High level secretory expression of recombinant human tissue factor in Pichia pastoris
10.3969/j.issn.1005-1678.2016.04.09
- VernacularTitle:重组人组织因子在毕赤酵母中的高效分泌表达的研究
- Author:
Xiuhua ZHANG
;
Qianqian ZHAO
;
Fei LIU
;
Xiqiang ZHU
- Publication Type:Journal Article
- Keywords:
recombinant human tissue factor;
Pichia pastoris;
Western blot;
high yield
- From:
Chinese Journal of Biochemical Pharmaceutics
2016;36(4):37-40
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct a eukaryotic expression vector in Pichia pastoris containing human tissue factor( hTF) gene,in order to achieve high level secretory expression in extracellular.Methods Expression plasmid, pGAPZaA-hTF, was constructed by inserting the synthesized sequence encoding human extracellular tissue factor into yeast expression vector pGAPZaA and transformed into Pichia pastoris SMD1168H with electroporation.Having been selected by Zeocin, transformants containing hTF cDNA were expressed in YPD, extracellular proteins were detected by SDS-PAGE and confirmed by Western bolt.Results Successfully constructed the recombinant pGAPZaA-hTF expression system in Pichia pastoris.SDS-PAGE showed that the molecular weight of the expression product was about 37 -40 kDa.Western-blot indicated that it was human extracellular tissue factor.The crude yield of total protein in medium was up to 1 g/L, more than 80% of which was hTF.Conclusion Truncated rTF gene is expressed in Pichia pastoris and the active products are secreted into the medium which have the same activation as the native TF.
