title: Detection and characterization of SUMO protease activity using a sensitive enzyme-based reporter assay.
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.
authors.affiliation: Progenra Inc., Malvern, PA, USA.
authors.forename: Craig A
authors.initials: CA
authors.lastname: Leach
authors.forename: Xufan
authors.initials: X
authors.lastname: Tian
authors.forename: Michael R
authors.initials: MR
authors.lastname: Mattern
authors.forename: Benjamin
authors.initials: B
authors.lastname: Nicholson
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D002384:Catalysis; D019411:Clinical Laboratory Techniques; D003546:Cysteine Endopeptidases / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D010447:Peptide Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D054467:Phospholipases A2 / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D025842:SUMO-1 Protein / Q000378:metabolism*; D012680:Sensitivity and Specificity; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism; D051742:Transcription Factor CHOP / Q000737:chemistry / Q000378:metabolism / Q000502:physiology
chemical_list: D011993:Recombinant Fusion Proteins; D025842:SUMO-1 Protein; C117909:SUMO2 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins; D051742:Transcription Factor CHOP; D054467:Phospholipases A2; D010450:Endopeptidases; D010447:Peptide Hydrolases; C404512:SENP1 protein, human; D003546:Cysteine Endopeptidases; C118083:Ulp1 protease