1. Improved expression and purification of highly-active 3 chymotrypsin-like protease from SARS-CoV-2.
- Author
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Nguyen HT, Nguyen NT, Le TT, Pham XT, Pham HL, Le HT, Phan TN, and Dinh NT
- Subjects
- Humans, Chymases, Pandemics, Prospective Studies, Peptide Hydrolases metabolism, Protease Inhibitors, Antiviral Agents therapeutic use, Molecular Docking Simulation, SARS-CoV-2 genetics, SARS-CoV-2 metabolism, COVID-19
- Abstract
Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is the causative pathogen of coronavirus disease-19 (COVID-19). The COVID-19 pandemic has resulted in millions of deaths and widespread socio-economic damage worldwide. Therefore, numerous studies have been conducted to identify effective measures to control the spreading of the virus. Among various potential targets, the 3 chymotrypsin-like protease (3CLpro), also known as Mpro, stands out as the key protease of SARS-CoV-2, playing an essential role in virus replication and assembly, is the most prospective. In this study, we modified the commercial vector, pETM33-Nsp5-Mpro (plasmid # 156475, Addgene, USA), by inserting an autocleavage site (AVLQ) of 3CLpro and 6 × His-tag encoding sequences before and after the Nsp5-Mpro sequence, respectively. This modification enabled the expression of 3CLpro as an authentic N terminal protease (au3CLpro), which was purified to electrophoretic homogeneity by a single-step chromatography using two tandem Glutathione- and Ni-Sepharose columns. The enzyme au3CLpro demonstrated significantly higher activity (3169 RFU/min/μg protein) and catalytic efficiency (K
cat /Km of 0.007 μM-1 .s-1 ) than that of the 3CLpro (com3CLpro) expressed from the commercial vector (pETM33-Nsp5-Mpro) with specific activity 889 RFU/min/μg and Kcat /Km of 0.0015 μM-1 .s-1 , respectively. Optimal conditions for au3CLpro activity included a 50 mM Tris-HCl buffer at pH 7, containing 150 mM NaCl and 0.1 mg/ml BSA at 37 °C., Competing Interests: Declaration competing of interest The authors declare that they have no conflict of interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)- Published
- 2024
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