Expression of anti-gp96 scFv fragment in Pichia pastoris and identification of its biological activity.
- Author:
Mingming GUI
;
Huiying WU
;
Lu SUN
;
Yaxing XU
;
Bao ZHAO
;
Xin LI
;
Changfei LI
;
Xidong WANG
;
Songdong MENG
- Publication Type:Journal Article
- MeSH:
Blotting, Western;
Chromatography, Affinity;
Electrophoresis, Polyacrylamide Gel;
Enzyme-Linked Immunosorbent Assay;
Membrane Glycoproteins;
immunology;
Pichia;
metabolism;
Plasmids;
Recombinant Proteins;
biosynthesis;
Single-Chain Antibodies;
biosynthesis
- From:
Chinese Journal of Biotechnology
2014;30(4):595-604
- CountryChina
- Language:Chinese
-
Abstract:
Secretory anti-gp96 scFv fragment was expressed in Pichia pastoris to obtain a small molecule antibody that specifically recognizes heat shock protein gp96. The gp96-scFv fragment gene was synthesized and cloned to Pichia pastoris expression plasmid pPICZa-A. Pichia pastoris X33 was electroporated with the linearized recombinant expression vector, and expression of gp96-scFv fragment was induced by methanol. The His-tagged recombinant protein was then purified by affinity chromatography and analyzed with SDS-PAGE and Western blotting assays. The biological activities of recombinant gp96-scFv fragment were determined by Western blotting, Immunofluorescence, ELISA and FACS assays. The gp96-scFv fragment was expressed successfully in Pichia pastoris. About 50 mg of recombinant protein could be purified from 1 liter of the Pichia pastoris culture supernatant. Its molecular weight was about 15 kDa. The gp96-scFv fragment could specifically bind to gp96 protein by Western blotting, immunofluorescence, ELISA and FACS analyses. Pichia pastoris-expressed gp96-scFv fragment specifically recognizes gp96 protein, which could be used for Western blotting, Immunofluorescence, ELISA and FACS analyses.
