High level expression of recombinant human fibrinogen in Pichia pastoris
10.3969/j.issn.1005-1678.2016.11.001
- VernacularTitle:重组人纤维蛋白原基因在毕赤酵母中的高效表达
- Author:
Ronghua HAO
;
Xiaoyuan ZHANG
;
Fei LIU
;
Mian CHEN
;
Fengshan WANG
;
Xiqiang ZHU
;
Peixue LING
- Keywords:
recombinant human fibrinogen;
Pichia pastoris;
secretory expression;
purification
- From:
Chinese Journal of Biochemical Pharmaceutics
2016;36(11):1-4
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct a eukaryotic expression vector in Pichia pastoris containing human fibrinogen gene, in order to achieve high level secretory expression in extracellular.Methods Expression plasmid,pGAPZαA-FGB-FGG-FGA-AOX1,was constructed by inserting the synthesized sequence encoding human fibrinogen(FGA, FGB,FGG) and then introduced into Pichia pastoris SMD1168H by electroporation.Transformants were availably screened by Zeocin resistance,the expression of recombinant protein was identified by SDS-PAGE and Western blot analysis, the protein yield was tested by ELISA assay.After ultrafiltration and purification, the biological activity of protein was detected.Results The crude yield of human fibrinogen in Pichia pastoris supernatant reached 15 mg/L in flask and the biological aggregation activity was determined.Conclusion The human fibrinogen gene was obtained and successfully expressed in Pichia pastoris and the active products were secreted into the medium.
