On-column tris(2-carboxyethyl)phosphine reduction and IC5-maleimide labeling during purification of a RpoC fragment on a nickel-nitrilotriacetic acid Column
On-column tris(2-carboxyethyl)phosphine reduction and IC5-maleimide labeling during purification of a RpoC fragment on a nickel-nitrilotriacetic acid Column
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DOI：
10.1016/s0003-2697(02)00061-1
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发表时间：
2002-08-15
影响因子：
2.9
通讯作者：
Burgess, RR
中科院分区：
文献类型：
--
作者：
Bergendahl, V;Anthony, LC;Burgess, RR
蛋白质的荧光标记变得越来越重要，因为荧光技术如FRET和荧光偏振现在通常用于蛋白质结合研究、蛋白质组学和药物发现中的高通量筛选。在我们研究大肠杆菌RNA聚合酶（RNAP）的β '-亚基与sigma 70的结合的努力中，我们以非常方便的方式合成了荧光标记的β'-片段（残基100-309），其可用作六组氨酸标记蛋白质的一般方案。通过执行所有以下步骤，纯化，还原，用IC 5-马来酰亚胺衍生化，以及在蛋白质结合到柱上时去除游离染料，我们能够显著减少操作时间，同时实现更好的标记效率和质量。从包涵体中纯化具有N-末端His（6）-标签的β '-片段，并且可以在结合到Ni-NTA亲和柱之前或之后重折叠。在标记前用不干扰Ni-NTA化学的TCEP实现还原。在电泳迁移率变动测定中，用标记有铕基荧光团的sigma 70测试标记的β ′-片段的结合。细菌RNA聚合酶中的σ-核心蛋白相互作用为药物发现提供了潜在的特异性靶点，因为它在真细菌中高度保守，但与真核生物显著不同。(C)2002 Elsevier Science（美国）。All rights reserved.
Fluorescence labeling of proteins has become increasingly important since fluorescent techniques like FRET and fluorescence polarization are now commonly used in protein binding studies, proteomics, and for high-throughput screening in drug discovery. In our efforts to study the binding of the beta'-subunit from Escherichia coli RNA polymerase (RNAP) to sigma70, we synthesized a fluorescent-labeled beta'-fragment (residues 100-309) in a very convenient way, that could be used as a general protocol for hexahistidine-tagged proteins. By performing all the following steps, purification, reduction, derivatization with IC5-maleimide, and free dye removal while the protein was bound to the column, we were able to reduce the procedure time significantly and at the same time achieve better labeling efficiency and quality. The beta'-fragment with a N-terminal His(6)-tag was purified from inclusion bodies and could be refolded prior to or after binding to a Ni-NTA affinity column. Reduction prior to labeling was achieved with TCEP that does not interfere with Ni-NTA chemistry. The labeled beta'-fragment was tested with sigma70 that was labeled with an europium-based fluorophore for binding in a electrophoretic mobility-shift assay. The sigma-to-core protein interaction in bacterial RNA polymerase offers a potentially specific target for drug discovery, since it is highly conserved among the eubacteria, but differs significantly from eukaryotes. (C) 2002 Elsevier Science (USA). All rights reserved.