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(pro)s 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-GPH(6) (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.