Production of Authentic SARS-CoV M ^pro with Enhanced Activity: Application as a Novel Tag-cleavage Endopeptidase for Protein Overproduction

Abstract

The viral proteases have proven to be the most selective and useful for removing the fusion tags in fusion protein expression systems. As a key enzyme in the viral life-cycle, the main protease (M ^pro) is most attractive for drug design targeting the SARS coronavirus (SARS-CoV), the etiological agent responsible for the outbreak of severe acute respiratory syndrome (SARS) in 2003. In this study, SARS-CoV M ^pro was used to specifically remove the GST tag in a new fusion protein expression system. We report a new method to produce wild-type (WT) SARS-CoV M ^pro with authentic N and C termini, and compare the activity of WT protease with those of three different types of SARS-CoV M ^pro with additional residues at the N or C terminus. Our results show that additional residues at the N terminus, but not at the C terminus, of M ^pro are detrimental to enzyme activity. To explain this, the crystal structures of WT SARS-CoV M ^pro and its complex with a Michael acceptor inhibitor were determined to 1.6 Å and 1.95 Å resolution respectively. These crystal structures reveal that the first residue of this protease is important for sustaining the substrate-binding pocket and inhibitor binding. This study suggests that SARS-CoV M ^pro could serve as a new tag-cleavage endopeptidase for protein overproduction, and the WT SARS-CoV M ^pro is more appropriate for mechanistic characterization and inhibitor design.