重组巴曲酶在毕赤酵母中的高效表达

Recombinant Batroxobin Expressed Highly in Pichia pastoris

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10 mg重组巴曲酶, 其比活为238 NIH units/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。

Abstract:

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.