title: An in vitro FRET-based assay for the analysis of SUMO conjugation and isopeptidase cleavage.
abstract: To measure rates of sumoylation and isopeptidase cleavage in vitro, we developed an enzyme assay that is based on fluorescence resonance energy transfer (FRET). FRET is a process by which the excited state energy of a fluorescent donor molecule is transferred to an acceptor molecule. Efficient 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 purified 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 microl volume, set up in 384-wells plates, give sufficient signal for analysis. Consequently, this assay requires very low amounts of recombinant proteins and allows measurement of time courses in high-throughput format.
authors.affiliation: Department of Biochemistry I, Faculty of Medicine, Georg-August University of Göttingen, Germany.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Stankovic-Valentin
authors.forename: Lukasz
authors.initials: L
authors.lastname: Kozaczkiewicz
authors.forename: Katja
authors.initials: K
authors.lastname: Curth
authors.forename: Frauke
authors.initials: F
authors.lastname: Melchior
mesh_terms: D000818:Animals; D019759:Carbon-Nitrogen Lyases / Q000378:metabolism*; D019411:Clinical Laboratory Techniques; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D008954:Models, Biological; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism; D025842:SUMO-1 Protein / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D025842:SUMO-1 Protein; D019759:Carbon-Nitrogen Lyases; C034300:isopeptidase