An in vitro FRET-based assay for the analysis of SUMO conjugation and isopeptidase cleavage.
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.
Stankovic-Valentin
Nicolas
N
Department of Biochemistry I, Faculty of Medicine, Georg-August University of Göttingen, Germany.
Kozaczkiewicz
Lukasz
L
Curth
Katja
K
Melchior
Frauke
F
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*
D011993:Recombinant Fusion Proteins; D025842:SUMO-1 Protein; D019759:Carbon-Nitrogen Lyases; C034300:isopeptidase