Isolation of transcription factor complexes by in vivo biotinylation tagging and direct binding to streptavidin beads.
Efficient tagging methodologies are an integral aspect of protein complex characterization by proteomic approaches. Owing to the very high affinity of biotin for avidin and streptavidin, biotinylation tagging offers an attractive approach for the efficient purification of protein complexes. The very high affinity of the biotin/(strept)avidin system also offers the potential for the single-step capture of lower abundance protein complexes, such as transcription factor complexes. The identification of short peptide tags that are efficiently biotinylated by the bacterial BirA biotin ligase led to an approach for the single-step purification of transcription factor complexes by specific in vivo biotinylation tagging. A short sequence tag fused N-terminally to the transcription factor of interest is very efficiently biotinylated by BirA coexpressed in the same cells, as was demonstrated by the tagging of the essential hematopoietic transcription factor GATA-1. The direct binding to streptavidin of biotinylated GATA-1 in nuclear extracts resulted in the single-step capture of the tagged factor and associated proteins, which were eluted and identified by mass spectrometry. This led to the characterization of several distinct GATA-1 complexes with other transcription factors and chromatin remodeling cofactors, which are involved in activation and repression of gene targets. Thus, BirA-mediated tagging is an efficient approach for the direct capture and characterization of transcription factor complexes.
Rodriguez
Patrick
P
Department of Cell Biology, Erasmus Medical Center, Rotterdam, The Netherlands.
Braun
Harald
H
Kolodziej
Katarzyna E
KE
de Boer
Ernie
E
Campbell
Jennifer
J
Bonte
Edgar
E
Grosveld
Frank
F
Philipsen
Sjaak
S
Strouboulis
John
J
D000595:Amino Acid Sequence; D000818:Animals; D001710:Biotin; D015153:Blotting, Western; D019731:Carbon-Nitrogen Ligases; D002460:Cell Line; D002847:Chromatography, Agarose; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D029968:Escherichia coli Proteins; D050982:GATA1 Transcription Factor / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes; D040901:Proteomics / Q000379:methods; D012097:Repressor Proteins; D012685:Sepharose; D019809:Streptavidin; D014157:Transcription Factors / Q000737:chemistry* / Q000302:isolation & purification*
D029968:Escherichia coli Proteins; D050982:GATA1 Transcription Factor; C494689:Gata1 protein, mouse; D046912:Multiprotein Complexes; D012097:Repressor Proteins; D014157:Transcription Factors; D001710:Biotin; C043127:Superose 6b; D012685:Sepharose; D019809:Streptavidin; D010450:Endopeptidases; C099488:TEV protease; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli