Site-specific protein double labeling by expressed protein ligation: applications to repeat proteins
Site-specific protein double labeling by expressed protein ligation: applications to repeat proteins
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DOI：
10.1039/c1ob06397a
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发表时间：
2012-01-01
影响因子：
3.2
通讯作者：
D'Andrea, Luca Domenico
中科院分区：
文献类型：
--
作者：
De Rosa, Lucia;Cortajarena, Aitziber L.;D'Andrea, Luca Domenico
在过去的几年里，标记蛋白质的使用在生命科学中得到了显著的扩展。现在，标记蛋白质是广泛的生物物理和化学生物学应用中不可缺少的工具。特别是，对更复杂的实验设置的追求需要开发新的合成方法，特别是对于多个特定部位的标记。在本文中，我们描述了一种基于表达蛋白质连接的合成策略来制备高纯度和均一的蛋白质，其中两个不同的分子探针被特定地掺入到任何想要的位置。蛋白质在溶液中被顺序标记，优点是不需要大量过量的探针，标记的片段不受肽合成长度的限制。这一策略被应用于在蛋白质序列的不同位置用荧光团对选择性地标记重复蛋白质。根据单分子FRET研究的要求，以高纯度和均一性制备了双标记蛋白。值得注意的是，这种方法可以适用于引入两个以上的分子探针。
In the last few years, the use of labeled proteins has significantly expanded in the life sciences. Now, labeled proteins are indispensable tools for a wide spectrum of biophysical and chemical biology applications. In particular, the quest for more sophisticated experimental setups requires the development of new synthetic methodology, especially for multiple site-specific labeling. In this paper, we describe a synthetic strategy based on expressed protein ligation to prepare proteins in high purity and homogeneity, in which two different molecular probes are incorporated specifically at any desired position. Proteins are sequentially labeled in solution, with the advantage that a large excess of probes is not required and the labeled fragments are not restricted to peptide synthesis length limitations. This strategy was applied to selectively label a repeat protein with a fluorophores pair in different positions along the protein sequence. The doubly labeled proteins were prepared at high purity and homogeneity, as required for single molecule FRET studies. Remarkably, this approach can be adapted to the introduction of more than two molecular probes.