重组巴曲酶在毕赤酵母中的高效表达  

Recombinant Batroxobin Expressed Highly in Pichia pastoris

作　　者：李招发[1] 于学玲[1] 黄金路[1] 方宏清[1] 陈惠鹏[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071

出　　处：《生物工程学报》2007年第3期483-486,共4页Chinese Journal of Biotechnology

摘　　要：以毕赤酵母为表达系统,建立生产重组巴曲酶的技术工艺路线。通过递归式PCR的方法,人工合成了巴曲酶基因,将其插入pPIC9表达质粒中,转化至毕赤酵母GS115(his4),筛选出的表达株经甲醇诱导,表达了重组巴曲酶,并得以纯化。从每升发酵液中可纯化得到10mg重组巴曲酶,其比活为238NIHunits/mg,分子量为30.55kD。重组巴曲酶在体外可使纤维蛋白凝固,在体内缩短小鼠出血时间。为开发重组的蛇毒类凝血酶止血剂打下了基础。Methylotrophic yeast, Pichia pastoris was used to express recombinant batroxobin, and a technology route of producing recombinant protein was finally established. We synthesized batroxobin gene artificially by means of recursive PCR. pPIC9-batroxobin was constructed and transformed into Pichia pastoris GS115 （ his4）. Recombinant batroxobin was expressed in yeast engineering strain and it was purified from the culture supernatant. 10mg of recombinant batroxobin was purified from 1 liter fermentation media, it exhibited specific activity of 238 NIH units/mg and had molecular weight of 30.55 kD. The purified recombinant protein converted fibrinogen into fibrin clot in vitro, and shortened bleeding time in vivo. This study laid a foundation of development of hemostatic of recombinant snake venom thrombin-like enzyme.

关 键 词：毕赤酵母 重组巴曲酶 蛇毒类凝血酶 止血剂 

分 类 号：Q78[生物学—分子生物学]