Development of a tandem protein trans-splicing system based on native and engineered split inteins
Development of a tandem protein trans-splicing system based on native and engineered split inteins
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DOI：
10.1021/ja042287w
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发表时间：
2005-05-04
影响因子：
15
通讯作者：
Muir, TW
中科院分区：
文献类型：
--
作者：
Shi, JX;Muir, TW
涉及天然或人工分裂的内含肽的蛋白质转拼导致两个多肽通过肽键连接在一起。虽然这种现象在化学生物学和生物技术中得到了广泛的应用，但人们对控制初始片段关联步骤的分子识别事件知之甚少。在本研究中，荧光方法已用于测量 Ssp DnaE 分裂内含肽相互作用的解离常数，并确定片段关联的开启和关闭速率。 DnaE 片段以低纳摩尔亲和力结合，我们的数据表明静电对于生理 pH 下片段的快速结合做出了重要贡献。该信息用于开发基于天然和工程化的分裂内含肽的串联转拼系统。这种新颖的系统允许在天然条件下一锅组装三种多肽，并且可以在粗制细胞裂解物中进行。该技术应该提供一种方便的方法，在大型多结构域蛋白质的背景下对特定结构域进行分段同位素或荧光标记。
Protein trans-splicing involving naturally or artificially split inteins results in two polypeptides being linked together by a peptide bond. While this phenomenon has found a variety of applications in chemical biology and biotechnology, precious little is known about the molecular recognition events governing the initial fragment association step. In this study, fluorescence approaches have been used to measure the dissociation constant for the Ssp DnaE split intein interaction and to determine the on and off rates of fragment association. The DnaE fragments bind with low nanomolar affinity, and our data suggest that electrostatics make an important contribution to the very rapid association of the fragments at physiological pH. This information was used to develop a tandem trans-splicing system based on native and engineered split inteins. This novel system allows the one-pot assembly of three polypeptides under native conditions and can be performed in crude cell lysates. The technology should provide a convenient approach to the segmental isotopic or fluorogenic labeling of specific domains within the context of large multidomain proteins.