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.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, U.S. Gov't, Non-P.H.S.
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Review
MeSH terms
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Biosensing Techniques / methods*
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Catalysis
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Clinical Laboratory Techniques
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Cysteine Endopeptidases / metabolism
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Endopeptidases / metabolism
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Humans
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Kinetics
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Peptide Hydrolases / chemistry
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Peptide Hydrolases / isolation & purification*
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Peptide Hydrolases / metabolism*
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Phospholipases A2 / metabolism
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Protein Processing, Post-Translational
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / metabolism
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Recombinant Fusion Proteins / pharmacology
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SUMO-1 Protein / metabolism*
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Sensitivity and Specificity
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Small Ubiquitin-Related Modifier Proteins / metabolism
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Transcription Factor CHOP / chemistry
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Transcription Factor CHOP / metabolism
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Transcription Factor CHOP / physiology
Substances
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Recombinant Fusion Proteins
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SUMO-1 Protein
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SUMO2 protein, human
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Small Ubiquitin-Related Modifier Proteins
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Transcription Factor CHOP
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Phospholipases A2
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Endopeptidases
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Peptide Hydrolases
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SENP1 protein, human
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Cysteine Endopeptidases
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Ulp1 protease