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
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.
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
Type of study:
Prognostic study
Language:
Chinese
Journal:
Chinese Journal of Biotechnology
Year:
2014
Type:
Article
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