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

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 ͞ERM, 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.caspase-3 ͉ enteropeptidase ͉ FACS ͉ peptidase P roteolytic enzymes play central roles in most biological processes. At the same time, proteases are implicated in the pathogenesis of diverse diseases ranging from viral and bacterial infections (1) to cancer and neurodegenerative diseases (2, 3). The biological functions of proteases, or peptidases, are largely determined by the spectrum of biologically occurring substrates. Frequently, substrate specificity has been inferred by comparison and alignment of known, physiological substrates (4). Given this information, individually synthesized fluorogenic peptide substrates have been used extensively to measure protease activity (5). More recently, the substrate specificity of a small group of proteases has been characterized in greater detail by using combinatorial peptide libraries (6, 7). Library-based approaches to characterize specificity have contributed substantially to our understanding of protease function (6, 7). Nevertheless, quantitative characterization of protease specificity and substrate cleavage kinetics remains a bottleneck in basic and applied biological research (6,8).Among combinatorial methods, the ''substrate phage'' approach has been used to define the substrate specificities of Ϸ30 proteases and has been recently reviewed (6). In this approach, a library of peptide substrates is displayed on the surface of filamentous bacteriophage (9) as fusions between a surface-anchoring affinity domain and the display scaffold protein, typically the phage minor coat protein GPIII. The substrate library then is allowed to bind to a solid surface via the affinity domain. Phage liberated from the surface by proteolytic cleavage are rec...

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