An In Vitro FRET-Based Assay for the Analysis of SUMO Conjugation and Isopeptidase Cleavage
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To measure rates of sumoylation and isopeptidase cleavage in vitro, we developed an enzyme assay that is based on fl uorescence resonance energy transfer (FRET). FRET is a process by which the excited state energy of a fl uorescent donor molecule is transferred to an acceptor molecule. Effi cient energy transfer requires very close proximity, and can therefore be used as a read-out for covalent and non-covalent protein interactions. The assay described here uses bacterially expressed and purifi ed YFP-SUMO-1 and CFP-RanGAP1 as model substrates that are covalently coupled in the presence of recombinant SUMO E1 and E2 enzymes and ATP. Reactions of 25 μl volume, set up in 384-wells plates, give suffi cient signal for analysis. Consequently, this assay requires very low amounts of recombinant proteins and allows measurement of time courses in high-throughput format.
Key wordsSUMO E1 activating enzyme E2 conjugating enzyme Aos1/Uba2 Ubc9 yellow fluorescent protein fluorescence resonance energy transfer FRET isopeptidase
We would like to acknowledge previous and current lab members for contributing to the assay development, for sharing reagents and for many stimulating discussions. Funding by the EU (Rubicon and UbiRegulator), and fellowships by the Fondation pour la Recherche Médicale (to NS) and the Niedersachsen Lich-tenberg Program (to LK) are gratefully acknowledged.