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  • Cystatin Activity–Based Protease Profiling to Select Protease Inhibitors Useful in Plant Protection

    Author(s)
    Goulet, MC
    Sainsbury, F
    Michaud, D
    Griffith University Author(s)
    Sainsbury, Frank
    Year published
    2020
    Metadata
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    Abstract
    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 ...
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    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.
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    Journal Title
    Methods in Molecular Biology
    Volume
    2139
    DOI
    https://doi.org/10.1007/978-1-0716-0528-8_26
    Subject
    Other chemical sciences
    Biochemistry and cell biology
    Biotinylated cystatins
    Cys protease capture
    Cystatin activity–based protease profiling
    Herbivorous insect digestive proteases
    Plant protease inhibitors
    Publication URI
    http://hdl.handle.net/10072/395186
    Collection
    • Journal articles

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