巴曲酶在毕赤酵母中的高效表达

目的研究巴曲酶在毕赤酵母菌中的表达。方法按Pichiapastoris偏好密码子人工合成巴曲酶全基因，克隆到酵母分泌型表达载体pPICZaA中，将重组载体酶切线性化后经电转化转入X-33。筛选鉴定转化子．经摇瓶发酵甲醇诱导，酵母菌分泌表达有凝血活性的巴曲酶。经SDS-PAGE电泳确定其分子量为33．0 kDa．免疫印迹证明重组巴曲酶具有天然巴曲酶的免疫活性。结果经发酵条件的优化．发酵罐的表达量达到25000Ku／L发酵液。从每升发酵液中可纯化出11．0mg重组巴曲酶。结论巴曲酶毕氏酵母菌成功的构建．为重组巴曲酶止血药的开发奠定了基础。

High Leel expression of Batroxobin,a Thrombin-like Enzyme, in Pichia pastoris

Objective Batroxobin was expressed in Pichia pastoris in order to be developed as haemostat. Methods Batroxobin gene was synthetic based on codon usage bias of Pichia pastoris. The gene was cloned into expression vector pPICZaA and transformed into Pichia pastoris X-33. The recombinant batroxobin was purified from fermentation broth,showing immunological and enzyme activity by western-blotting analysis and plasma clotting assay respec- tively. Results The molecular weight is about 33.0 kDa by SDS-PAGE analysis. The a- mount of recombinant batroxobin could achieve 25 Ku/ml fermentation broth by 30 L fermentor,and 11.0 mg recombinant batroxobin was purified from 1 L fermentation broth. Conclu- sion The successful construction of Pichia pastoris expressing recombinant batroxobin,establish the foundation of recombinant batroxobin being developed as haemostat.