新型冠状病毒木瓜样蛋白酶在大肠杆菌中的可溶表达与酶活性测定  

Soluble Expression and Enzymatic Activity Analysis of SARS-CoV-2 Papain-like Protease in Escherichia Coli

作　　者：闫干干 李淼 戚海燕 刘志成 闫浩浩 刘晓丽 刘晓平[1] 陈云雨 YAN Gangan;LI Miao;QI Haiyan;LIU Zhicheng;YAN Haohao;LIU Xiaoli;LIU Xiaoping;CHEN Yunyu(Institute for Drug Screening and Evaluation,Wannan Medical College,Wuhu 241002,China;Changchun Institute of Biological Products,Changchun 130012,China)

机构地区：[1]皖南医学院/药物筛选与评价研究所,安徽芜湖241002 [2]长春生物制品研究所,长春130012

出　　处：《中国现代应用药学》2022年第1期5-11,共7页Chinese Journal of Modern Applied Pharmacy

基　　金：国家自然科学基金项目(81703546);安徽省自然科学基金(1808085QH265);安徽省高校自然科学研究项目(KJ2019ZD30,KJ2021A0839,YJS20210549);安徽省重点研究与开发计划项目(202004a07020041)。

摘　　要：目的制备高活性的新型冠状病毒木瓜样蛋白酶(papain-like protease,PLpro),为PLpro小分子抑制剂高通量筛选模型的建立奠定基础。方法将密码子优化的新型冠状病毒PLpro基因连接到pET-28a载体中,构建重组质粒pET-28a-PLpro。将重组质粒转化到Escherichia coli Rosetta(DE3)感受态细胞中,以十二烷基磺酸钠-聚丙烯酰胺凝胶电泳法(SDS-PAGE)鉴定PLpro的原核表达。采用低温诱导策略进行PLpro的可溶表达后,以HisTrap^(TM)亲和层析柱分离纯化PLpro,再以荧光共振能量转移(fluorescence resonance energy transfer,FRET)法测定PLpro的米氏常数(Michaelis constant,K_(m))值及其水解活性。结果基因测序与双酶切试验结果表明,成功构建了重组质粒pET-28a-PLpro。SDS-PAGE结果表明,PLpro在大肠杆菌中呈可溶表达,且纯化的PLpro纯度>90%。FRET结果表明,纯化的PLpro具有良好的水解活性,其K_(m)值为25.38μmol·L^(−1)。结论新型冠状病毒PLpro在大肠杆菌中成功地进行了可溶表达,且具有良好的酶活性。OBJECTIVE To prepare the highly active SARS-CoV-2 papain-like protease(PLpro)for the development of a high-throughput screening assay to rapidly identify novel PLpro inhibitors.METHODS A codon-optimized SARS-CoV-2 PLpro gene was ligated into a pET-28a vector to construct a recombinant plasmid named by pET-28a-PLpro.After transformation into Escherichia coli Rosetta(DE3)competent cells,the soluble PLpro was expressed under a low condition and further identified by a sodium dodecyl sulfonate polyacrylamide gel electrophoresis(SDS-PAGE)assay.The soluble PLpro was purified by a HisTrap^(TM) column,and then the enzymatic activity and Michaelis constant(K_(m))value of purified PLpro were determined by fluorescence resonance energy transfer(FRET)assay.RESULTS The DNA sequence alignment and double digestion assay results showed that a recombinant plasmid pET-28a-PLpro was constructed successfully.SDS-PAGE assay showed that the soluble PLpro was soluble expressed in E.coli,and the purity of purified PLpro was>90%.Importantly,the purified PLpro exhibited a perfect proteolytic activity in the FRET assay,and the K_(m) value was 25.38μmol·L^(−1).CONCLUSION The soluble SARS-CoV-2 PLpro is successfully expressed in E.coli cells,and has an ideal enzymatic activity.

关 键 词：新型冠状病毒 木瓜样蛋白酶 原核表达 亲和层析 荧光共振能量转移 

分 类 号：R915[医药卫生—微生物与生化药学]