title: Recombinant reconstitution of sumoylation reactions in vitro.
abstract: Reconstituting posttranslational modification with SUMO in vitro is an essential tool in the analysis of sumoylation. In this article, we provide detailed protocols that allow to set up and perform sumoylation reactions using a purified recombinant sumoylation machinery. The protocols include purification of the SUMO E1 enzyme His-Aos1/Uba2, untagged E2 enzyme Ubc9, untagged SUMO, and the RanBP2 E3 ligase fragment IR1 + M. Using these components, we provide step-by-step instructions to set up sumoylation reactions. Two established SUMO model substrates, His-RanGAPtail and HisYFP-Sp100, complement the described tool box; these proteins serve as positive controls in E3 ligase-independent and -dependent sumoylation reactions and are valuable instruments to adjust the reaction conditions if necessary.
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Im Neuenheimer Feld 282, Heidelberg 69120, Germany.
authors.forename: Annette
authors.initials: A
authors.lastname: Flotho
authors.forename: Andreas
authors.initials: A
authors.lastname: Werner
authors.forename: Tobias
authors.initials: T
authors.lastname: Winter
authors.forename: Andrea S
authors.initials: AS
authors.lastname: Frank
authors.forename: Heidi
authors.initials: H
authors.lastname: Ehret
authors.forename: Frauke
authors.initials: F
authors.lastname: Melchior
mesh_terms: D020690:GTPase-Activating Proteins / Q000378:metabolism; D006801:Humans; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism*; D058207:Sumoylation; D044764:Ubiquitin-Activating Enzymes / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D000098843:Ubiquitin-Conjugating Enzyme UBC9
chemical_list: D020690:GTPase-Activating Proteins; C092317:RANGAP1 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins; C470301:UBA2 protein, human; D044763:Ubiquitin-Conjugating Enzymes; C470299:SAE1 protein, human; D044764:Ubiquitin-Activating Enzymes; D000098843:Ubiquitin-Conjugating Enzyme UBC9