玉米赤霉烯酮降解酶毕赤酵母表达载体的构建及其表达

目的构建毕赤酵母表达载体pPIC9-ZEN-jjm,筛选高效分泌表达活性目的蛋白的菌株。方法克隆ZEN-jjm基因,经EcoRⅠ和NotⅠ双酶切连接至pPIC9中,电转化至毕赤酵母GS115。利用RDB培养基和甲醇诱导表达进行筛选。HPLC检测表达蛋白降解玉米赤霉烯酮的活性。结果测序表明ZEN-jjm成功插入pPIC9中,SDS-PAGE表明获得1株高效表达目的蛋白的重组酵母,其分子量约29 kDa。HPLC表明其能有效地降解玉米赤霉烯酮。结论玉米赤霉烯酮降解酶在毕赤酵母中获得了高效分泌表达。

Construction of an ZEN-jjm expressing vector and its expression in Pichia pastoris

Objective To construct an Pichia pastoris expressing vector pPIC9-ZEN-jjm and screen the strains which could express high-level active proteins.Method ZEN-jjm gene was spliced into pPIC9 after digested with EcoRⅠand NotⅠ,then transformed into Pichia pastoris GS115 by electroporation.The recombinant yeast strains were screened with RDB medium and methanol inducible expression.The ZEN degradation capabilities of expressed supernatant was verified by HPLC test.Result DNA sequencing demonstrated that ZEN-jjm was inserted into pPIC9.SDS-PAGE demonstrated that one yeast strain with high-level expression was obtained,and the molecular weight of the expressed protein was about 29 kDa.The HPLC result showed that the expressed protein could effectively degrade ZEN.Conclusion ZEN-degrading enzyme is highly expressed in Pichia pastoris.