Kashlev M, Martin E, Polyakov A, Severinov K, Nikiforov V, Goldfarb A
Public Health Research Institute, New York, NY 10016.
Gene. 1993 Aug 16;130(1):9-14. doi: 10.1016/0378-1119(93)90340-9.
A stretch of six histidine residues (His6) has been genetically fused to the C terminus of the beta' polypeptide of Escherichia coli RNA polymerase. The His6-tagged beta' subunit assembles into RNA polymerase molecules which perform all vital in vivo functions and behave qualitatively normally in vitro. The His6 tag permits rapid purification of the enzyme directly from crude cell extracts or from an in vitro reconstitution reaction by adsorption to Ni(2+)-chelating agarose resin, followed by elution with imidazole. The enzyme bound to the matrix remains transcriptionally active. The immobilized enzyme can withstand repeated buffer changes without substantial activity loss and permits controlled stepwise 'walking' of the transcriptional complex along the DNA template, and isolation of defined intermediates in the transcription cycle. The immobilized RNA polymerase provides a powerful experimental system for structural and functional analysis of RNA polymerase and its interaction with regulatory factors.
一段六个组氨酸残基（His6）已通过基因工程融合到大肠杆菌RNA聚合酶β'多肽的C末端。带有His6标签的β'亚基组装成RNA聚合酶分子，这些分子在体内执行所有重要功能，并且在体外定性表现正常。His6标签允许通过吸附到Ni(2+)螯合琼脂糖树脂上，然后用咪唑洗脱，直接从粗细胞提取物或体外重组反应中快速纯化该酶。与基质结合的酶保持转录活性。固定化的酶可以承受反复的缓冲液更换而不会有大量活性损失，并允许转录复合物沿着DNA模板进行可控的逐步“移动”，以及分离转录循环中确定的中间体。固定化的RNA聚合酶为RNA聚合酶及其与调节因子相互作用的结构和功能分析提供了一个强大的实验系统。
