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 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.
Leach
Craig A
CA
Progenra Inc., Malvern, PA, USA.
Tian
Xufan
X
Mattern
Michael R
MR
Nicholson
Benjamin
B
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
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