Nguyen, Hong Loan ThiHong Loan ThiNguyenNguyen, Nhu Quynh T.Nhu Quynh T.NguyenLe, The ThaiThe ThaiLePham, Xuan Dieu T.Xuan Dieu T.PhamPham, Hai LongHai LongPhamLe Thi Hong, NhungNhungLe Thi HongPhan, Tuan NghiaTuan NghiaPhanDinh, Nho ThaiNho ThaiDinh2025-09-142025-09-14202410.1016/j.pep.2023.106414https://scholar.vnu.edu.vn/handle/123456789/8652Severe 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<inf>cat</inf>/K<inf>m</inf> 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 K<inf>cat</inf>/K<inf>m</inf> 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. © 2023 Elsevier B.V., All rights reserved.English3 Chymotrypsin-like ProteaseC41(de3)Petm33PurificationSars-cov-2ChymasePeptide HydrolaseProteinase InhibitorViral ProteaseAntiviral AgentsChymasesPeptide HydrolasesProtease InhibitorsAntivirus AgentChymasePeptide HydrolaseProteinase InhibitorCoronavirus Disease 2019GeneticsHumanMetabolismMolecular DockingPandemicProspective StudySevere Acute Respiratory Syndrome Coronavirus 2Antiviral AgentsChymasesCovid-19HumansMolecular Docking SimulationPandemicsPeptide HydrolasesProspective StudiesProtease InhibitorsSars-cov-2Article