Li Zhao-Fa, Yu Xue-Ling, Huang Jin-Lu, Fang Hong-Qing, Chen Hui-Peng
Institute of Bioengineering, Academy of Military Medical Science, Beijing 100071, China.
Sheng Wu Gong Cheng Xue Bao. 2007 May;23(3):483-6.
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. 10 mg 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.
利用甲基营养型酵母毕赤酵母表达重组巴曲酶，最终建立了一条重组蛋白的生产技术路线。我们通过递归PCR人工合成了巴曲酶基因。构建了pPIC9-巴曲酶并将其转化到毕赤酵母GS115（his4）中。重组巴曲酶在酵母工程菌中表达，并从培养上清中纯化得到。从1升发酵培养基中纯化得到10毫克重组巴曲酶，其比活性为238 NIH单位/毫克，分子量为30.55 kD。纯化的重组蛋白在体外能将纤维蛋白原转化为纤维蛋白凝块，在体内能缩短出血时间。本研究为重组蛇毒类凝血酶止血剂的开发奠定了基础。
