Publication date: Oct 01, 2024

The SARS-CoV-2 main protease (M) plays a crucial role in virus amplification and is an ideal target for antiviral drugs. Currently, authentic M is prepared through two rounds of proteolytic cleavage. In this method, M carries a self-cleavage site at the N-terminus and a protease cleavage site followed by an affinity tag at the C-terminus. This article proposes a novel method for producing authentic M through single digestion. M was constructed by fusing a His tag containing TEV protease cleavage sites at the N-terminus. The expressed recombinant protein was digested by TEV protease, and the generated protein had a decreased molecular weight and significantly increased activity, which was consistent with that of authentic M generated by the previous method. These findings indicated that authentic M was successfully obtained. Moreover, the substrate specificity of M was investigated. M had a strong preference for Phe at position the P2, which suggested that the S2 subsite was an outstanding target for designing inhibitors. This article also provides a reference for the preparation of M for sudden coronavirus infection in the future.

Semantics

Original Article