Purification of transcription factor protein complexes traditionally involves several pre-purification steps thus making them laborious and costly. We have applied an in vivo biotinylation tagging methodology as a simple approach for the efficient direct purification of transcription factor complexes from crude nuclear extracts ^{1, 2}.

The biotinylation tagging approach is outlined in Figure 1. It is based on previous work on the screening of a combinatorial synthetic peptide library for efficient biotinylation by the bacterial BirA biotin ligase, an enzyme responsible for the covalent attachment of biotin to metabolic enzymes ³. This led to the identification of a number of short sequence tags that can be very efficiently biotinylated in vitro ^{3, 4}. The main advantage of biotinylation tagging is that biotinylated substrates can be bound very tightly by avidin and streptavidin proteins (K_d = 10^-15), a fact that has been widely exploited in many applications in molecular biology. In addition, there are few (mostly cytoplasmic) naturally biotinylated proteins, ensuring that non-specific background binding remains low.

We have used one such short tag to fuse it N-terminally to hematopoietic transcription factors, such as GATA-1, and to co-express them with the bacterial BirA biotin ligase in cultured mouse erythroleukemic (MEL) cells. Screening nuclear extracts with streptavidin-horseradish peroxidase (HRP) conjugate showed robust biotinylation of tagged GATA-1, with biotinylation efficiency reaching almost 100% (Fig. 2, ref. 1). Background of nuclear extracts from cells expressing BirA bound to streptavidin beads consists mostly of endogenous biotinylated proteins such as carboxylases, as evidenced by Coomasie staining of SDS-PAGE gels (Fig. 3, ref. 1). The staining pattern after binding to beads of nuclear extracts from cells expressing biotinylated GATA-1 was significantly different to the background binding pattern showing enrichment in proteins co-eluting with GATA-1 (lane 3 versus lane 5, Fig. 3). Background binding proteins were further confirmed by mass spectrometry as consisting mostly of carboxylases and their associated proteins, of proteins involved in mRNA processing and of ribosomal proteins (Fig. 4). We have found that pre-treatment of extracts with RNase or benzonase reduces background of mRNA processing proteins and of ribosomal proteins, whereas inclusion of a protease cleavage sequence in the expression construct of the fusion protein may also help reduce background from endogenous biotinylated proteins ⁵. By contrast, streptavidin pulldown of extracts expressing biotinylated transcription factors are enriched for other transcription factors and chromatin remodeling and modification complexes (Fig. 5, ref 2, 6). The identity of proteins co-purifying with the tagged transcription factor can be determined by mass spectrometry and confirmed by immunoprecipitations.

Biotinylation tagging has also been shown to work well in transgenic mice ^{1, 7} and also as an alternative to antibodies in chromatin immunoprecipitation (ChIP) assays ^{1, 8, 9}.

References

1. de Boer, Ε., Rodriguez, P., Bonte, E., Krijgsveld, J., Katsantoni, E., Heck, A., Grosveld, F. and Strouboulis, J. 2003 “Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice”. Proc Natl Acad Sci U S A. 2003 Jun 24;100(13):7480-5. PubMed

2. Rodriguez, P., Bonte, E., Krijgsveld, J., Guyot, B., Heck, A., Vyas, P., de Boer, E., Grosveld, F. and Strouboulis, J. 2005 “GATA-1 forms distinct activating and repressive complexes in erythroid cells”. EMBO J. 2005 Jul 6;24(13):2354-66. PubMed

3. Schatz, P.J. 1993 “Use of peptide libraries to map the substrate specificity of a peptide-modifying enzyme: a 13 residue consensus peptide specifies biotinylation in Escherichia coli”. Biotechnology (N Y), 11, 1138-43. PubMed

4. Beckett, D., Kovaleva, E. and Schatz, P.J. 1999 “A minimal peptide substrate in biotin holoenzyme synthetase-catalyzed biotinylation”. Protein Sci, 8, 921-9. PubMed

5. Rodriguez, P., Braun, H., Kolodziej, K.E., de Boer, E., Campbell, J., Bonte, E., Grosveld, F., Philipsen, S. and Strouboulis, J. 2006 “Isolation of transcription factor complexes by in vivo biotinylation tagging and direct binding to streptavidin beads”. Methods Mol Biol. 2006;338:305-23. PubMed

6. Meier, N., Krpic, S., Rodriguez, P., Strouboulis, J., Monti, M., Krijgsveld, J., Gering, M., Patient, R., Hostert, A. and Grosveld,F. 2006 “Novel binding partners of Ldb1 are required for hematopoietic development”. Development. 2006 Dec;133(24):4913-23. PubMed

7. Driegen, S., Ferreira, R., van Zon, A., Strouboulis, J., Jaegle, M., Grosveld, F., Philipsen, S. and Meijer, D. 2005 “A generic tool for biotinylation of tagged proteins in transgenic mice”. Transgenic Res. 2005 Aug;14(4):477-82. PubMed

8. Horsman, S., Moorhouse, M., de Jager, V., van der Spek, P., Grosveld, F., Strouboulis, J., and Katsantoni, E. 2006 “TFT Mapper: A BLAST search tool for identification of Transcription Factors Target Genes”. BMC Bioinformatics. 2006 Mar 8;7:120. PubMed

9. van Werven, F.J. and Timmers, H.T. 2006 “The use of biotin tagging in Saccharomyces cerevisiae improves the sensitivity of chromatin immunoprecipitation”. Nucleic Acids Res. 2006 Feb 25;34(4):e33. PubMed


Figure 1	Figure 2

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Figure 5