Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice
Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice
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DOI：
10.1073/pnas.1332608100
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发表时间：
2003-06-24
影响因子：
11.1
通讯作者：
Strouboulis, J
中科院分区：
文献类型：
--
作者：
de Boer, E;Rodriguez, P;Strouboulis, J
蛋白质组学方法需要简单高效且适用于高通量的蛋白质纯化方法。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，因为亲和素/链霉亲和素对生物素化模板具有极高的亲和力。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的一步纯化方法。我们在哺乳动物细胞中表达了细菌的BirA生物素连接酶，并证明了带有一个小的（23个氨基酸）人工肽标签的造血转录因子能非常高效地被生物素化。带标签的转录因子的生物素化既没有改变该因子在体内的蛋白质相互作用或DNA结合特性，也没有改变其亚核分布。利用这种方法，我们通过直接结合链霉亲和素磁珠从粗核提取物中分离出了生物素标记的转录因子以及至少一种其他已知的相互作用蛋白。最后，这种方法在转基因小鼠中高效起作用，从而提高了直接从动物组织中利用生物素化标签进行蛋白质复合物纯化的可能性。因此，BirA介导的带标签蛋白质的生物素化为从哺乳动物细胞中一步纯化蛋白质提供了基础。
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.