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.
出处
《高技术通讯》
EI
CAS
CSCD
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
关键词
甜蛋白
毕赤酵母
基因重组
基因工程
Sweet protein, Monellin, Yeast, Pichia pastoris, Expression
