Expression Of Dioscin-glycosidase Gene

In this paper, gene Dioscin-glycosidase was expressed in E.coli BL21,and subcloned into expression vector pPIC9K, then transformed into Pichia pastoris GS115.Containing gene Dioscin-glycosidase, E.coli BL21 was inductive cultivated.After separating the expression products by breaking the cell wall, the products were detected by TLC and SDS-PAGE.The gene was subcloned into expression vector of pPIC9K to construct the recombinant plasmid. The recombinant plasmid was transformed into Pichia pastoris GS115 cells by. electroporation after linearizing and ethanol refining.Then the positive clones were filtered with PCR.The filtered Pichia pastoris was cultivated in BMGY media for growing and cultivated in BMMY media for constructing.The inducer was methanol.The cultivated condiction was optimized and compared in blank.The results showed that, in E. coli expression, the expressed gene was no activity, and in Pichia pastoris expression, the protein not only has the activity, its molecular weight determination was also in line with the theoretical value. The most of content of expressed protein outside the cell was 144h and the best density of the methanol was 0.5%. The result showed that the dioscin-glycosidase in the Pichia pastoris is expressed successfully...