Site specific labeling of two proteins in one system by atypical split inteins
Site specific labeling of two proteins in one system by atypical split inteins
复制标题
通过非典型分裂内含子对一个系统中的两种蛋白质进行位点特异性标记
DOI：
10.1016/j.ijbiomac.2017.11.077
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发表时间：
2018-04-01
影响因子：
8.2
通讯作者：
Meng，Qing
中科院分区：
文献类型：
--
作者：
Li，Xue;Zhang，Xiao-ling;Meng，Qing
非典型内含肽已被用于蛋白质位点特异性标记和修饰。S1或S11内含肽含有小的N-内含肽或C-内含肽，其可以化学合成并添加所需的标记或修饰。然而，在一个系统中同时进行多蛋白质特异性标记的报道很少。在此，我们建立了基于三对非典型内含肽RBS 1（RmaDnaB S1）/SGS 1（SspGyrB S1）内含肽、TE 3S 11（TerDnaE-3 S11）/SGS 11（SspGyrB S11）内含肽和RBS 1/TE 3S 11内含肽的两种蛋白质的特异性标记方法。首先，将内含肽片段分别与模型蛋白融合，作为前体蛋白进行表达和纯化。然后通过Western印迹分析剪接反应来确定内含肽之间的非交叉反应性。最后，在一个介导这些内含肽对的系统中，用荧光基团特异性地标记模型蛋白，并且甚至可以在细胞裂解物中标记。我们的研究结果首次报道了一种基于上述内含肽对在同一系统中同时标记两种蛋白质N/C末端的方法，该方法具有显著的剪接效率，可能用于蛋白质结构和功能的研究或某些特定的应用。
Atypical inteins have been used for protein site-specific labeling and modification. S1 or S11 intein contain a small N-intein or C-intein which can be chemically synthesized and added with desired labels or modifications. However, seldom reports have been found to develop multi-protein specific labeling in one system at the same time. Here, we established a specific labeling method for two proteins based on three pairs of atypical inteins, RBS1 (RmaDnaB S1)/SGS1 (SspGyrB S1) intein, TE3S11 (TerDnaE-3 S11)/SGS11 (SspGyrB S11) intein, and RBS1/TE3S11 intein. Firstly, intein fragments were respectively fused with model proteins, expressed and purified as precursors. The non-cross reactivity between inteins was then determined by splicing reactions analysis through Western-blot. Finally, the model proteins were specifically labeled with fluorescent groups in one system mediating these intein pairs, and can be labeled even in the cell lysate. Our results for the first time report a method labeling the N/C-terminal of two proteins altogether in the same system based on above intein pairs with a marked splicing efficiency, which could potentially be used for protein structural and functional research or some specific applications.