Protease substrate profiling using bacterial display of self-blocking affinity proteins and flow-cytometric sorting

Proteases are involved in fundamental biological processes and are important tools in both biotechnological and biomedical research. An important property of proteases is to discriminate among potential substrates. Here, a new method for substrate profiling of proteases is presented. The substrates are displayed between two anti-idiotypic affinity domains on the Gram-positive bacterium Staphylococcus carnosus. The first domain functions as a reporter tag and has affinity for a labeled reporter protein, whereas the second domain blocks the reporter tag from interacting with the reporter protein. Site-specific proteolysis of the substrate results in release of the blocking domain, enabling the reporter tag to bind the labeled reporter protein. Proteolysis is therefore reflected in reporter binding, which is quantified by flow cytometry. First, the method with tobacco etch virus protease (TEVp) is evaluated and then the substrate preference of matrix metalloprotease- 1 (MMP-1) is determined using two libraries of around three million substrates each. Identified substrate peptides contained the previously reported motif (PXXXHy) and on-cell determination of apparent k(cat)/K-M revealed that the enriched substrate peptides are hydrolyzed six to eightfold more efficiently than a previously reported substrate peptide. The method thus works as intended and the authors believe it has potential as an efficient tool for substrate profiling.