PMID- 21708266
OWN - NLM
STAT- MEDLINE
DCOM- 20120209
LR  - 20161125
IS  - 1096-0279 (Electronic)
IS  - 1046-5928 (Linking)
VI  - 80
IP  - 2
DP  - 2011 Dec
TI  - Purification of SUMO-1 modified IkappaBalpha and complex formation with NF-kappaB.
PG  - 211-6
LID - 10.1016/j.pep.2011.06.009 [doi]
AB  - 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 IkappaBalpha, its 
      sumoylation, the subsequent separation and purification of the modified and the 
      unmodified forms and the purification of the complex IkappaBalpha-SUMO-1/NF-kappaB. 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 IkappaBalpha. Our pure SUMO-1 modified IkappaB/NF-kappaB complex could be a 
      useful tool to identify new interaction partner specific of the SUMO-1 modified 
      IkappaBalpha form. This approach may be extended to other SUMO substrates not isolable by 
      classical chromatography techniques.
CI  - Copyright (c) 2011 Elsevier Inc. All rights reserved.
FAU - Lens, Zoe
AU  - Lens Z
AD  - IRI USR 3078 CNRS, Parc CNRS de la Haute Borne, 50 Avenue de Halley, BP 70478, 
      59658 Villeneuve d'Ascq Cedex, France.
FAU - Dewitte, Frederique
AU  - Dewitte F
FAU - Van Lint, Carine
AU  - Van Lint C
FAU - de Launoit, Yvan
AU  - de Launoit Y
FAU - Villeret, Vincent
AU  - Villeret V
FAU - Verger, Alexis
AU  - Verger A
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20110625
PL  - United States
TA  - Protein Expr Purif
JT  - Protein expression and purification
JID - 9101496
RN  - 0 (I-kappa B Proteins)
RN  - 0 (NF-kappa B p50 Subunit)
RN  - 0 (NFKB1 protein, human)
RN  - 0 (NFKBIA protein, human)
RN  - 0 (Recombinant Proteins)
RN  - 0 (SUMO-1 Protein)
RN  - 0 (SUMO1 protein, human)
RN  - 0 (Transcription Factor RelA)
RN  - 139874-52-5 (NF-KappaB Inhibitor alpha)
SB  - IM
MH  - Catalytic Domain
MH  - Chromatography, Affinity
MH  - Electrophoresis, Polyacrylamide Gel
MH  - Escherichia coli/genetics/metabolism
MH  - Genetic Vectors/genetics/metabolism
MH  - Humans
MH  - I-kappa B Proteins/genetics/*isolation & purification/metabolism
MH  - NF-KappaB Inhibitor alpha
MH  - NF-kappa B p50 Subunit/genetics/*isolation & purification/metabolism
MH  - Protein Interaction Mapping
MH  - Recombinant Proteins/genetics/isolation & purification/metabolism
MH  - SUMO-1 Protein/genetics/*metabolism
MH  - Sumoylation
MH  - Transcription Factor RelA/genetics/*isolation & purification/metabolism
MH  - Ultrafiltration
EDAT- 2011/06/29 06:00
MHDA- 2012/02/10 06:00
CRDT- 2011/06/29 06:00
PHST- 2011/05/24 00:00 [received]
PHST- 2011/06/09 00:00 [revised]
PHST- 2011/06/10 00:00 [accepted]
PHST- 2011/06/29 06:00 [entrez]
PHST- 2011/06/29 06:00 [pubmed]
PHST- 2012/02/10 06:00 [medline]
AID - S1046-5928(11)00156-2 [pii]
AID - 10.1016/j.pep.2011.06.009 [doi]
PST - ppublish
SO  - Protein Expr Purif. 2011 Dec;80(2):211-6. doi: 10.1016/j.pep.2011.06.009. Epub 
      2011 Jun 25.