Optimization of the expression of the main protease from SARS-CoV-2

The main protease (M^pro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a vital role in viral replication. To study the function of M^pro and screen inhibitors targeting M^pro, it is necessary to prepare high-purity and high-activity M^pro. In this study, four types of SARS-CoV-2 M^pros containing different termini were prepared, and their activities were determined successfully. The results showed that the activity of wild-type (WT) M^pro was the highest, and the additional residues at the N-terminus but not at the C-terminus had a major effect on the enzyme activity. To explain this, the alignment of structures of different forms of M^pro was determined, and the additional residues at the N-terminus were found to interfere with the formation of the substrate binding pocket. This study confirms the importance of the natural N-terminus to the activity of M^pro and suggests that WT-GPH₆ (M^pro with eight additional residues at the C-terminus) can be used as a substitute for authentic M^pro to screen inhibitors. In short, this study provides a reference for the expression and purification of new coronaviruses confronted in the future.