Engineering substrate specificity of O6-alkylguanine-DNA alkyltransferase for specific protein labeling in living cells.

Fusion proteins of human O(6)-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.