title: [Expression of anti-gp96 scFv fragment in Pichia pastoris and identification of its biological activity].
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.
authors.forename: Mingming
authors.initials: M
authors.lastname: Gui
authors.forename: Huiying
authors.initials: H
authors.lastname: Wu
authors.forename: Lu
authors.initials: L
authors.lastname: Sun
authors.forename: Yaxing
authors.initials: Y
authors.lastname: Xu
authors.forename: Bao
authors.initials: B
authors.lastname: Zhao
authors.forename: Xin
authors.initials: X
authors.lastname: Li
authors.forename: Changfei
authors.initials: C
authors.lastname: Li
authors.forename: Xidong
authors.initials: X
authors.lastname: Wang
authors.forename: Songdong
authors.initials: S
authors.lastname: Meng
mesh_terms: D015153:Blotting, Western; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D008562:Membrane Glycoproteins / Q000276:immunology; D010843:Pichia / Q000378:metabolism*; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis; D057127:Single-Chain Antibodies / Q000096:biosynthesis*
chemical_list: D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; C053387:endoplasmin