Detection and Characterization of SUMO Protease Activity Using a Sensitive Enzyme-Based Reporter Assay

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 K_m values and to characterize inhibitors of SUMO proteases.