RESUMO
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.
