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
em Zh
| WPRIM
| ID: wpr-279480
Biblioteca responsável:
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.
Texto completo:
1
Índice:
WPRIM
Assunto principal:
Pichia
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Plasmídeos
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Proteínas Recombinantes
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Ensaio de Imunoadsorção Enzimática
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Glicoproteínas de Membrana
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Western Blotting
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Cromatografia de Afinidade
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Eletroforese em Gel de Poliacrilamida
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Alergia e Imunologia
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Anticorpos de Cadeia Única
Tipo de estudo:
Prognostic_studies
Idioma:
Zh
Revista:
Chinese Journal of Biotechnology
Ano de publicação:
2014
Tipo de documento:
Article