定点突变巴曲酶在毕赤酵母中的克隆与表达

目的 为避免毕赤酵母分泌的Kex2蛋白酶对发酵液中所表达的巴曲酶的降解.利用重叠PCR方法对巴曲酶基因进行定点突变,将巴曲酶基因第45位的Arg突变为Lys.方法 将突变后的基因克隆到酵母分泌型表达载体pPICZαA中,将重组载体酶切线性化后经电转化转入巴斯德毕赤酵母细胞,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定点突变巴曲酶,经SDS-PAG电泳、免疫印迹确定其分子量为32 kD.结果发酵罐的表达量达到52 KU/ml发酵液,较重组天然巴曲酶的表达量提高了73.3%.结论 定点突变巴曲酶的表达量比重组天然巴曲酶的表达量有显著提高,表达的突变巴曲酶同样具有凝血活性.

Cloning and Expression of mutant Batroxobin,a Thrombin-like Enzyme,in Pichia pastoris by Overlap Extension PCR

Objective In order to avoid secreted batroxobin expressed in p. pichia X-33 being hydrolysis by Kex2 protease,the gene of batroxobin,a thrombin-like enzyme of bathrops atrox moojeni venom, was site-directed mutated by overlap extension of PCR. The arginine residues at positions 45 of the protein product are replaced by basic amino acid residues Lys, which is Kex2 protease site. Methods The mutant gene of batroxobin was cloned into expression vector pPICZaA and transfered into Pichia pastoris X-33. The recombinant mutant batroxobin was separated and purified from fermentation liquid, showing immunological and enzyme activity by western-blotting analysis and fibrinogen-clotting assay respectively. The molecular weight is about 32.0 kD by SDS-PAGE analysis. Results The production of mutant recombinant batroxobin could achieve 52 KU/ml fermentation liquide by fermentor, the yield rise by 73.3% compared with recombinant batroxobin. Conclusion Compared with recombinant batroxobin,the production of site-directed mutant batroxobin significantly increased. The production of mutant batroxobin＇also possessed coagulation activity.