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PMID: 16672368 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S.

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

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 103 ·No. 20 ·2006-05-16 ·Pages 7583-8

Boulware KT, Daugherty PS

Abstract

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.

MeSH Terms
Amino Acid Sequence Caspase 3 Caspases/metabolism Cell Separation Enteropeptidase/metabolism Flow Cytometry Fluorescent Dyes/metabolism Peptide Library Peptides/genetics,metabolism Recombinant Fusion Proteins/genetics,metabolism Substrate Specificity
Chemicals
Fluorescent Dyes Peptide Library Peptides Recombinant Fusion Proteins Enteropeptidase Caspase 3 Caspases
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Boulware Kevin T
Department of Chemical Engineering, University of California, Santa Barbara, CA 93106, USA.
Daugherty Patrick S
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.0511108103
Published
2006-05-16
Epub
2006-00-03
Pages
7583-8
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC1456804
Subset
IM
External Links
PubMed source DOI: 10.1073/pnas.0511108103 PMC Full Text View journal details
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