| 引用本文: | 闫干干,李淼,戚海燕,刘志成,闫浩浩,刘晓丽,刘晓平,陈云雨.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[J].中国现代应用药学,2022,39(1):5-11. |
| YAN Gangan,LI Miao,QI Haiyan,LIU Zhicheng,YAN Haohao,LIU Xiaoli,LIU Xiaoping,CHEN Yunyu.Soluble Expression and Enzymatic Activity Analysis of SARS-CoV-2 Papain-like Protease in Escherichia Coli[J].Chin J Mod Appl Pharm(中国现代应用药学),2022,39(1):5-11. |
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| 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HisTrapTM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，Km)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Km值为25.38 μmol·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。 |
| 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 |
| DOI：10.13748/j.cnki.issn1007-7693.2022.01.002 |
| 分类号:R915 |
| 基金项目: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) |
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| Soluble Expression and Enzymatic Activity Analysis of SARS-CoV-2 Papain-like Protease in Escherichia Coli |
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YAN Gangan1, LI Miao2, QI Haiyan1, LIU Zhicheng1, YAN Haohao1, LIU Xiaoli1, LIU Xiaoping1, CHEN Yunyu1
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1.Institute for Drug Screening and Evaluation, Wannan Medical College, Wuhu 241002, China;2.Changchun Institute of Biological Products, Changchun 130012, China
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| Abstract: |
| 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 HisTrapTM column, and then the enzymatic activity and Michaelis constant(Km) 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 Km value was 25.38 μmol·L-1.CONCLUSION The soluble SARS-CoV-2 PLpro is successfully expressed in E. coli cells, and has an ideal enzymatic activity. |
| Key words: SARS-CoV-2 papain-like protease prokaryotic expression affinity chromatography fluorescence resonance energy transfer |
