title: Purification of SUMO-1 modified IκBα and complex formation with NF-κB.
abstract: Covalent modification of proteins with SUMO (Small Ubiquitin-like MOdifier) affects many cellular processes, including transcriptional regulation, DNA repair and signal transduction. Although hundreds of SUMO targets have been identified, many biological outcomes of protein sumoylation remain poorly understood. In particular, biochemical and structural analysis can only be easily conducted if highly pure sumoylated substrates are available. Purification of sumoylated substrates in vitro or in bacteria have been previously reported but separating the sumoylated protein from the undesired unmodified fraction is often technically challenging, inefficient and time consuming. Here we develop a new vector system for in vivo sumoylation in Escherichia coli which improves purification of sumoylated proteins. We describe the purification of IκBα, its sumoylation, the subsequent separation and purification of the modified and the unmodified forms and the purification of the complex IκBα-SUMO-1/NF-κB. After a first GST affinity chromatography and GST-tag removal, a unique metal-ion affinity chromatography using a 6xHis-SUMO-1 tag results in mgs of highly pure SUMO-1 modified IκBα. Our pure SUMO-1 modified IκB/NF-κB complex could be a useful tool to identify new interaction partner specific of the SUMO-1 modified IκBα form. This approach may be extended to other SUMO substrates not isolable by classical chromatography techniques.
authors.affiliation: IRI USR 3078 CNRS, Parc CNRS de la Haute Borne, 50 Avenue de Halley, BP 70478, 59658 Villeneuve d'Ascq Cedex, France.
authors.forename: Zoé
authors.initials: Z
authors.lastname: Lens
authors.forename: Frédérique
authors.initials: F
authors.lastname: Dewitte
authors.forename: Carine
authors.initials: C
authors.lastname: Van Lint
authors.forename: Yvan
authors.initials: Y
authors.lastname: de Launoit
authors.forename: Vincent
authors.initials: V
authors.lastname: Villeret
authors.forename: Alexis
authors.initials: A
authors.lastname: Verger
mesh_terms: D020134:Catalytic Domain; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020672:I-kappa B Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000072000:NF-KappaB Inhibitor alpha; D052002:NF-kappa B p50 Subunit / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D025941:Protein Interaction Mapping; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D025842:SUMO-1 Protein / Q000235:genetics / Q000378:metabolism*; D058207:Sumoylation; D051996:Transcription Factor RelA / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D014462:Ultrafiltration
chemical_list: D020672:I-kappa B Proteins; D052002:NF-kappa B p50 Subunit; C489548:NFKB1 protein, human; C000606204:NFKBIA protein, human; D011994:Recombinant Proteins; D025842:SUMO-1 Protein; C495501:SUMO1 protein, human; D051996:Transcription Factor RelA; D000072000:NF-KappaB Inhibitor alpha