Detection and characterization of SUMO protease activity using a sensitive enzyme-based reporter assay.

Craig A Leach, Xufan Tian, Michael R Mattern, Benjamin Nicholson

Journal: Methods in molecular biology (Clifton, N.J.) 2009;497():269-81

PMID: 19107424

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 His(6)-tagged small ubiquitin-like modifier (SUMO) to the amino terminus of the reporter enzyme phospholipase A(2) (PLA(2)). Following cleavage by a member of the sentrin specific proteases (SENPs), free PLA(2) is able to turn over its substrate, resulting in the release of a fluorescent product which is readily quantifiable using a fluorimeter or a fluorescence 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.

Address: Progenra Inc., Malvern, PA, USA.

DOI: 10.1007/978-1-59745-566-4_18