Chen Yunyu, Fu Zhenghao, Yan Gangan, Lin Yuan, Liu Xiaoping
Institute for Drug Screening and Evaluation, Wannan Medical College, Wuhu 241002, Anhui, China.
State Key Laboratory of Bioactive Substances and Function of Natural Medicine, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
Sheng Wu Gong Cheng Xue Bao. 2021 Apr 25;37(4):1334-1345. doi: 10.13345/j.cjb.200416.
The main protease (Mpro) of SARS-CoV-2 is a highly conserved and mutation-resistant coronaviral enzyme, which plays a pivotal role in viral replication, making it an ideal target for the development of novel broad-spectrum anti-coronaviral drugs. In this study, a codon-optimized Mpro gene was cloned into pET-21a and pET-28a expression vectors. The recombinant plasmids were transformed into E. coli Rosetta(DE3) competent cells and the expression conditions were optimized. The highly expressed recombinant proteins, Mpro and Mpro-28, were purified by HisTrapTM chelating column and its proteolytic activity was determined by a fluorescence resonance energy transfer (FRET) assay. The FRET assay showed that Mpro exhibits a desirable proteolytic activity (25 000 U/mg), with Km and kcat values of 11.68 μmol/L and 0.037/s, respectively. The specific activity of Mpro is 25 times that of Mpro-28, a fusion protein carrying a polyhistidine tag at the N and C termini, indicating additional residues at the N terminus of Mpro, but not at the C terminus, are detrimental to its proteolytic activity. The preparation of active SARS-CoV-2 Mpro through codon-optimization strategy might facilitate the development of the rapid screening assays for the discovery of broad-spectrum anti-coronaviral drugs targeting Mpro.
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的主要蛋白酶（Mpro）是一种高度保守且抗突变的冠状病毒酶，在病毒复制中起关键作用，这使其成为开发新型广谱抗冠状病毒药物的理想靶点。在本研究中，将密码子优化的Mpro基因克隆到pET-21a和pET-28a表达载体中。将重组质粒转化到大肠杆菌Rosetta(DE3)感受态细胞中，并优化表达条件。通过HisTrapTM螯合柱纯化高表达的重组蛋白Mpro和Mpro-28，并通过荧光共振能量转移（FRET）测定法测定其蛋白水解活性。FRET测定法表明，Mpro表现出理想的蛋白水解活性（25000 U/mg），Km和kcat值分别为11.68 μmol/L和0.037/s。Mpro的比活性是Mpro-28（一种在N端和C端带有多组氨酸标签的融合蛋白）的25倍，这表明Mpro的N端而非C端的额外残基对其蛋白水解活性有害。通过密码子优化策略制备活性SARS-CoV-2 Mpro可能有助于开发针对Mpro的广谱抗冠状病毒药物的快速筛选测定法。
