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Fusion proteins of human O6-alkylguanine-DNA alkyltransferase (AGT) can be specifically labeled with a wide variety of synthetic probes in mammalian cells; this makes them an attractive tool for studying protein function. However, to avoid undesired labeling of endogenous wild-type AGT (wtAGT), the specific labeling of AGT fusion proteins has been restricted to AGT-deficient mammalian cell lines. We present here the synthesis of an inhibitor of wtAGT and the generation of AGT mutants that are resistant to this inhibitor. This enabled the inactivation of wtAGT and specific labeling of fusion proteins of the AGT mutant in vitro and in living cells. The ability to specifically label AGT fusion proteins in the presence of endogenous AGT, after brief incubation of the cells with a small-mol. inhibitor, should significantly broaden the scope of application of AGT fusion proteins for studying protein function in living cells. [on SciFinder (R)]
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Sophie Bouchet, Jean Cevey, David Hacker, Stéphanie Tissot, Sarah Wulhfard, Florian Maria Wurm
Christopher Chidley, Thomas Gronemeyer, Christian Heinis, Kai Johnsson, Alexandre Juillerat, Antje Keppler