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


CONCLUSIONS
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.
Zhang
Yi-bang
YB
Beijing Institute of Biotechnology, Beijing 100071, China.
Shi
Li-nan
LN
Chang
Shao-hong
SH
Gong
Xin
X
Xiong
Ling-shuang
LS
Wu
Jun
J
D015153:Blotting, Western; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D000068800:Etanercept; D015870:Gene Expression; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009281:Naphthalenes / Q000737:chemistry; D009844:Oligosaccharides / Q000737:chemistry; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D018124:Receptors, Tumor Necrosis Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D007074:Immunoglobulin G; D009281:Naphthalenes; D009844:Oligosaccharides; D018124:Receptors, Tumor Necrosis Factor; C046465:8-amino-1,3,6-naphthalenetrisulfonic acid; D000068800:Etanercept