基于密码子优化策略的新型冠状病毒主蛋白酶在大肠杆菌中的表达条件优化与活性鉴定

Optimization of expression conditions and determination the proteolytic activity of codon-optimized SARS-CoV-2 main protease in Escherichia coli

摘要:

新型冠状病毒主蛋白酶 (Main protease，Mpro) 在调控新冠病毒RNA复制中具有重要的生物学功能，且Mpro在冠状病毒中的进化高度保守并不易突变，已成为新型广谱抗冠状病毒药物开发的理想靶标之一。为了制备高纯度、高活性的Mpro，根据密码子偏爱性原则，将优化的Mpro基因分别连接到pET-21a与pET-28a表达载体中构建重组质粒。将重组质粒转化到大肠杆菌Escherichia coli Rosetta(DE3) 感受态细胞中，分别进行原核表达条件优化，所表达的重组蛋白质命名为Mpro与Mpro-28。Mpro与Mpro-28经HisTrapTM亲和层析法分离纯化后，以荧光共振能量转移 (Fluorescence resonance energy transfer，FRET) 实验进行生物学活性鉴定。FRET实验结果表明，纯化的Mpro具有良好的水解活性，Km值为11.68 μmol/L，kcat值为0.037/s，比活力不低于25 000 U/mg，约为Mpro-28的25倍，说明天然的氨基端对Mpro的生物学功能是必需的，羧基端残留的多聚组氨酸标签对其水解活性影响较小。文中基于密码子优化策略，成功地进行了新冠病毒Mpro在大肠杆菌中的表达条件优化与活性鉴定，为靶向Mpro广谱抗冠状病毒药物高通量筛选模型的建立奠定了实验基础。

Abstract:

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.