongyang; Zhang, Shuqin; Liu, Zhiyi; Sun, Fei

    2014-02-01

    The fungal immunomodulatory proteins (FIPs) are a new protein family identified from several edible and medical mushrooms and play an important role in antitumor, anti-allergy and immunomodulating activities. A gene encoding the FIP-vvo was cloned from the mycelia of Volvariella volvacea and recombinant expressed in the Pichia pastoris expression system. SDS-PAGE, amino acid composition and circular dichroism analyses of the recombinant FIP-vvo (reFIP-vvo) indicated that the gene was correctly and successfully expressed. In vitro assays of biological activities revealed that the reFIP-vvo exhibited similar immunomodulating capacities as native form. The reFIP-vvo significantly stimulated the proliferation of mouse spleen lymphocytes and apparently enhanced the expression level of IFN-γ released from the mouse splenocytes. Taken together, the FIP-vvo gene from V. volvacea has been integrated into the yeast genome and expressed effectively at a high level (about 410mg/L), it was capable of agglutinating sheep and rat red blood cells. The reFIP-vvo possessed very similar biological activities to native FIPs, suggesting its potential application as a food supplement or immunomodulating agent in pharmaceuticals and even medical studies. PMID:24262209

  14. Optimization of synergism of a recombinant auxiliary activity 9 from Chaetomium globosum with cellulase in cellulose hydrolysis.

    PubMed

    Kim, In Jung; Nam, Ki Hyun; Yun, Eun Ju; Kim, Sooah; Youn, Hak Jin; Lee, Hee Jin; Choi, In-Geol; Kim, Kyoung Heon

    2015-10-01

    Auxiliary activity family 9 (AA9, formerly known as glycoside hydrolase family 61 or polysaccharide monooxygenase) is a group of fungal proteins that were recently found to have a significant synergism with cellulase in cellulose hydrolysis via the oxidative cleavage of glycosidic bonds of cellulose chains. In this study, we report the active expression of a recombinant fungal AA9 from Chaetomium globosum (CgAA9) in a bacterial host, Escherichia coli, and the optimization of its synergistic activity in cellulose hydrolysis by using cellulase. The recombinant CgAA9 (0.9 mg/g cellulose) exhibited 1.7-fold synergism in the hydrolysis of Avicel when incubated with 0.9 filter paper units of Celluclast 1.5 L/g cellulose. The first study of the active expression of AA9 using a bacterial host and its synergistic optimization could be useful for the industrial application of AA9 for the saccharification of lignocellulose. PMID:25936375

  15. Functional analysis of N-linked glycosylation mutants of the measles virus fusion protein synthesized by recombinant vaccinia virus vectors.

    PubMed Central

    Alkhatib, G; Shen, S H; Briedis, D; Richardson, C; Massie, B; Weinberg, R; Smith, D; Taylor, J; Paoletti, E; Roder, J

    1994-01-01

    The role of N-linked glycosylation in the biological activity of the measles virus (MV) fusion (F) protein was analyzed by expressing glycosylation mutants with recombinant vaccinia virus vectors. There are three potential N-linked glycosylation sites located on the F2 subunit polypeptide of MV F, at asparagine residues 29, 61, and 67. Each of the three potential glycosylation sites was mutated separately as well as in combination with the other sites. Expression of mutant proteins in mammalian cells showed that all three sites are used for the addition of N-linked oligosaccharides. Cell surface expression of mutant proteins was reduced by 50% relative to the wild-type level when glycosylation at either Asn-29 or Asn-61 was abolished. Despite the similar levels of cell surface expression, the Asn-29 and Asn-61 mutant proteins had different biological activities. While the Asn-61 mutant was capable of inducing syncytium formation, the Asn-29 mutant protein did not exhibit any significant cell fusion activity. Inactivation of the Asn-67 glycosylation site also reduced cell surface transport of mutant protein but had little effect on its ability to cause cell fusion. However, when the Asn-67 mutation was combined with mutations at either of the other two sites, cleavage-dependent activation, cell surface expression, and cell fusion activity were completely abolished. Our data show that the loss of N-linked oligosaccharides markedly impaired the proteolytic cleavage, stability, and biological activity of the MV F protein. The oligosaccharide side chains in MV F are thus essential for optimum conformation of the extracellular F2 subunit that is presumed to bind cellular membranes. Images PMID:8107215

  16. Recombinant CHIK virus E1 coat protein of 11 KDa with antigenic domains for the detection of Chikungunya.

    PubMed

    Yathi, Krishna Kammara; Joseph, Julia Mary; Bhasker, Salini; Kumar, Ramesh; Chinnamma, Mohankumar

    2011-09-30

    Chikungunya is an acute febrile illness caused by an alpha virus technically called as CHIK virus. A smaller size of CHIK virus E1 coat protein -11 kDa was expressed in prokaryotic expression system. The recombinant protein was purified and confirmed by western blot analysis. The positions of the antigenic domain in the protein were identified and the immunoreactivity of recombinant protein with anti-CHIK IgM antibodies was ascertained. The antigen showed an 88% sensitivity and 100% specificity by Indirect ELISA. No cross reactivity of the antigen was observed with anti-Dengue virus serum samples. The results strongly support that the recombinant CHIK coat protein could be used as a diagnostic antigen for the detection of Chikungunya by Indirect ELISA. The relevance of a smaller size recombinant antigen highlights its large scale application in serodiagnosis of CHIK virus since bacterial expression is more simple and cost effective than eukaryotic system. PMID:21798263

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

    PubMed

    Bill, Roslyn M; von der Haar, Tobias

    2015-06-01

    Membrane protein structural biology is critically dependent upon the supply of high-quality protein. Over the last few years, the value of crystallising biochemically characterised, recombinant targets that incorporate stabilising mutations has been established. Nonetheless, obtaining sufficient yields of many recombinant membrane proteins is still a major challenge. Solutions are now emerging based on an improved understanding of recombinant host cells; as a 'cell factory' each cell is tasked with managing limited resources to simultaneously balance its own growth demands with those imposed by an expression plasmid. This review examines emerging insights into the role of translation and protein folding in defining high-yielding recombinant membrane protein production in a range of host cells. PMID:26037971

  18. Affinity Purification of a Recombinant Protein Expressed as a Fusion with the Maltose-Binding Protein (MBP) Tag

    PubMed Central

    Duong-Ly, Krisna C.; Gabelli, Sandra B.

    2015-01-01

    Expression of fusion proteins such as MBP fusions can be used as a way to improve the solubility of the expressed protein in E. coli (Fox and Waugh, 2003; Nallamsetty et al., 2005; Nallamsetty and Waugh, 2006) and as a way to introduce an affinity purification tag. The protocol that follows was designed by the authors as a first step in the purification of a recombinant protein fused with MBP, using fast protein liquid chromatography (FPLC). Cells should have been thawed, resuspended in binding buffer, and lysed by sonication or microfluidization before mixing with the amylose resin or loading on the column. Slight modifications to this protocol may be made to accommodate both the protein of interest and the availability of equipment. PMID:26096500

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

    PubMed Central

    2016-01-01

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

  20. Mutant strains of Pichia pastoris with enhanced secretion of recombinant proteins.

    PubMed

    Larsen, Sasha; Weaver, Jun; de Sa Campos, Katherine; Bulahan, Rhobe; Nguyen, Jackson; Grove, Heather; Huang, Amy; Low, Lauren; Tran, Namphuong; Gomez, Seth; Yau, Jennifer; Ilustrisimo, Thomas; Kawilarang, Jessica; Lau, Jonathan; Tranphung, Maivi; Chen, Irene; Tran, Christina; Fox, Marcia; Lin-Cereghino, Joan; Lin-Cereghino, Geoff P

    2013-11-01

    Although Pichia pastoris is a popular protein expression system, it exhibits limitations in its ability to secrete heterologous proteins. Therefore, a REMI (restriction enzyme mediated insertion) strategy was utilized to select mutant beta-g alactosidase s upersecretion (bgs) strains that secreted increased levels of a β-galactosidase reporter. Many of the twelve BGS genes may have functions in intracellular signaling or vesicle transport. Several of these strains also appeared to contain a more permeable cell wall. Preliminary characterization of four bgs mutants showed that they differed in the ability to enhance the export of other reporter proteins. bgs13, which has a disruption in a gene homologous to Saccharomyces cerevisiae protein kinase C (PKC1), gave enhanced secretion of most recombinant proteins that were tested, raising the possibility that it has the universal super-secreter phenotype needed in an industrial production strain of P. pastoris. PMID:23881328

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

    PubMed

    Jia, Baolei; Jeon, Che Ok

    2016-08-01

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

  2. Successful Interference with Cellular Immune Responses to Immunogenic Proteins Encoded by Recombinant Viral Vectors

    PubMed Central

    Sarukhan, Adelaida; Camugli, Sabine; Gjata, Bernard; von Boehmer, Harald; Danos, Olivier; Jooss, Karin

    2001-01-01

    Vectors derived from the adeno-associated virus (AAV) have been successfully used for the long-term expression of therapeutic genes in animal models and patients. One of the major advantages of these vectors is the absence of deleterious immune responses following gene transfer. However, AAV vectors, when used in vaccination studies, can result in efficient humoral and cellular responses against the transgene product. It is therefore important to understand the factors which influence the establishment of these immune responses in order to design safe and efficient procedures for AAV-based gene therapies. We have compared T-cell activation against a strongly immunogenic protein, the influenza virus hemagglutinin (HA), which is synthesized in skeletal muscle following gene transfer with an adenovirus (Ad) or an AAV vector. In both cases, cellular immune responses resulted in the elimination of transduced muscle fibers within 4 weeks. However, the kinetics of CD4+ T-cell a