A tobacco etch virus protease with increased substrate tolerance at the P1' position

PLoS One. 2013 Jun 24;8(6):e67915. doi: 10.1371/journal.pone.0067915. Print 2013.

Abstract

Site-specific proteases are important tools for in vitro and in vivo cleavage of proteins. They are widely used for diverse applications, like protein purification, assessment of protein-protein interactions or regulation of protein localization, abundance or activity. Here, we report the development of a procedure to select protease variants with altered specificity based on the well-established Saccharomyces cerevisiae adenine auxotrophy-dependent red/white colony assay. We applied this method on the tobacco etch virus (TEV) protease to obtain a protease variant with altered substrate specificity at the P1' Position. In vivo experiments with tester substrates showed that the mutated TEV protease still efficiently recognizes the sequence ENLYFQ, but has almost lost all bias for the amino acid at the P1' Position. Thus, we generated a site-specific protease for synthetic approaches requiring in vivo generation of proteins or peptides with a specific N-terminal amino acid.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acids / metabolism*
  • Endopeptidases / chemistry
  • Endopeptidases / metabolism*
  • Models, Molecular
  • Molecular Sequence Data
  • Mutant Proteins / metabolism
  • Mutation / genetics
  • Proteolysis
  • Substrate Specificity

Substances

  • Amino Acids
  • Mutant Proteins
  • Endopeptidases
  • TEV protease

Grants and funding

This work was supported by the DFG grants GK1216 "Intra- and Intercellular Transport and Communication" and TA320/3-1. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.