转甲状腺素蛋白基因在毕赤氏酵母中的非融合表达  

Direct Expression of Transthyretin Gene in Yeast Pichia pastoris

作　　者：翟杰[1] 高卜渝[1] 裴黎霞 吴俊 宋大新[1] 

机构地区：[1]复旦大学生命科学学院,上海200433

出　　处：《复旦学报（自然科学版）》2002年第6期646-649,共4页Journal of Fudan University：Natural Science

基　　金：国家自然科学基金资助项目(39970019)

摘　　要：用限制酶将pSK TTR质粒上TTR基因切下,分别插入分泌型载体pPIC9和pPIC9K.将重组的pPIC9K TTR用BglⅡ酶切后,转化PichiapastorisGS115菌株,筛选获得G418高抗性转化子.转化子经摇瓶发酵,上清液用SDS PAGE检测,有明显的rTTR表达.经DEAE sepharoseF.F.离子交换柱和Superdex75分子筛层析,得到蛋白纯度大于95%的rTTR.经Westernblotting,分子质量和等电点测定等证明,毕赤氏酵母表达的rTTR与人血浆提取的TTR极为相似.TTR gene was cut off from the plasmid pSKTTR by restriction enzymes, and was inserted into secretion vectors pPIC9 and pPIC9K. The recombined pPIC9KTTR was digested by BglⅡ and transformed into Pichia pastoris strain GS115, then the transformants with high G418 resistance were selected. After the transformants grew in the shaking flask, the SDSPAGE analysis of supernatant showed that they could express rTTR in a high level. rTTR was isolated and purified by DEAESepharose F. F. and Superdex75 gel permeation chromatography with more than 95% purity. The results obtained through experiments such as Western blotting and Mr and PI determination indicated that purified rTTR protein expressed by Pichia pastoris had very similar characteristics compared with those of natural TTR protein purified from human serum.

关 键 词：转甲状腺素蛋白基因 分泌型载体 毕赤氏酵母 蛋白质印迹 

分 类 号：Q51[生物学—生物化学] Q786