Journal
PROTEIN EXPRESSION AND PURIFICATION
Volume 80, Issue 2, Pages 211-216Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2011.06.009
Keywords
SUMO; Sumoylation; I kappa B alpha; NF-kappa B; On-column tag cleavage
Categories
Funding
- FNRS, Belgium
- 'Fondation pour la Recherche Medicale' (FRM, Comite Nord-Pas de Calais, France)
- 'Association pour la Recherche contre le Cancer' (ARC, France)
- 'Ligue Nationale contre le Cancer' (Comite Nord, France)
- Ministry of Higher Education and Research
- FNRS
- Nord-Pas de Calais Regional Council
- FEDER through the 'Contrat de Projets Etat Region (CPER)
- Belgian National Fund for Scientific Research (FNRS)
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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 I kappa B alpha, its sumoylation, the subsequent separation and purification of the modified and the unmodified forms and the purification of the complex I kappa B alpha-SUMO-1/NF-kappa B. 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 I kappa B alpha. Our pure SUMO-1 modified I kappa B/NF-kappa B complex could be a useful tool to identify new interaction partner specific of the SUMO-1 modified I kappa B alpha form. This approach may be extended to other SUMO substrates not isolable by classical chromatography techniques. (C) 2011 Elsevier Inc. All rights reserved.
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