定点突变巴曲酶在毕赤酵母中的克隆与表达  

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

作　　者：王宏英 徐梅 兰海英 杨宇 张宏杰 李娜 薛雁 薛百忠 

机构地区：[1]辽宁诺康生物制药有限责任公司,辽宁沈阳110071

出　　处：《蛇志》2011年第2期105-110,共6页Journal of Snake

摘　　要：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。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.

关 键 词：突变巴曲酶 类凝血酶 巴斯德毕赤酵母 克隆和表达 偏好密码子 套叠PCR 

分 类 号：Q78[生物学—分子生物学] R977.3[医药卫生—药品]