Efficient system for biotinylated recombinant Ets-1 production in Escherichia coli: A useful tool for studying interactions between Ets-1 and its partners

Clélia Laitem, Souhaila Choul-li, David Baillat, Agnès Bègue, Marc Aumercier

Research output: Contribution to journalArticle

10 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)53-63
Number of pages11
JournalProtein Expression and Purification
Volume62
Issue number1
DOIs
StatePublished - Nov 1 2008

Keywords

  • Affinity purification
  • Biotinylated recombinant Ets-1
  • Escherichia coli biotinylated protein production system
  • Oncogenes
  • Transcriptional regulation

ASJC Scopus subject areas

  • Biotechnology

Fingerprint Dive into the research topics of 'Efficient system for biotinylated recombinant Ets-1 production in Escherichia coli: A useful tool for studying interactions between Ets-1 and its partners'. Together they form a unique fingerprint.

  • Cite this