title: 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 Angstroms and 1.95 Angstroms 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.
authors.affiliation: Tsinghua-Nankai-IBP Joint Research Group for Structural Biology, Tsinghua University, Beijing, China.
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Xue
authors.forename: Haitao
authors.initials: H
authors.lastname: Yang
authors.forename: Wei
authors.initials: W
authors.lastname: Shen
authors.forename: Qi
authors.initials: Q
authors.lastname: Zhao
authors.forename: Jun
authors.initials: J
authors.lastname: Li
authors.forename: Kailin
authors.initials: K
authors.lastname: Yang
authors.forename: Cheng
authors.initials: C
authors.lastname: Chen
authors.forename: Yinghua
authors.initials: Y
authors.lastname: Jin
authors.forename: Mark
authors.initials: M
authors.lastname: Bartlam
authors.forename: Zihe
authors.initials: Z
authors.lastname: Rao
mesh_terms: D064026:Calbindins; D000086862:Coronavirus M Proteins; D010450:Endopeptidases / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D064030:S100 Calcium Binding Protein G / Q000302:isolation & purification; D045473:Severe acute respiratory syndrome-related coronavirus; D014763:Viral Matrix Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism*; D014764:Viral Proteins / Q000096:biosynthesis*
chemical_list: D064026:Calbindins; D000086862:Coronavirus M Proteins; C000706237:M protein, SARS-CoV; D011993:Recombinant Fusion Proteins; D064030:S100 Calcium Binding Protein G; D014763:Viral Matrix Proteins; D014764:Viral Proteins; D005982:Glutathione Transferase; D010450:Endopeptidases