一种铜锌超氧化物歧化酶突变体的原核表达、纯化及酶活性测定  

Prokaryotic expression,purification and enzyme activity of a copper-zinc superoxide dismutase(Cu,Zn-SOD) mutant

作　　者：唐剑光[1] 张健锋[1] 常东英[1] 曹玉锋[1] 乔宏雷[1] 高洪喜[1] 迟春萍[1] TANG Jian-guang;ZHANG Jian-feng;CHANG Dong-ying;CAO Yu-feng;QIAO Hong-lei;GAO Hong-xi;CHI Chun-ping(Changchun Institute of Biological Products,Changchun 130062,Jilin Province,China)

机构地区：[1]长春生物制品研究所,吉林长春130062

出　　处：《中国生物制品学杂志》2020年第6期614-618,共5页Chinese Journal of Biologicals

基　　金：吉林省科学技术发展重点项目(20070924-02)。

摘　　要：目的原核表达、纯化一种铜锌超氧化物歧化酶(Cu,Zn-SOD)突变体,并检测其酶活性。方法将经热力学优化的Cu,Zn-SOD突变体基因亚克隆至pET-28a载体中,构建重组表达质粒SOD1-pET-28a,转化入大肠埃希菌BL21(DE3)中,IPTG诱导表达;在500 mL摇瓶培养条件下,通过改变加入Cu、Zn离子的量优化诱导表达条件;表达的重组蛋白经初步纯化后,测定酶活性。结果重组表达质粒SOD-pET-28a经双酶切及测序证明构建正确;表达的重组蛋白相对分子质量约为15000,主要以可溶性形式表达;工程菌株在500 mL摇瓶培养条件下培养至10 h左右,A600至3.5左右时加入终浓度为0.1 mmol/L IPTG、0.2 mmol/L ZnCl2、0.5 mmol/L CuSO4,可获得最佳表达量(29.2%);初步纯化获得的重组蛋白纯度为97.32%,比活为5.08×103U/mg。结论成功利用大肠埃希菌表达系统表达了一种经热力学优化的Cu,Zn-SOD突变体,初步纯化的突变体具有较好的酶活性,为进一步摸索大规模生产工艺及实际应用奠定了基础。Objective To express a copper-zinc superoxide dismutase(Cu,Zn-SOD)mutant in prokaryotic cells,purify the expressed product and determine its enzyme activity.Methods The thermodynamically optimized SOD mutant gene was subcloned into vector pET-28 a,and the constructed recombinant plasmid SOD1-pET-28 a was transformed to E.coli BL21(DE3)for expression under induction of IPTG.The condition for expression was optimized by changing the amounts of copper and zinc ions added into 500 mL shake flask culture.The expressed recombinant protein was purified and determined for enzyme activity.Results Restriction analysis and sequencing proved that recombinant plasmid SOD-pET-28 a was constructed correctly.The expressed recombinant protein,with a relative molecular mass of about 15000,mainly existed in a soluble form.The expression level reached a maximum of 29.2%after the engineered strain was cultured in a 500 mL shake flask for about 10 h and IPTG,zinc chloride and copper sulphate were added to final concentrations of 0.1,0.2 and 0.5 mmol/L respectively when the A600 value reached about 3.5.The purity and specific activity of purified recombinant protein were 97.32%and 5.08×103 U/mg respectively.Conclusion A thermodynamically optimized Cu,Zn-SOD mutant was successfully expressed by using E.coli expression system,and showed good enzymatic activity after preliminary purification,which laid a foundation of further study on large-scale production process and practical application.

关 键 词：铜锌超氧化物歧化酶 原核表达 层析 酶活性 

分 类 号：Q786[生物学—分子生物学]