Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice

摘要: Proteomic approaches require simple and efficient protein purification methodologies that are amenable to high throughput. Biotinylation is an attractive approach for complex due the very affinity of avidin/streptavidin biotinylated templates. Here, we describe single-step transcription factor complex(es) based on specific in vivo biotinylation. We expressed bacterial BirA biotin ligase mammalian cells demonstrated biotinylation a hematopoietic bearing small (23-aa) artificial peptide tag. tagged altered neither factor's interactions or DNA binding properties nor its subnuclear distribution. Using this approach, isolated biotin-tagged at least one other known interacting from crude nuclear extracts by direct streptavidin beads. Finally, method works efficiently transgenic mice, thus raising prospect using tagging directly animal tissues. Therefore, BirA-mediated proteins provides basis cells.