Protein In Prokaryotic System Research Articles

Cloning and Expression of Partial CDS of Leptospiral Beta Propeller Repeat Protein in Prokaryotic System

Cloning and Expression of Partial CDS of Leptospiral Beta Propeller Repeat Protein in Prokaryotic System

Prokaryotic Expression of H1N1 Influenza A Virus Haemagglutinin Protein Globular Domain (HA1)

Abstract Background: Influenza viruses are a significant cause of morbidity and mortality .The influenza virus pandemics, 1918, 1977, and especially the most recent one, A/H1N1/2009, made evident the need for generating recombinant Influenza H1N1 antigens which are essential to develop both basic and applied research programs. Among influenza virus proteins, haemagglutinin (HA) is a major surface antigen of influenza virus, thus it is highly topical in influenza research and vaccine engineering programs. Alternatively, expression of fragments of the HA (HA1 and HA2) proteins in prokaryotic systems can potentially be the most efficacious strategy for manufacture of large quantities of influenza vaccine in a short period of time. Methodology: The gene encoding the HA1 protein from the influenza A/Puerto Rico/8/34 was amplified by PCR, then cloned into pTZ57R/T cloning vector. The fidelity of HA1 open reading frame was confirmed by bidirectional sequencing, then sub-cloned into pET28a prokaryotic expression plasmid, and proteins containing HA1 N-terminally fused to His-Tag were produced in Escherichia coli BL21 through IPTG inducing. The accuracy of the expression was confirmed by running time coursed fraction samples taken before and after the IPTG induction in SDS-PAGE, Western blot analysis was also used for confirmation of the recombinant protein. Conclusion: The HA1 protein produced here could be considered and evaluated as a protective antigen which its immunogenicity potential needs to be assessed in animal models along with proper control groups. Moreover, it could be subjected for polyclonal antibody preparation, which, in turn, may be used as an essential material in western blot analyses, as well as in other immunological applications, such as ELISA, immunocytochemistry, immunohistochemistry, and other immunological and serological studies. Key words: Influenza A, H1N1 , Hemagglutinin , Prokaryotic Expression

Prokaryotic Expression of H1N1 Influenza A Virus Haemagglutinin Protein Globular Domain (HA1)

Background: Influenza viruses are a significant cause of morbidity and mortality. The influenza virus pandemics, 1918, 1977, and especially the most recent one, A/H1N1/2009, made evident the need for generating recombinant Influenza H1N1 antigens which are essential to develop both basic and applied research programs. Among influenza virus proteins, haemagglutinin (HA) is a major surface antigen of influenza virus, thus it is highly topical in influenza research and vaccine engineering programs. Alternatively, expression of fragments of the HA (HA1 and HA2) proteins in prokaryotic systems can potentially be the most efficacious strategy for manufacture of large quantities of influenza vaccine in a short period of time. Materials and Methods: The gene encoding the HA1 protein of the influenza A/Puerto Rico/8/34 was amplified by PCR, then cloned into pTZ57R/T cloning vector. The fidelity of the HA1 open reading frame was confirmed by bidirectional sequencing, then sub-cloned into pET28a prokaryotic expression plasmid, and proteins containing HA1 N-terminally fused to His-Tag were produced in Escherichia coli BL21 through IPTG inducing. The accuracy of the expression was confirmed by running time coursed fraction samples taken before and after the IPTG induction in SDS-PAGE, Western blot analysis were also used for confirmation of the recombinant protein. Results and Conclusion: The HA1 protein produced here could be considered and evaluated as a protective antigen, which its immunogenicity potential needs to be assessed in animal models along with proper control groups. Moreover, it could be subjected for polyclonal antibody preparation, which, in turn, may be used as an essential material in western blot analyses, as well as in other immunological applications, such as ELISA, immunocytochemistry, immunohistochemistry, and other immunological and serological studies. K e y w o r ds: Influenza A, H1N1, Hemagglutinin, Prokaryotic Expression

Expression of Erythroblastic Leukemia Viral Oncogene Homolog 2 (ERBB2) from Canine Mammary Tumor

The ERBB2 oncogene encodes a trans-membrane glycoprotein (p185) with tyrosine kinase activity, which is closely related to epidermal growth factor receptor (EGFR). All four members of the family have been reported to be expressed in breast cancer cells in vitro. ERBB2 expression predicts poor prognosis in cancer. The present study has evaluated the association of the expression levels of ERBB2 with mammary tumor development in dogs and checked the heterologous expression of ERBB2 protein in prokaryotic system. Gene expression was measured using qRT-PCR. Mammary tissue samples were collected from healthy canine mammary gland (n = 10) and from tumor subjects (n = 10). ERBB2 expression was found to be upregulated (5.659 times) in canine mammary tumor state suggesting malignant behavior of canine mammary tumor association with increased transcription of ERBB2 gene. Ectodomain encoding ERBB2 gene was cloned in E. coli and the recombinant protein produced was successfully purified by Ni–NTA affinity chromatography which was further confirmed by western blotting. Recombinant ERBB2 protein can be used as a marker for prognosis of canine mammary tumor.

Tat Peptide-Mediated Soluble Expression of the Membrane Protein LSECtin-CRD in Escherichia coli

The human liver and lymph node sinusoidal endothelial cell C-type lectin (hLSECtin), a type II integral membrane protein, containing a Ca2+-dependent carbohydrate recognition domain (CRD), has a well-established biological activity, yet its three-dimensional structure is unknown due to low expression yields and aggregation into inclusion bodies. Previous study has demonstrated that the HIV-1 virus-encoded Tat peptide (‘YGRKKRRQRRR’) can increase the yields and the solubility of heterologous proteins. However, whether the Tat peptide could promote the high-yield and soluble expression of membrane proteins in Escherichia coli is not known. Therefore, the prokaryotic expression vector pET28b-Tat-hLSECtin-CRD (using pET28b and pET28b-hLSECtin-CRD as controls) was constructed, and transformed into E. coli BL21 (DE3) cells and induced with isopropyl-β-d-thiogalactoside (IPTG) followed with identifying by SDS-PAGE and Western blot. Subsequently, the bacterial subcellular structure, in which overexpressed the heterologous proteins Tat-hLSECtin-CRD and Tat-free hLSECtin-CRD, was analyzed by transmission electron microscope (TEM) respectively, and the mannose-binding activity of Tat-hLSECtin-CRD was also determined. Expectedly, the solubility of Tat-LSECtin-CRD significantly increased compared to Tat-free LSECtin-CRD (**p < 0.01) with prolonged time, and the Tat-LSECtin-CRD had a significant mannose-binding activity. The subcellular structure analysis indicated that the bacterial cells overexpressed Tat-hLSECtin-CRD exhibited denser region compared with controls, while dot denser region aggregated in the two ends of bacterial cells overexpressed Tat-free hLSECtin-CRD. This study provided a novel method for improving the soluble expression of membrane proteins in prokaryotic systems by fusion with the Tat peptide, which may be potentially expanded to the expression of other membrane proteins.

Re-designed N-terminus enhances expression, solubility and crystallizability of mitochondrial protein

Mitochondrial aminoacyl-tRNA synthetases are key enzymes in translation. They are encoded by the nuclear genome, synthesized as precursors in the cytosol and imported. Most are matured by cleavage of their N-terminal targeting sequence. The poor expression of mature proteins in prokaryotic systems, along with their low solubility and stability after purification are major obstacles for biophysical and crystallographic studies. The purpose of the present work was to analyze the influence of additives on a slightly soluble aspartyl-tRNA synthetase and of the N-terminal sequence of the protein on its expression and solubility. On the one hand, the solubility of the enzyme was augmented to some extent in the presence of a chemical analog of the intermediary product aspartyl-adenylate, 5'-O-[N-(L aspartyl) sulfamoyl] adenosine. On the other hand, expression was enhanced by extending the N-terminus by seven natural amino acids from the predicted targeting sequence. The re-designed enzyme was active, monodisperse, more soluble and yielded crystals that are suitable for structure determination. This result underlines the importance of the N-terminal residue sequence for solubility. It suggests that additional criteria should be taken into account for the prediction of cleavage sites in mitochondrial targeting sequences.

Construction and characterization of a novel bispecific antibody against both IL-1β and IL-17A

Objective To construct bispecific antibody BsAb1/17 against both IL-1β and IL-17A,express and purify the biologically active BsAbl/17 protein in prokaryotic system for further studies and applications. Methods VH1VL17-CL and VL1VH17-CH1 gene segments were constructed by overlap-PCR.Restriction enzyme sites Nco Ⅰ and BamH Ⅰ were designed at the 5'and 3' end primers respectively. The products of overlap-PCR were ligated to the Nco Ⅰ/BamH Ⅰ -prepared pET-27b vector. The recombinant plasmids pET-27b-VH1 VL17-CL(petA) and pET-27b-VL1 VH17-CH1 ( petB ) were transformed into E. coliRosetta separately. The expressing products were analyzed by SDS-PAGE and Western blot. Neutralization activity of the bispecific antibody for blocking the induction of IL-18 expression by IL-1β in human T cells was determined by real-time PCR. Neutralization activity of the bispecific antibody for blocking the induction of IL-6 expression by IL-17A in HeLa cells was determined by ELISA assay. Results The structure of the plasmids pET-27b-VH1 VL17-CL(petA) and pET-27b-VL1 VH17-CH1 (petB)was confirmed by DNA sequencing. After induction, the fusion proteins were expressed mainly as inclusion bodies. The purity of the both proteins exceeded 90%. SDS-PAGE analysis suggests the relative molecular mass of both products expressed by petA and petB were approximately 38× 103, which is in accordance with the theoretical value. The results of Western blot and ELISA test demonstrated that BsAb1/17 molecule had binding ability to both IL-1β and IL-17A. The BsAb1/17 could block IL-1β to stimulate human T cell to express IL-18 and block IL-17A to stimulate HeLa cell to express IL-6. Conclusion We successfully constructed a novel bispecific antibody BsAb1/17 against both IL-1 β and IL-17A, and expressed biologically active BsAb1/17 protein in prokaryotic system. Key words: Bispecific antibody; Prokaryotic expression; Real-time PCR

The production of soluble and correctly folded recombinant bovine β-lactoglobulin variants A and B in Escherichia coli for NMR studies

The production of soluble and correctly folded eukaryotic proteins in prokaryotic systems has always been hampered by the difference in or lack of cell machinery responsible for folding, post-translation modification and secretion of the proteins involved. In the case of bovine β-lactoglobulin (BLG), a major cow’s milk allergen and a protein widely used for protein folding studies, a eukaryotic yeast expression system has been the preferred choice of many researchers, particularly for the production of isotopically labeled protein required for NMR studies. Although this system yields high amounts of recombinant protein, the BLG produced is usually associated with extracellular polysaccharides, which is problematic for NMR analysis. In our study we show that when co-expressed with the signal-sequence-less disulfide bond isomerase (ΔssDsbC) in the dual expression vector, pETDUET-1, both BLG A and BLG B can be reproducibly produced in a soluble form. Expression was carried out in Escherichia coli Origami(DE3) , a trxB/ gor mutant for thioredoxin- and glutathione reductase, which allows for proper formation of disulfide bonds in the cytoplasm. The protein was purified by anion exchange chromatography followed by salting-out at low pH and size exclusion chromatography. Our expression system is able to consistently produce milligram quantities of correctly folded BLG A and B with no additional amino acid residues at the N-terminus, except for a methionine. 15N-labeled BLG A and B, prepared and purified using this method, produced HSQC spectra typical of native bovine BLG.

Expression of Peste des petits ruminants virus nucleocapsid protein in prokaryotic system and its potential use as a diagnostic antigen or immunogen

In this study, both partial and full-length nucleocapsid (N) gene of Peste des petits ruminants virus (PPRV) were cloned into pET33b vector and expressed in Escherichia coli (BL21) with the objective of replacing live PPRV antigen with recombinant protein in ELISA. The expressed proteins were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blot by using a PPRV N protein specific monoclonal antibody. The expressed histidine-tagged fusion proteins were purified using affinity Ni-NTA column and were assessed for their conformation in terms of reactivity by ELISA. The immunogenicity of recombinant proteins was also assessed in rabbits and anti-N antibody response against PPRV was observed in all the immunized rabbits, when tested by competitive and indirect ELISAs. In sandwich ELISA, a mean OD 492 nm of 1.4 and 0.90 was obtained for crude lysate having expressed the N protein and the PPRV antigen, respectively. Further, the N protein was tested as a coating antigen in competitive ELISA instead of PPRV antigen for serological diagnosis of PPR infection. This indicates the diagnostic potential of the PPRV recombinant N proteins, which are safe and better alternatives to live PPRV antigen in ELISA for clinical or sero-surveillance of PPR in enzootic or non-enzootic countries.

Expression of porcine circovirus 2 ORF2 gene requires codon optimized E. coli cells

Expression and purification of whole and nuclear localization signal (NLS) deleted ORF2 capsid protein of porcine circovirus 2 (PCV2) is demonstrated in the present study. Gene coding for both protein forms were cloned into pDest17 vector and expressed in BL21 (DE3)AI cells and in BL21-CodonPlus (DE3)-RIPL E. coli cells. The later cells were used to overcome difficulties with the heterologous expression of viral proteins in prokaryotic systems. Whole 30 kDa recombinant ORF2 protein was successfully expressed in BL21-CodonPlus (DE3)-RIPL cells only, 3 mg of pure protein was consistently obtained per liter of bacterial culture. NLS deleted ORF2 protein was expressed in both cell types. Resulting proteins reacted with PCV2 positive swine serum in immunofluorescent test and immunoblot.

Beyond antibodies ? lessons from bacterial ?immunity?

Isolation and production of highly specific protein-based binding molecules are crucial to the ever expanding diagnostics, therapeutics and protein array fields. Traditionally, such reagents have been sourced from vertebrate immune systems, where antibodies have evolved over millennia into highly effective molecules of immune surveillance capable of targeting a huge range of targets in response to infection and disease. Now, a growing number of alternative protein scaffolds are being investigated as specific binding molecules incorporating a diverse and powerful range of binding and recognition interfaces. These are being sourced from human proteins, from alternative immune molecules present in evolutionarily old vertebrates, and from highly evolved binding proteins in prokaryotic systems.

Secretory expression of thermostable T1 lipase through bacteriocin release protein

The extracellular production of T1 lipase was performed by co-expression of pJL3 vector encoding bacteriocin release protein in prokaryotic system. Secretory expression was optimized by considering several parameters, including host strains, inducer (IPTG) concentration, media, induction at A 600 nm , temperature, and time of induction. Among the host strains tested, Origami B excreted out 18,100 U/ml of lipase activity into culture medium when induced with 50 μM IPTG for 12 h. The Origami B harboring recombinant plasmid pGEX/T1S and pJL3 vector was chosen for further study. IPTG at 0.05 mM, YT medium, induction at A 600 nm of 1.25, 30 °C, and 32 h of induction time were best condition for T1 lipase secretion with Origami B as a host.

Anion-dependent modulations of DNA topoisomerase II-mediated reactions in potassium-containing solutions

DNA binding proteins operate in an intracellular environment of low chloride concentration, yet in vitro assays of the activities of these proteins are often performed in isotonic chloride-containing solutions. Previously, the activity of bacterial DNA-binding proteins was found to be enhanced in potassium-containing solutions in which the anion glutamate (Glu) was substituted for chloride. We have extended this observation to include eukaryotic topoisomerase I and II activities. The concentration ranges over which DNA strand passing activities of these enzymes were observed was broader in KGlu than in KCl. This was also true for the topoisomerase II-mediated DNA strand passage and antineoplastic drug-dependent DNA cleavage produced by nuclear extracts from HL-60 human leukemia cells. The rate of topoisomerase II-mediated DNA strand passage was also dependent on anion moiety and concentration in potassium-containing buffers. Drug-dependent topoisomerase II-mediated DNA cleavage in intact HL-60 cell nuclei was also anion-dependent, suggesting that anion type and concentration may influence topoisomerase II-mediated events in mammalian cells as had been described for other DNA binding proteins in prokaryotic systems. This should be considered in developing biochemical assays of topoisomerase activities to reproduce intracellular conditions.