Engineering a novel endopeptidase based on SARS 3CL(pro)

Biotechniques. 2009 Dec;47(6):1029-32. doi: 10.2144/000113303.


A 3C-like protease (3CLpro) from the severe acute respiratory syndrome-coronavirus (SARS-CoV) is required for viral replication, cleaving the replicase polyproteins at 11 sites with the conserved Gln [downward arrow](Ser, Ala, Gly) sequences. In this study, we developed a mutant 3CLpro (T25G) with an expanded S1' space that demonstrates 43.5-fold better k(cat)/K(m) compared with wild-type in cleaving substrates with a larger Met at P1' and is suitable for tag removal from recombinant fusion proteins. Two vectors for expressing fusion proteins with the T25G recognition site (Ala-Val-Leu-Gln [downward arrow]Met) in Escherichia coli and yeast were constructed. Identical cloning sites were used in these vectors for parallel cloning. PstI was chosen as a 5' cloning site because it overlapped the nucleotide sequence encoding the protease site and avoided addition of extra amino acids at the N terminus of recombinant proteins. 3CL(pro) (T25G) was found to have a 3-fold improvement over TEV(pro) in tag cleavage at each respective preferred cleavage site.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Coronavirus 3C Proteases
  • Cysteine Endopeptidases / chemistry
  • Cysteine Endopeptidases / genetics*
  • Cysteine Endopeptidases / metabolism
  • Endopeptidases / metabolism
  • Escherichia coli
  • Genetic Vectors / genetics
  • Kinetics
  • Molecular Sequence Data
  • Mutant Proteins / chemistry
  • Mutant Proteins / metabolism
  • Mutation / genetics
  • Protein Engineering / methods*
  • Protein Structure, Secondary
  • Recombinant Fusion Proteins / metabolism
  • Saccharomyces cerevisiae
  • Substrate Specificity
  • Viral Proteins / chemistry
  • Viral Proteins / genetics*
  • Viral Proteins / metabolism


  • Mutant Proteins
  • Recombinant Fusion Proteins
  • Viral Proteins
  • Endopeptidases
  • TEV protease
  • Cysteine Endopeptidases
  • Coronavirus 3C Proteases