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Expression of anti-gp96 scFv fragment in Pichia pastoris and identification of its biological activity / 生物工程学报
Chinese Journal of Biotechnology ; (12): 595-604, 2014.
Article in Chinese | WPRIM | ID: wpr-279480
Responsible library: WPRO
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.
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Full text: Available Index: WPRIM (Western Pacific) Main subject: Pichia / Plasmids / Recombinant Proteins / Enzyme-Linked Immunosorbent Assay / Membrane Glycoproteins / Blotting, Western / Chromatography, Affinity / Electrophoresis, Polyacrylamide Gel / Allergy and Immunology / Single-Chain Antibodies Language: Chinese Journal: Chinese Journal of Biotechnology Year: 2014 Type: Article

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Full text: Available Index: WPRIM (Western Pacific) Main subject: Pichia / Plasmids / Recombinant Proteins / Enzyme-Linked Immunosorbent Assay / Membrane Glycoproteins / Blotting, Western / Chromatography, Affinity / Electrophoresis, Polyacrylamide Gel / Allergy and Immunology / Single-Chain Antibodies Language: Chinese Journal: Chinese Journal of Biotechnology Year: 2014 Type: Article