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 Citations (Scopus)

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 2008
Externally publishedYes

Fingerprint

Biotinylation
Matrix Metalloproteinase 3
Binding Sites
Escherichia coli
DNA
New England
Streptavidin
Surface Plasmon Resonance
Biotin
Proteins
Transcription Factors
collagenase 1

Keywords

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

ASJC Scopus subject areas

  • Biotechnology

Cite this

Efficient system for biotinylated recombinant Ets-1 production in Escherichia coli : A useful tool for studying interactions between Ets-1 and its partners. / Laitem, Clélia; Choul-li, Souhaila; Baillat, David; Bègue, Agnès; Aumercier, Marc.

In: Protein Expression and Purification, Vol. 62, No. 1, 11.2008, p. 53-63.

Research output: Contribution to journalArticle

Laitem, Clélia ; Choul-li, Souhaila ; Baillat, David ; Bègue, Agnès ; Aumercier, Marc. / Efficient system for biotinylated recombinant Ets-1 production in Escherichia coli : A useful tool for studying interactions between Ets-1 and its partners. In: Protein Expression and Purification. 2008 ; Vol. 62, No. 1. pp. 53-63.
@article{635fae0367fe4531b2da995d9d2970ca,
title = "Efficient system for biotinylated recombinant Ets-1 production in Escherichia coli: A useful tool for studying interactions between Ets-1 and its partners",
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{\circledR}), 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.",
keywords = "Affinity purification, Biotinylated recombinant Ets-1, Escherichia coli biotinylated protein production system, Oncogenes, Transcriptional regulation",
author = "Cl{\'e}lia Laitem and Souhaila Choul-li and David Baillat and Agn{\`e}s B{\`e}gue and Marc Aumercier",
year = "2008",
month = "11",
doi = "10.1016/j.pep.2008.06.010",
language = "English (US)",
volume = "62",
pages = "53--63",
journal = "Protein Expression and Purification",
issn = "1046-5928",
publisher = "Academic Press Inc.",
number = "1",

}

TY - JOUR

T1 - Efficient system for biotinylated recombinant Ets-1 production in Escherichia coli

T2 - A useful tool for studying interactions between Ets-1 and its partners

AU - Laitem, Clélia

AU - Choul-li, Souhaila

AU - Baillat, David

AU - Bègue, Agnès

AU - Aumercier, Marc

PY - 2008/11

Y1 - 2008/11

N2 - 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.

AB - 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.

KW - Affinity purification

KW - Biotinylated recombinant Ets-1

KW - Escherichia coli biotinylated protein production system

KW - Oncogenes

KW - Transcriptional regulation

UR - http://www.scopus.com/inward/record.url?scp=53149092050&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=53149092050&partnerID=8YFLogxK

U2 - 10.1016/j.pep.2008.06.010

DO - 10.1016/j.pep.2008.06.010

M3 - Article

VL - 62

SP - 53

EP - 63

JO - Protein Expression and Purification

JF - Protein Expression and Purification

SN - 1046-5928

IS - 1

ER -