Abstract
In this chapter we describe a novel, sensitive, homogenous high throughput reporter-based in vitro assay for SUMO protease activity developed by Progenra, Inc. A reporter construct was created by fusing His6-tagged small ubiquitin-like modifi er (SUMO) to the amino terminus of the reporter enzyme phospholipase A2 (PLA2). Following cleavage by a member of the sentrin specifi c proteases (SENPs), free PLA2 is able to turn over its substrate, resulting in the release of a fl uorescent product which is readily quantifi able using a fl uorimeter or a fl uorescence plate reader. The utility of this SUMO-CHOP-Reporter assay platform is demonstrated by its ability to determine Km values and to characterize inhibitors of SUMO proteases.
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Acknowledgments
The authors thank Keith Wilkinson (Emory University) and current and past employees of Progenra for their contributions to the development of the CHOP-Reporter assay platform. This work was supported in part by grants 1R43 CA115205, 1R43 HL083527, and 1R43 DK071391 from the National Institutes of Health, US Department of Health and Human Services, to Progenra, Inc.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Leach, C.A., Tian, X., Mattern, M.R., Nicholson, B. (2009). Detection and Characterization of SUMO Protease Activity Using a Sensitive Enzyme-Based Reporter Assay. In: Ulrich, H.D. (eds) SUMO Protocols. METHODS IN MOLECULAR BIOLOGY™, vol 497. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-566-4_18
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DOI: https://doi.org/10.1007/978-1-59745-566-4_18
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-934115-80-0
Online ISBN: 978-1-59745-566-4
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