Segmental isotopic labeling of a 140 kDa dimeric multi-domain protein CheA from Escherichia coli by expressed protein ligation and protein trans-splicing.
Segmental isotopic labeling of a 140 kDa dimeric multi-domain protein CheA from Escherichia coli by expressed protein ligation and protein trans-splicing.
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DOI：
10.1007/s10858-012-9628-3
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发表时间：
2012-07
影响因子：
2.7
通讯作者：
Iwaï H
中科院分区：
文献类型：
--
作者：
Minato Y;Ueda T;Machiyama A;Shimada I;Iwaï H
片段同位素标记是一种强大的标记工具，不仅可以缓解信号重叠问题，还可以保留均匀同位素标记的特征，从而促进较大蛋白质的 NMR 研究。尽管表达蛋白连接（EPL）和蛋白反式剪接（PTS）这两种方法主要用于片段同位素标记，但还没有这两种方法直接用于相同蛋白质的例子。在这里，我们将 EPL 和 PTS 方法应用于 140 kDa 二聚体多结构域蛋白大肠杆菌 CheA，并通过这两种方法成功产生了连接的 CheA 二聚体。在 EPL 方法中，需要对连接位点和条件进行广泛优化，以获得足够量的 CheA NMR 样品，因为 CheA 含有二聚体形成结构域，并且不可能在理想的 EPL 条件下获得高反应物浓度 (1–5 mM) 的 CheA 片段，从而导致分段标记的 CheA 二聚体的产量低。 PTS 方法无需大量优化即可在体内和体外充分产生片段标记的连接 CheA。这可能是因为 CheA 具有与长接头连接的独立结构域，可容纳七残基突变而不丧失功能，这是 PTS 引入的以实现高产率。对于片段同位素标记的 CheA 二聚体的常规制备，PTS 方法比 EPL 方法省力。这两种方法仍有待进一步开发，以促进片段同位素标记样品的制备，而无需对连接进行广泛优化。本文的在线版本 (doi:10.1007/s10858-012-9628-3) 包含补充材料，可供授权用户使用。
Segmental isotopic labeling is a powerful labeling tool to facilitate NMR studies of larger proteins by not only alleviating the signal overlap problem but also retaining features of uniform isotopic labeling. Although two approaches, expressed protein ligation (EPL) and protein trans-splicing (PTS), have been mainly used for segmental isotopic labeling, there has been no single example in which both approaches have been directly used with an identical protein. Here we applied both EPL and PTS methods to a 140 kDa dimeric multi-domain protein E. coli CheA, and successfully produced the ligated CheA dimer by both approaches. In EPL approach, extensive optimization of the ligation sites and the conditions were required to obtain sufficient amount for an NMR sample of CheA, because CheA contains a dimer forming domain and it was not possible to achieve high reactant concentrations (1–5 mM) of CheA fragments for the ideal EPL condition, thereby resulting in the low yield of segmentally labelled CheA dimer. PTS approach sufficiently produced segmentally labeled ligated CheA in vivo as well as in vitro without extensive optimizations. This is presumably because CheA has self-contained domains connected with long linkers, accommodating a seven-residue mutation without loss of the function, which was introduced by PTS to achieve the high yield. PTS approach was less laborious than EPL approach for the routine preparation of segmentally-isotope labeled CheA dimer. Both approaches remain to be further developed for facilitating preparations of segmental isotope-labelled samples without extensive optimizations for ligation. The online version of this article (doi:10.1007/s10858-012-9628-3) contains supplementary material, which is available to authorized users.
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影响因子：
3.4
作者：
Kinoshita, Eiji;Kinoshita-Kikuta, Emiko
通讯作者：
Kinoshita-Kikuta, Emiko
DOI：
10.1073/pnas.1104824108
发表时间：
2011-06-07
影响因子：
11.1
作者：
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通讯作者：
Hazelbauer, Gerald L.
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作者：
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通讯作者：
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64.5
作者：
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影响因子：
3.5
作者：
Ellila, Simo;Jurvansuu, Jaana Maria;Iwai, Hideo
通讯作者：
Iwai, Hideo