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

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Plant Proteomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2139))

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 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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Acknowledgments

Work supported by Discovery and Discovery Accelerator Supplement grants from the Natural Science and Engineering Research Council of Canada to D.M.

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Authors and Affiliations

  1. Centre de Recherche et d’Innovation sur les Végétaux, Université Laval, Québec, QC, Canada

    Marie-Claire Goulet, Frank Sainsbury & Dominique Michaud

  2. Griffith Institute for Drug Discovery, Griffith University, Brisbane, QLD, Australia

    Frank Sainsbury

Authors
  1. Marie-Claire Goulet
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  2. Frank Sainsbury
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  3. Dominique Michaud
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Corresponding author

Correspondence to Dominique Michaud .

Editor information

Editors and Affiliations

  1. Agroforestry and Plant Biochemistry, Proteomics and Systems Biology, Department of Biochemistry and Molecular Biology, University of Cordoba, Cordoba, Spain

    Jesus V. Jorrin-Novo

  2. Department of Organisms and Systems Biology, Institute of Biotechnology of Asturias, University of Oviedo, Oviedo, Asturias, Spain

    Luis Valledor

  3. Agroforestry and Plant Biochemistry, Proteomics and Systems Biology, Department of Biochemistry and Molecular Biology, University of Cordoba UCO-CeiA3, Cordoba, Cordoba, Spain

    Mari Angeles Castillejo

  4. Agroforestry and Plant Biochemistry, Proteomics and Systems Biology, Department of Biochemistry and Molecular Biology, University of Cordoba, Cordoba, Spain

    Maria-Dolores Rey

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Goulet, MC., Sainsbury, F., Michaud, D. (2020). Cystatin Activity–Based Protease Profiling to Select Protease Inhibitors Useful in Plant Protection. In: Jorrin-Novo, J., Valledor, L., Castillejo, M., Rey, MD. (eds) Plant Proteomics. Methods in Molecular Biology, vol 2139. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0528-8_26

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  • DOI: https://doi.org/10.1007/978-1-0716-0528-8_26

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0527-1

  • Online ISBN: 978-1-0716-0528-8

  • eBook Packages: Springer Protocols

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