Protease inhibitors of the cystatin protein superfamily show potential in plant protection for the control of herbivorous pests. Here, we describe a cystatin activity-based profiling procedure for the selection of potent cystatin candidates, using single functional variants of tomato cystatin SlCYS8 and digestive Cys proteases of the herbivore insect Colorado potato beetle as a case study. The procedure involves the capture of target Cys proteases with biotinylated versions of the cystatins, followed by the identification and quantitation of captured proteases by mass spectrometry. An example is given to illustrate usefulness of the approach as an alternative to current procedures for recombinant inhibitor selection based on in vitro assays with synthetic peptide substrates. A second example is given showing its usefulness as a tool to compare the affinity spectra of inhibitor variants toward different subsets of target protease complements.
Keywords: Biotinylated cystatins; Cys protease capture; Cystatin activity–based protease profiling; Herbivorous insect digestive proteases; Plant protease inhibitors.