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

Abstract

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 (PXXX ) and on-cell determination of apparent k /K revealed that the enriched substrate peptides are hydrolyzed six to eight-fold 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.

Staphylococcus carnosus: from starter culture to protein engineering platform

“…Upon addition of protease, variants with a functional substrate will be cleaved within the linker, resulting in release of the blocking domain and binding of the fluorescent reporter molecule. Using substrate libraries for tobacco etch virus (TEV) protease and matrix metalloprotease (MMP)-1, the substrate profiles for the respective protease were identified and several new peptides were isolated for MMP-1 that were processed with up to eightfold higher catalytic activity compared with previously reported substrates (Sandersjoo et al 2017 ).…”

Editorial: Methods and advances for systems and synthetic biology, nanobiotech and medicine

“…Proteases play important roles in fundamental biological processes and are critical important in biotechnological and biomedical research. Dr. John Löfblom and colleagues [5] introduce a novel method for protease substrate profiling using bacterial display of self-blocking affinity proteins and flow-cytometric sorting, and they have successfully demonstrated that this method has potential as an efficient tool for substrate profiling.…”

Profiling Substrate Specificity of the SUMO Protease Ulp1 by the YESS–PSSC System to Advance the Conserved Mechanism for Substrate Cleavage

“…Obviously, the diversity of substrates makes the mapping of one or more sites technically difficult and time-consuming. Several in vivo methods have been exploited to investigate the substrate specificity of peptide–substrate proteases in a high-throughput manner, such as the phage display system [ 21 ], the bacterial display system [ 22 ] and the yeast display system based analysis methods [ 18 , 23 ], whereas protein–substrate proteases, like the SUMO protease, have never been reported in these systems.…”