title: Reconstitution of the Recombinant RanBP2 SUMO E3 Ligase Complex.
abstract: One of the few proteins that have SUMO E3 ligase activity is the 358 kDa nucleoporin RanBP2 (Nup358). While small fragments of RanBP2 can stimulate SUMOylation in vitro, the physiologically relevant E3 ligase is a stable multi-subunit complex comprised of RanBP2, SUMOylated RanGAP1, and Ubc9. Here, we provide a detailed protocol to in vitro reconstitute the RanBP2 SUMO E3 ligase complex. With the exception of RanBP2, reconstitution involves untagged full-length proteins. We describe the bacterial expression and purification of all complex components, namely an 86 kDa His-tagged RanBP2 fragment, the SUMO E2-conjugating enzyme Ubc9, RanGAP1, and SUMO1, and we provide a protocol for quantitative SUMOylation of RanGAP1. Finally, we present details for the assembly and final purification of the catalytically active RanBP2/RanGAP1*SUMO1/Ubc9 complex.
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ - ZMBH Alliance, Im Neuenheimer Feld 282, 69120, Heidelberg, Germany.|Department of Biochemistry, University of Washington, Seattle, Washington, USA.
authors.forename: Tobias
authors.initials: T
authors.lastname: Ritterhoff
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ - ZMBH Alliance, Im Neuenheimer Feld 282, 69120, Heidelberg, Germany.
authors.forename: Hrishikesh
authors.initials: H
authors.lastname: Das
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ - ZMBH Alliance, Im Neuenheimer Feld 282, 69120, Heidelberg, Germany.
authors.forename: Yuqing
authors.initials: Y
authors.lastname: Hao
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ - ZMBH Alliance, Im Neuenheimer Feld 282, 69120, Heidelberg, Germany.|Department of Infectious Diseases and Virology, University Hospital of Heidelberg, Heidelberg, Germany.
authors.forename: Volkan
authors.initials: V
authors.lastname: Sakin
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ - ZMBH Alliance, Im Neuenheimer Feld 282, 69120, Heidelberg, Germany.
authors.forename: Annette
authors.initials: A
authors.lastname: Flotho
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ - ZMBH Alliance, Im Neuenheimer Feld 282, 69120, Heidelberg, Germany. andreas.werner@zmbh.uni-heidelberg.de.
authors.forename: Andreas
authors.initials: A
authors.lastname: Werner
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ - ZMBH Alliance, Im Neuenheimer Feld 282, 69120, Heidelberg, Germany. f.melchior@zmbh.uni-heidelberg.de.
authors.forename: Frauke
authors.initials: F
authors.lastname: Melchior
mesh_terms: D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D028861:Nuclear Pore Complex Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D025842:SUMO-1 Protein / Q000235:genetics / Q000378:metabolism*; D058207:Sumoylation; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism*; D000098843:Ubiquitin-Conjugating Enzyme UBC9
chemical_list: D020690:GTPase-Activating Proteins; D018832:Molecular Chaperones; D028861:Nuclear Pore Complex Proteins; C092317:RANGAP1 protein, human; D011994:Recombinant Proteins; D025842:SUMO-1 Protein; C495501:SUMO1 protein, human; C094493:ran-binding protein 2; D044763:Ubiquitin-Conjugating Enzymes; D000098843:Ubiquitin-Conjugating Enzyme UBC9
keywords: Complex reconstitution; In vitro SUMOylation; RanBP2; RanBP2 complex; RanGAP1; SUMO; SUMO E3 ligase; Ubc9