de Boer Ernie, Rodriguez Patrick, Bonte Edgar, Krijgsveld Jeroen, Katsantoni Eleni, Heck Albert, Grosveld Frank, Strouboulis John
Department of Cell Biology, Erasmus Medical Center, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands.
Proc Natl Acad Sci U S A. 2003 Jun 24;100(13):7480-5. doi: 10.1073/pnas.1332608100. Epub 2003 Jun 11.
Proteomic approaches require simple and efficient protein purification methodologies that are amenable to high throughput. Biotinylation is an attractive approach for protein complex purification due to the very high affinity of avidin/streptavidin for biotinylated templates. Here, we describe an approach for the single-step purification of transcription factor complex(es) based on specific in vivo biotinylation. We expressed the bacterial BirA biotin ligase in mammalian cells and demonstrated very efficient biotinylation of a hematopoietic transcription factor bearing a small (23-aa) artificial peptide tag. Biotinylation of the tagged transcription factor altered neither the factor's protein interactions or DNA binding properties in vivo nor its subnuclear distribution. Using this approach, we isolated the biotin-tagged transcription factor and at least one other known interacting protein from crude nuclear extracts by direct binding to streptavidin beads. Finally, this method works efficiently in transgenic mice, thus raising the prospect of using biotinylation tagging in protein complex purification directly from animal tissues. Therefore, BirA-mediated biotinylation of tagged proteins provides the basis for the single-step purification of proteins from mammalian cells.
蛋白质组学方法需要简单高效的蛋白质纯化方法，且这些方法要适用于高通量操作。由于抗生物素蛋白/链霉抗生物素蛋白与生物素化模板具有极高的亲和力，生物素化是一种用于蛋白质复合物纯化的有吸引力的方法。在此，我们描述了一种基于特异性体内生物素化的转录因子复合物单步纯化方法。我们在哺乳动物细胞中表达了细菌BirA生物素连接酶，并证明了带有小的（23个氨基酸）人工肽标签的造血转录因子能够非常有效地进行生物素化。标记转录因子的生物素化在体内既不改变该因子的蛋白质相互作用或DNA结合特性，也不改变其核内分布。使用这种方法，我们通过直接与链霉抗生物素蛋白磁珠结合，从粗核提取物中分离出了生物素标记的转录因子和至少一种其他已知的相互作用蛋白。最后，该方法在转基因小鼠中有效发挥作用，从而增加了直接从动物组织中使用生物素化标记进行蛋白质复合物纯化的可能性。因此，BirA介导的标记蛋白生物素化提供了从哺乳动物细胞中单步纯化蛋白质的基础。
