ABSTRACT
Coronavirus disease-19 (COVID-19) pandemic caused millions of deaths and socio-economics damage worldwide. Corresponding to this, many studies on antiviral drugs exploration are rising to investigate the potential of drug compounds that can repress the replication of the SARS-CoV-2 virus by targeting its main protease named 3-chymotrypsin like protease (3CLpro). Without 3CLpro splicing, polyproteins of SARS-CoV-2 will not function to form new virions. We conducted an in silico thermodynamic study, and found several already known compounds from natural sources such as lovastatin, quinidine, and quinine were potential in inhibiting 3CLpro. However, most of the findings still need further wet-lab experiments to confirm their activity against it. To facilitate rapid screening of protease inhibitors, we aim to develop a screening kit for researchers with focus studies on herbal sources for COVID-19 treatments. Hence, the development of 3CLpro as the target of inhibition screening is essential. Our current work focused on the expression of the SARS-CoV-2 3CL Pro gene harbored by pET-32b(+) in E. coli BL21(DE3) and its purification. The recombinant E.coli were cultivated in LB media at 37 °C with 5 hours IPTG-induced phase followed by crude protein lysate extraction by sonication, and then Ni-NTA purification. A band of recombinant 3CLpro protein (33.8 kDa) detected by SDS PAGE and Western blotting after Ni-NTA purification was specific and clear, showing the right size of our protein interest. A final concentrated protein mixture obtained by 10-kDa membrane filtration displayed >90% purity with a total protein of 2.497 mg mL-1. Further functional assays and inhibitory tests with several natural compounds showed that 3CLpro functioned well as a drug target. © Published under licence by IOP Publishing Ltd.
ABSTRACT
The SARS-CoV-2 virus that caused pandemic COVID-19 has spread rapidly to humans and causes a very serious health and social problem around the world. In order to develop vaccine and therapeutic approaches, a comprehensive study of viral structure and viral biological infection needs to be understood. In addition to four structural proteins;spike glycoprotein (S), envelope (E), membrane protein (M), and nucleocapsid protein (N), SARS-CoV-2 viral genome, consist of two open reading frame (ORF) which is located in N-terminus, ORF1a, and ORF1b. The ORF1a consist of Main protease (3CLpro) and together with papain-like protease (PLpro) play a role in the cleavage of polyproteins and translated from viral RNA to mature protein. Here we describe a method of production of 6xHis-tagged Papain-like protease fragment in E. coli RIPL, including gene expression, solubilization of inclusion bodies by different methods, and detection of gene product target by Western Blot Analyses. © Published under licence by IOP Publishing Ltd.