玉米赤霉烯酮降解酶毕赤酵母表达载体的构建及其表达  

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

作　　者：谭强来[1] 徐锋[1] 黎鹏[1] 程波财[1] 罗洁[1] 魏华[1] 

机构地区：[1]南昌大学食品科学与技术国家重点实验室,江西南昌330047

出　　处：《中国微生态学杂志》2010年第12期1061-1064,共4页Chinese Journal of Microecology

基　　金：国家自然科学基金项目(30860008);江西省国际科技合作项目(2010EHA02200);江西省科技支撑计划(2010BNA09300)

摘　　要：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。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.

关 键 词：ZEN-jjm基因 玉米赤霉烯酮 毕赤酵母 表达 降解 

分 类 号：Q786[生物学—分子生物学]