Abstract：
The main protease (Mpro) of SARS-CoV-2 is a essential enzyme that facilitates viral transcription and replication. Furthermore, the conservation of Mpro across different variants and its non-overlapping nature with human proteases make it an appealing target for therapeutic interventions against SARS-CoV-2. Multiple inhibitors specifically target Mpro to mitigate the infection caused by SARS-CoV-2. In the current study, successful cloning and expression of SARS-CoV-2 Mpro were achieved using two E. coli hosts, namely BL21-DE3 and BL21-DE3-RIL. By optimizing the conditions for induction, the expression of Mpro in the soluble fraction of E. coli was improved. Subsequently, Mpro was purified using affinity chromatography, yielding significantly higher quantities from the BL21-DE3-RIL strain compared to the BL21-DE3 strain, with the former producing nearly twice as much as the latter. The purified Mpro was further characterized by mass spectrometry, fluorescence spectroscopy and circular dichroism (CD). Through fluorescence quenching studies, it was discovered that both GC376 and chitosan, which are inhibitors of Mpro, induced structural changes in the purified Mpro protein. This indicates that the protein retained its functional activity even after being expressed in a bacterial host. Further, FRET-based assay highlighted that the enzymatic activity of Mpro was significantly reduced in presence of both GC376 and chitosan. Consequently, the utilization of optimal conditions and the BL21-DE3-RIL bacterial host facilitates the cost-effective production of Mpro on a large scale, enabling high yields. This production approach can be applied for the screening of potent therapeutic drugs, making it a valuable resource for drug development endeavors.
摘要：
SARS-CoV-2的主要蛋白酶(Mpro)是促进病毒转录和复制的必需酶。此外，Mpro在不同变体中的保守性及其与人类蛋白酶的非重叠性质使其成为针对SARS-CoV-2的治疗干预的有吸引力的靶标。多种抑制剂特异性靶向Mpro以减轻由SARS-CoV-2引起的感染。在目前的研究中,使用两个大肠杆菌宿主成功克隆和表达SARS-CoV-2Mpro，即BL21-DE3和BL21-DE3-RIL。通过优化诱导条件，Mpro在大肠杆菌可溶性部分的表达得到改善。随后，使用亲和层析纯化Mpro,与BL21-DE3菌株相比，BL21-DE3-RIL菌株的产量明显更高，前者的产量几乎是后者的两倍。纯化的Mpro通过质谱进一步表征，荧光光谱和圆二色性。通过荧光猝灭研究，发现GC376和壳聚糖，是Mpro的抑制剂，诱导纯化的Mpro蛋白的结构变化。这表明该蛋白质即使在细菌宿主中表达后仍保持其功能活性。Further,基于FRET的测定强调了在GC376和壳聚糖存在下Mpro的酶活性显著降低。因此,利用最佳条件和BL21-DE3-RIL细菌宿主有助于大规模生产Mpro，实现高收益。这种生产方法可用于筛选有效的治疗药物，使其成为药物开发工作的宝贵资源。
