Production of authentic SARS-CoV M(pro) with enhanced activity: application as a novel tag-cleavage endopeptidase for protein overproduction

J Mol Biol. 2007 Feb 23;366(3):965-75. doi: 10.1016/j.jmb.2006.11.073. Epub 2006 Dec 1.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Calbindins
  • Coronavirus M Proteins
  • Endopeptidases / metabolism*
  • Gene Expression
  • Genetic Vectors
  • Glutathione Transferase / metabolism
  • Recombinant Fusion Proteins / metabolism
  • S100 Calcium Binding Protein G / isolation & purification
  • SARS Virus
  • Viral Matrix Proteins / antagonists & inhibitors
  • Viral Matrix Proteins / biosynthesis*
  • Viral Matrix Proteins / chemistry
  • Viral Matrix Proteins / metabolism*
  • Viral Proteins / biosynthesis*

Substances

  • Calbindins
  • Coronavirus M Proteins
  • M protein, SARS-CoV
  • Recombinant Fusion Proteins
  • S100 Calcium Binding Protein G
  • Viral Matrix Proteins
  • Viral Proteins
  • Glutathione Transferase
  • Endopeptidases

Associated data

  • PDB/2HOB