Isolation of transcription factor complexes by in vivo biotinylation tagging and direct binding to streptavidin beads.
Isolation of transcription factor complexes by in vivo biotinylation tagging and direct binding to streptavidin beads.
复制标题
通过体内生物素化标记和直接结合链霉亲和素珠来分离转录因子复合物。
DOI：
10.1385/1-59745-097-9:305
复制
发表时间：
2006
影响因子：
--
通讯作者：
J. Strouboulis
中科院分区：
文献类型：
--
作者：
Patrick Rodriguez;H. Braun;Katarzyna E. Kolodziej;E. de Boer;Jennifer Campbell;E. Bonte;F. Grosveld;S. Philipsen;J. Strouboulis
高效的标记方法是蛋白质组学方法表征蛋白质复合物的一个组成部分。由于生物素对亲和素和链霉亲和素具有很高的亲和力，生物素标记为蛋白质复合物的有效纯化提供了一种有吸引力的方法。生物素/（链霉）抗生物素蛋白系统的非常高的亲和力也提供了单步捕获较低丰度蛋白质复合物（例如转录因子复合物）的潜力。通过细菌BirA生物素连接酶有效地生物素化的短肽标签的鉴定导致了通过特异性体内生物素化标记对转录因子复合物进行单步纯化的方法。与目的转录因子N-末端融合的短序列标签被在相同细胞中共表达的BirA非常有效地生物素化，如通过标记必需造血转录因子加塔-1所证明的。核提取物中生物素化的加塔-1与链霉亲和素的直接结合导致标记因子和相关蛋白的单步捕获，其被洗脱并通过质谱法鉴定。这导致了几种不同的加塔-1与其他转录因子和染色质重塑辅因子的复合物的表征，这些因子参与了基因靶标的激活和抑制。因此，BirA介导的标签是一种有效的方法，用于直接捕获和表征的转录因子复合物。
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.