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As the number of identified proteases continues to grow—to date, nearly 700 have been found in the human proteome—the need for a reliable and high-throughput method for characterizing target-sequence specificity has become more and more apparent. A variety of screens have been tested in recent years, including methods based on viral display, pooled substrate libraries and peptide arrays, and these have yielded some valuable information regarding the target specificity and kinetics of previously uncharacterized proteases.

University of California at Santa Barbara researcher Patrick Daugherty and graduate student Kevin Boulware have now added another promising method to the proteomics toolbox, based on the fluorescence-assisted cell sorting (FACS) of a bacterial display library. Their library is composed of Escherichia coli cells expressing chimeric cell-surface proteins, which consist of a streptavidin-binding peptide linked to a variable six-residue 'substrate' region. The library is labeled with a fluorescent streptavidin-conjugated phycoerythrin probe, which binds to the chimeric display protein; then fluorescently tagged cells are treated with a protease of interest. Cells expressing suitable target sequences lose their fluorescence as a result of protease cleavage, and can be sorted via FACS for expansion and characterization. Boulware and Daugherty have termed this method of screening with cellular libraries of peptide substrates 'CLiPS'.

They tested CLiPS with two proteases for which canonical substrates have been identified, caspase-3 and enteropeptidase. After multiple rounds of sorting, they identified several individual clones as containing appropriate substrate sequences for each protease. DNA sequencing of clones with the most efficient cleavage kinetics revealed a caspase-3 consensus target sequence, which closely matched the previously identified canonical target, DxVDG; in contrast, the team was surprised to identify several enteropeptidase substrates that differed substantially from but were cleaved more efficiently than the established DDDDK target sequence. They subsequently confirmed the relative kinetics of the different target sequences in additional assays with synthetic fluorogenic peptide substrates.

The authors tout their method as a rapid means for examining the specificity and cleavage kinetics of proteases, and suggest that it should be likewise extensible for use with mammalian or yeast cells, which could potentially allow investigators to examine the impact of post-translational modifications on cleavage.