National Institute of Immunology, New Delhi, 110067, India.
National Institute of Immunology, New Delhi, 110067, India.
Protein Expr Purif. 2023 Nov;211:106337. doi: 10.1016/j.pep.2023.106337. Epub 2023 Jul 14.
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.
新冠病毒主要蛋白酶（Mpro）是一种至关重要的酶，它促进病毒转录和复制。此外，Mpro 在不同变体中的保守性以及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。多种抑制剂专门针对 Mpro，以减轻由 SARS-CoV-2 引起的感染。本研究成功地使用两种大肠杆菌宿主 BL21-DE3 和 BL21-DE3-RIL 对 SARS-CoV-2 Mpro 进行了克隆和表达。通过优化诱导条件，提高了 Mpro 在大肠杆菌可溶性部分中的表达。随后，通过亲和层析对 Mpro 进行纯化，与 BL21-DE3 菌株相比，BL21-DE3-RIL 菌株产生的 Mpro 数量明显更高，前者的产量几乎是后者的两倍。通过质谱、荧光光谱和圆二色性（CD）进一步对纯化的 Mpro 进行了表征。通过荧光猝灭研究发现，Mpro 的抑制剂 GC376 和壳聚糖均诱导了纯化的 Mpro 蛋白的结构变化。这表明即使在细菌宿主中表达，该蛋白仍保留其功能活性。此外，基于 FRET 的测定突出表明，GC376 和壳聚糖的存在显著降低了 Mpro 的酶活性。因此，利用最佳条件和 BL21-DE3-RIL 细菌宿主可以在大规模上促进 Mpro 的经济高效生产，实现高产量。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。
