甜蛋白Monellin在毕赤酵母中的分泌表达  

Secretion Expression of Monellin in Pichia pastoris

作　　者：吴刚[1] 汪亚平[1] 朱作言[1] 

机构地区：[1]中国科学院水生生物研究所淡水生态与生物技术国家重点实验室,武汉430072

出　　处：《高技术通讯》2003年第11期20-23,共4页Chinese High Technology Letters

基　　金：863计划(2001AA212281);973规划(2001CB109006);国家自然科学基金(200102006)资助项目

摘　　要：在西非热带植物Dioscoreophyllum cumminsii中存在着一种由两条多肽构成的甜度极高的蛋白Monellin。本研究根据已知的Monellin晶体衍射分析结果和毕赤酵母(Pichia pastoris)基因偏爱密码子设计并合成了连接两条多肽的重组基因,插入到毕赤酵母分泌表达质粒pGAPZαA中。扩增后,电击穿孔转化毕赤酵母GS115,筛选高产菌株放大培养。以5升罐发酵培养,表达量为150mg/L。经纯化,可以获得大于130mg/L的具有高甜度的活性蛋白。Monellin is a kind of natural sweet-tasting protein existing in the West African plant Dioscoreophyllum cumminsii. According to the crystal structure of natural protein a linker was designed to fuse the peptides. An artificial gene was synthesized and subcloned into a Pichia pastoris secretory expression vector pGAPZαA. The recombined plasmid was then integrated into the Pichia pastoris stain GS115 chromosome by electroporation. High yield production stain was selected and fermented in a 5 liter fermentor. The expression level reached approximately 150mg/L. After purification more than 80% protein remained and kept the biologic activity.

关 键 词：甜蛋白 毕赤酵母 基因重组 基因工程 

分 类 号：Q786[生物学—分子生物学]