tetano
Editor, Senior Moderator
Protein Expr Purif
. 2023 Dec 8:106414.
doi: 10.1016/j.pep.2023.106414. Online ahead of print. Improved expression and purification of highly-active 3 chymotrypsin-like protease from SARS-CoV-2
Hong-Loan T Nguyen[SUP] 1 [/SUP], Nhu-Quynh T Nguyen[SUP] 2 [/SUP], The-Thai Le[SUP] 2 [/SUP], Xuan-Dieu T Pham[SUP] 2 [/SUP], Hai-Long Pham[SUP] 2 [/SUP], Hong-Nhung T Le[SUP] 1 [/SUP], Tuan-Nghia Phan[SUP] 1 [/SUP], Nho-Thai Dinh[SUP] 3 [/SUP]
Affiliations
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[SUB]cat[/SUB]/K[SUB]m[/SUB] of 0.007 μM[SUP]-1[/SUP].s-[SUP]1[/SUP]) than that of the 3CLpro (com3CLpro) expressed from the commercial vector (pETM33-Nsp5-Mpro) with specific activity 889 RFU/min/μg and K[SUB]cat[/SUB]/K[SUB]m[/SUB] of 0.0015 μM[SUP]-1[/SUP].s-[SUP]1[/SUP], 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.
Keywords: 3 chymotrypsin-like protease; C41(DE3); Purification; SARS-CoV-2; pETM33.
. 2023 Dec 8:106414.
doi: 10.1016/j.pep.2023.106414. Online ahead of print. Improved expression and purification of highly-active 3 chymotrypsin-like protease from SARS-CoV-2
Hong-Loan T Nguyen[SUP] 1 [/SUP], Nhu-Quynh T Nguyen[SUP] 2 [/SUP], The-Thai Le[SUP] 2 [/SUP], Xuan-Dieu T Pham[SUP] 2 [/SUP], Hai-Long Pham[SUP] 2 [/SUP], Hong-Nhung T Le[SUP] 1 [/SUP], Tuan-Nghia Phan[SUP] 1 [/SUP], Nho-Thai Dinh[SUP] 3 [/SUP]
Affiliations
- PMID: 38072143
- DOI: 10.1016/j.pep.2023.106414
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[SUB]cat[/SUB]/K[SUB]m[/SUB] of 0.007 μM[SUP]-1[/SUP].s-[SUP]1[/SUP]) than that of the 3CLpro (com3CLpro) expressed from the commercial vector (pETM33-Nsp5-Mpro) with specific activity 889 RFU/min/μg and K[SUB]cat[/SUB]/K[SUB]m[/SUB] of 0.0015 μM[SUP]-1[/SUP].s-[SUP]1[/SUP], 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.
Keywords: 3 chymotrypsin-like protease; C41(DE3); Purification; SARS-CoV-2; pETM33.