Protease specificity determination by using cellular libraries of peptide substrates (CLiPS)

Proc Natl Acad Sci U S A. 2006 May 16;103(20):7583-8. doi: 10.1073/pnas.0511108103. Epub 2006 May 3.


We report a general combinatorial approach to identify optimal substrates of a given protease by using quantitative kinetic screening of cellular libraries of peptide substrates (CLiPS). A whole-cell protease activity assay was developed by displaying fluorescent reporter substrates on the surface of Escherichia coli as N-terminal fusions. This approach enabled generation of substrate libraries of arbitrary amino acid composition and length that are self-renewing. Substrate hydrolysis by a target protease was measured quantitatively via changes in whole-cell fluorescence by using FACS. FACS enabled efficient screening to identify optimal substrates for a given protease and characterize their cleavage kinetics. The utility of CLiPS was demonstrated by determining the substrate specificity of two unrelated proteases, caspase-3 and enteropeptidase (or enterokinase). CLiPS unambiguously identified the caspase-3 consensus cleavage sequence DXVDG. Enteropeptidase was unexpectedly promiscuous, but exhibited a preference for substrates with the motif (D/E)RM, which were cleaved substantially faster than the canonical DDDDK recognition sequence, widely used for protein purification. CLiPS provides a straightforward and versatile approach to determine protease specificity and discover optimal substrates on the basis of cleavage kinetics.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Caspase 3
  • Caspases / metabolism*
  • Cell Separation
  • Enteropeptidase / metabolism*
  • Flow Cytometry
  • Fluorescent Dyes / metabolism
  • Peptide Library*
  • Peptides / genetics
  • Peptides / metabolism*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Substrate Specificity


  • Fluorescent Dyes
  • Peptide Library
  • Peptides
  • Recombinant Fusion Proteins
  • Enteropeptidase
  • Caspase 3
  • Caspases