Efficient system for biotinylated recombinant Ets-1 production in Escherichia coli: a useful tool for studying interactions between Ets-1 and its partners.
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.
Laitem
Clélia
C
CNRS Unité Mixte de Recherche 8161, Institut de Biologie de Lille, Institut Pasteur de Lille, Université de Lille 1, Lille Cedex, France.
Choul-Li
Souhaila
S
Baillat
David
D
Bègue
Agnès
A
Aumercier
Marc
M
D001665:Binding Sites; D019921:Biotinylation; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D007700:Kinetics; D008954:Models, Biological; D050784:Proto-Oncogene Protein c-ets-1 / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D020349:Surface Plasmon Resonance; D014162:Transfection
D050784:Proto-Oncogene Protein c-ets-1; D011994:Recombinant Proteins; D004247:DNA