Expression, purification and characterization of low-glycosylation influenza neuraminidase in α-1,6-mannosyltransferase defective Pichia pastoris.

Influenza A viruses expose two major surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Although N-glycosylation is essential for many glycoproteins, the glycoproteins expressed in yeast are sometimes hyper-glycosylated, which maybe a primary hindrance to the exploitation of therapeutic glycoprotein production because glycoproteins decorated with yeast-specific glycans are immunogenic and show poor pharmacokinetic properties in humans. To elucidate the NA with different glycosylation in interaction with immunogenicity, here we reported the heterologous expression of influenza NA glycoprotein derived from influenza virus A/newCaledonia/20/99(H1N1) in wide-type Pichia pastoris, α-1,6-mannosyltransferase (och1)-defective P. pastoris and Escherichia coli. We also assessed the immunogenicity of hyper-glycosylated NA expressed in the wide-type, low-glycosylated NA expressed in och1-defective P. pastoris strain and non-glycosylated NA produced in E. coli. Recombinant NA was expressed in wide-type P. pastoris as a 59-97 above kDa glycoprotein, 52-57 kDa in the och1 defective strain, and as a 45 kDa non-glycoprotein in E. coli. The antibody titers of Balb/c mice were tested after the mice were immunized three times with 0.2, 1, or 3 µg purified recombinant NA. Our results demonstrated that after the second immunization, the antibody titer elicited with 1 µg low-glycosylated NA was 1:5,500, while it was 1:10 and 1:13 when elicited by 1 µg hyper-glycosylated and non-glycosylated NA. In the 0.2 µg dose groups, a high antibody titer (1:4,900) was only found after third immunization by low-glycosylated NA, respectively. These results suggest that low-glycosylation in och1-defective P. pastoris enhances the immunogenicity of recombinant NA and elicits similar antibody titers with less antigen when compared with hyper- and non-glycosylated NA. Thus, och1-defective P. pastoris may be a better yeast expression system for production of glycoproteins to research immunogenic characterization.

[A Pichia pastoris with alpha-1, 6-mannosyltransferases deletion and its use in expression of HSA/GM-CSF chimera].

Yeast is a widely used host for recombinant protein expression. However, glycoproteins derived from yeast contain N-glycan of high mannose type and are usually hyperglycosylated. alpha-1,6-mannosyltransferases gene (och1) encodes the enzyme that initiates the first step of out-chain elongation of high mannose type N-glycan in yeast, which is different from that in human. So, a high efficient method to knockout target gene by two-step recombination was established and was used to delete och1. In the first recombinant, a plasmid with och1::ADE1 and ura3 gene was linearized in the downstream of och1 and inserted to the och1 site of P. pastoris genome, where the upstream and downstream of och1 were duplicated. In the second recombinant, the duplicated fragments of och1 were exchanged and the och1 deletion strains were selected on the plates containing 5-FOA, but no adenine. Then the och1 deletion strain was applied to express an human serum albumin (HSA) granulocyte-macrophage colony-stimulating factor (GM-CSF) chimera. Different with the hyperglycosylated HSA/GM-CSF chimera expressed in wild type P. pastoris, the chimera expressed in the och1 deletion strain, contained smaller N-glycan. The results suggested that the och1 mutant yeast may be more suitable for production of recombinant glycoproteins. And the och 1 deletion strain could be used for further re-engineering to produce complex human glycoproteins.

[Expression and characterization of human sTNFR II-IgG Fc fusion protein in Pichia pastoris].

AIM: To find if human soluble tumor necrosis factor receptor II (p75) fused IgG Fc protein (sTNFR II-IgG Fc) could be expressed in Pichia pastoris with an active dimmer form and characterize its N-linked oligosaccharides. METHODS: Two gene fragment, human sTNFR II and IgGFc, were got by RT-PCR from leucocytes stimulated with LPS. And the chimeric gene sTNFR II-IgG Fc achieved through gene splicing by over lap extension (SOE) method was cloned into pPIC9 and transformed into methanotropic yeast Pichia pastoris. The fusion protein purified by Protein A affinity column was analyzed with SDS-PAGE electrophoresis under reducing or non-reducing conditions and immunological methods. The anti-TNF-alpha biological activity assay of fusion protein was performed with L929 cells and detected with MTT colorimetry. The N-linked oligosaccharides hydrolyzed from fusion protein were labeled with 8-amino-1, 3, 6-naphthalene trisulfonic acid (ANTS) were analyzed with fluorophore-assisted carbohydrate eletrophoresis (FACE) as well. RESULTS: The recombinant P. pastoris strain that expressed human sTNFR II-IgG Fc fusion protein was constructed. The expression level of fusion protein in 2 L flask reached 2 mg/L. SDS-PAGE and Western blot showed the expressed fusion protein purified by protein was a dimer linked with inter-molecular disulfide linkage. The fusion protein neutralized cytotoxic activity of TNF-alpha to L929 cells, and the EC(50) of the fusion protein to inhibit 5 x 10(4) U/L of TNF-alpha was 170 microg/L. The FACE analysis showed there are 11 to 13 hexoses on each N-linked oligosaccharide. CONCLUSION: The human sTNFR II-IgG Fc fusion protein is expressed successfully in P. pastoris and it could be a reference for the future expression of other Fc fusion proteins or immunoglobulins in Pichia pastoris.

[Expression in Pichia pastoris and properties of human serum albumin-interferon alpha2b chimera].

To reduce the serum clearance of interferon alpha2b, a chimeric gene encoding an human serum albumin(HSA)--human interferon alpha2b(IFNalpha2b) fusion protein was overexpressed in Pichia pastoris. After fermentation in a 5L bioreactor, the fusion protein, capable of cross-reacting with anti-IFN alpha and anti-HSA antibody, was purified from the culture of the recombinant yeast by ultrafiltration, blue Sepharose affinity, phenyl hydrophobic interaction and Q ion exchange chromatography. Its IFNa2b moiety exhibits antiviral activity similar to that of recombinant human IFNa2b. In Cynomolgus monkeys model, The fusion protein was detectable in plasma, even 336h after a single does of 90 microg/kg injection intravenously or subcutaneously. The elimination phase half-life of the fusion protein was 101h after intravenous injection and 68.2h after subcutaneous injection. Its Subcutaneous bioavailability was 67.9%. The enhanced pharmacokinetics of interferon a2b fused to human serum albumin suggest its promissing application in clinic medicine.

[Cloning of hIL-1Ra gene and its expression in E.coli].

AIM: To clone human IL-1Ra gene and express it in E.coli. METHODS: Human IL-1Ra cDNA was obtained by RT-PCR with the the total RNA extracted from human peripheral blood leucocytes as template. The cDNA was cloned into pBV220 vector and expressed. The expressed product was renatured and purified. Separation, purification and bioactivity analysis of the expressed products were performed. RESULTS: IL-1Ra gene was successfully expressed in E.coli and the expression level reached to about 40% of total bacteria protein. The purity of the final product was over 98%. The product could obviously suppress the secretion of IL-2 by EL-4 cells stimulated with IL-1beta. CONCLUSION: The expression of hIL-1Ra gene with bioactivity in E.coli lays experimental foundation for further development and utilisation.

[In vitro evaluation of the safety and biological activity of recombinant Helicobacter pylori blood group antigen-binding adhesin].

OBJECTIVE: To evaluate the safety and biological activity of recombinant Helicobacter pylori (Hp) blood group antigen- binding adhesin (rBabA ) in vitro so as to investigate the feasibility of using rBabA as a Hp vaccine. METHODS: ELISA was used to measure rBabA-specific antibody in the serum of Hp-infected patients, and the proliferation of T lymphocytes in response to rBabA was examined by MTT assay. T cell apoptosis induced by rBabA was detected by diphenylamine assay. The effect of rBabA on Hp binding into human gastric carcinoma cell line(MGC-803) was determined by light microscopy. RESULTS: rBabA did not induce T cell apoptosis in BabA antibody- negative patients and was capable of stimulating T cell proliferation in rBabA antibody-positive patients. In the serum samples from 38 Hp-infected patients, the rBabA antibody positivity rate was 18.4%. rBabA could partially inhibit the binding of Hp to gastric epithelial cells. Under light microscope, the adhesion of Hp to MGC-803 was significantly inhibited by rBabA in comparison with negative control with PBS pretreatment. CONCLUSION: rBabA proves to be a safe and immunogenic bacterial component of Hp, which stimulates humoral and cellular immunity and can be a hopeful antigen targeting at BabA2 gene-positive Hp strain for the development of Hp vaccine.

Construction of the E.coli clone expressing adhesin BabA of Helicobacter pylori and evaluation of the adherence activity of BabA.

OBJECTIVE: To construct a recombinant E.coli strain that highly expresses blood group Ag-binding adhesin (BabA) of Helicobacter pylori (Hp) and to assess the adherence activity of Hp BabA. METHODS: The gene fragment encoding BabA was amplified from Hp chromosomal DNA by PCR technique and inserted into prokaryotic expression vector pET-22b (+), which was then transformed into BL21 (DE3) E.coli strain for the expression of BabA recombinant protein. The adherence activity of Hp BabA obtained was assayed by counting under light microscope. RESULTS: DNA sequence analysis showed that the sequence of babA2 DNA was in agreement with that published in GenBank. The BabA recombinant protein amounted to 34.8% of the total protein of the bacterium after IPTG induction for 3 h at 37 degrees Celsius, and BabA-mediated adherence was confirmed in vitro. CONCLUSION: A clone expressing biologically active Hp BabA has been obtained, which may facilitate further study of the function of the adhesin.

[Study of recombinant stem cell factor].

Stem cell factor is an important hematopoietic growth factor. In this study, the human stem cell factor was produced by recombinant E. coli, and the structure and biological activity of the recombinant stem cell factor(rhSCF) was studied. It was indicated that the rhSCF was a uncovalent dimer in phosphate buffer,and had the correct mass spectra, mass peptides spectra, composition of amino acid, N-terminal sequernce, C-terminal sequence and intrachain disulfide linkages, rhSCF alone or synergy with rhG-CSF could mobilze hematopoietic progenitors to blood in monkey.