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

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Proteases are crucial enzymes involved in numerous biological processes.
• Identifying specific protease substrates is essential for understanding enzyme function and for biotechnological applications.
• Current methods for substrate identification can be limited in scope and efficiency.

Purpose of the Study:

• To develop a general combinatorial approach for identifying optimal protease substrates.
• To establish a quantitative kinetic screening method using cellular libraries of peptide substrates (CLiPS).
• To characterize the substrate specificity and cleavage kinetics of different proteases.

Main Methods:

• Developed a whole-cell protease activity assay using fluorescent reporter substrates displayed on Escherichia coli.
• Generated self-renewing substrate libraries of arbitrary amino acid composition and length.
• Utilized fluorescence-activated cell sorting (FACS) for quantitative measurement of substrate hydrolysis and kinetic characterization.

Main Results:

• CLiPS successfully identified the consensus cleavage sequence for caspase-3 as DXVDG.
• Enteropeptidase showed promiscuous substrate specificity but preferred (D/E)RM motifs over the canonical DDDDK sequence.
• Demonstrated efficient screening and kinetic characterization of protease substrates using FACS and CLiPS.

Conclusions:

• CLiPS is a versatile and straightforward method for determining protease specificity.
• This approach enables the discovery of optimal protease substrates based on cleavage kinetics.
• CLiPS has significant potential for advancing protease research and applications.