Gautier Arnaud, Juillerat Alexandre, Heinis Christian, Corrêa Ivan Reis, Kindermann Maik, Beaufils Florent, Johnsson Kai
Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.
Chem Biol. 2008 Feb;15(2):128-36. doi: 10.1016/j.chembiol.2008.01.007.
The visualization of complex cellular processes involving multiple proteins requires the use of spectroscopically distinguishable fluorescent reporters. We have previously introduced the SNAP-tag as a general tool for the specific labeling of SNAP-tag fusion proteins in living cells. The SNAP-tag is derived from the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) and can be covalently labeled in living cells using O6-benzylguanine derivatives bearing a chemical probe. Here we report the generation of an AGT-based tag, named CLIP-tag, which reacts specifically with O2-benzylcytosine derivatives. Because SNAP-tag and CLIP-tag possess orthogonal substrate specificities, SNAP and CLIP fusion proteins can be labeled simultaneously and specifically with different molecular probes in living cells. We furthermore show simultaneous pulse-chase experiments to visualize different generations of two different proteins in one sample.
对涉及多种蛋白质的复杂细胞过程进行可视化，需要使用光谱可区分的荧光报告分子。我们之前已引入SNAP标签，作为在活细胞中特异性标记SNAP标签融合蛋白的通用工具。SNAP标签源自人类DNA修复蛋白O6-烷基鸟嘌呤-DNA烷基转移酶（AGT），可在活细胞中使用带有化学探针的O6-苄基鸟嘌呤衍生物进行共价标记。在此，我们报告了一种基于AGT的标签CLIP标签的产生，它能与O2-苄基胞嘧啶衍生物特异性反应。由于SNAP标签和CLIP标签具有正交的底物特异性，SNAP和CLIP融合蛋白可在活细胞中同时用不同的分子探针进行特异性标记。我们还展示了同时进行的脉冲追踪实验，以在一个样本中可视化不同代的两种不同蛋白质。
