Abstract
Fluorescent biosensors can report on the relative abundance, activity, or conformation of biomolecules and analytes through changes in fluorescence emission. A wide variety of genetically-encoded and synthetic biosensors have been developed to monitor protein kinase activity. We have focused on the design, engineering and characterization of fluorescent peptide biosensors of cyclin-dependent kinases (CDKs) that constitute attractive cancer biomarkers and pharmacological targets. In this chapter, we describe the CDKACT fluorescent peptide biosensor technology and its application to assess the relative kinase activity of CDKs in vitro, either using recombinant proteins or cell extracts as a more complex source of kinase. This technology offers a straightforward means of comparing CDK activity in different cell lines and evaluating the specific impact of treatments intended to target kinase activity in a physiologically relevant environment.
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Acknowledgments
This work was supported by the CNRS (Centre National de la Recherche Scientifique) and a grant from the Region Occitanie and European Regional Development Fund (READYNOV/FEDER n° 2018-003539-01) to M.C. Morris. We acknowledge GL Biochem (Shanghai, China) Ltd. and GENEPEP (St-Jean de Védas, France) for synthesis of peptide biosensors. We thank all former members of the “Kinase Biosensors & Inhibitors” group for their contributions to development of fluorescent peptide biosensors: T.N.N. Van, C. Prével, M. Peyressatre, J.A. Gonzalez Vera, C. Tilmaciu, Sebastien Diot, Jessica Soamalala and Arthur Laure.
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Pellerano, M., Morris, M.C. (2021). Fluorescent Peptide Biosensors for Probing CDK Kinase Activity in Cell Extracts. In: Coutts, A.S., Weston, L. (eds) Cell Cycle Oscillators . Methods in Molecular Biology, vol 2329. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1538-6_4
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DOI: https://doi.org/10.1007/978-1-0716-1538-6_4
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