Efficient system for biotinylated recombinant Ets-1 production in Escherichia coli: a useful tool for studying interactions between Ets-1 and its partners.
Protein Expr Purif
; 62(1): 53-63, 2008 Nov.
Article
em En
| MEDLINE
| ID: mdl-18639639
Identification of Ets-1 interaction partners is critical for understanding its properties. Ets-1 DNA-binding is governed by an intramolecular mechanism called autoinhibition. Ets-1 increases its DNA-binding affinity by counteracting autoinhibition through binding either to a particular organization of Ets binding sites (EBS) in palindrome, as in the Stromelysin-1 promoter, or to EBS adjacent to DNA-binding sites of its partners by combinatorial interactions, as in the Collagenase-1 promoter. Identification of new Ets-1 interaction partners should allow the identification of new functions for this transcription factor. To this end, we fused a biotin tag to Ets-1 protein in order to copurify it and its partners by affinity. For the first time, we cloned, produced in Escherichia coli and purified a biotinylated recombinant Ets-1 protein using the T7-Impact system (New England Biolabs), adapted to induce biotinylation. Nearly 100% biotinylation was attained without altering Ets-1 properties. Biotinylated Ets-1 bound to and transactivated the Stromelysin-1 promoter the same way as native Ets-1 did. It also conserved interactions with known Ets-1 partners such as c-Jun, Erk-2 and Runx-1. In addition, streptavidin pull-down and surface plasmon resonance assays demonstrated that biotinylated Ets-1 is a useful tool for qualitative and quantitative studies of Ets-1 interaction with its partners.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteínas Recombinantes
/
Escherichia coli
/
Proteína Proto-Oncogênica c-ets-1
Tipo de estudo:
Evaluation_studies
/
Prognostic_studies
/
Qualitative_research
Limite:
Humans
Idioma:
En
Ano de publicação:
2008
Tipo de documento:
Article