Improved expression and purification of highly-active 3 chymotrypsin-like protease from SARS-CoV-2.

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 /K _m 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 _cat /K _m 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.
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