抗自溶烟草蚀纹病毒蛋白酶的原核表达和纯化及活性分析  

Prokaryotic Expression,Purification and Activity Analysis of Auto-lysis Resistance Tobacco Etch Virus Protease

作　　者：陈鹏[1] 石小青[1] 

机构地区：[1]西北农林科技大学生命科学学院,陕西杨凌712100

出　　处：《西北农业学报》2011年第12期183-187,共5页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：国家自然科学基金(31171606);西北农林科技大学基本科研业务费(QN2009064)

摘　　要：利用QuikChangeSite-Directed Mutagenesis Kit将自溶性烟草蚀纹病毒蛋白酶(tobacco etch virus Protease,TEVP)219位的Ser(S)突变为Val(V),经测序验证的S219V突变质粒pMAL-TEVPS219V电转化大肠杆菌BL21Star(DE3),并优化异丙基硫代β-D半乳糖苷(IPTG)诱导浓度、温度、时间对TEVP表达的影响。结果表明,突变的TEVP表达最适条件为24℃,0.75mmol/L IPTG诱导11h。经金属离子螯合层析纯化得到高纯度,高活性的突变TEVP蛋白酶,比活力为1 048U/mg。每升菌液可纯化17.2mg目标蛋白。The amino acid 219 serine residue of auto-lysis liable tobacco etch virus protease（TEVP）was mutated to valine by QuikChange Site-Directed Mutagenesis Kit.The verified mutant plasmid pMAL-TEVPS219V was transformed into Escherichia coli BL21 Star（DE3）and the expression condition were optimized.The results showed recombinant prokaryotic expression vector of mutant TEVP was successfully constructed.The optimal expression conditions were 24 ℃,0.75 mmol/L IPTG with 11 hours induction.The recombinant TEVP was purified with Talon resin.From 1 L culture medium 17.2 mg TEVP could be purified with a specific activity of 1 048 U.

关 键 词：原核表达 TEV蛋白酶 定点突变 纯化 生物活性 

分 类 号：Q943.2[生物学—植物学]