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. 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-molecule inhibitor, should significantly broaden the scope of application of AGT fusion proteins for studying protein function in living cells.
Juillerat
Alexandre
A
Ecole Polytechnique Fédérale de Lausanne, Institute of Chemical Sciences and Engineering, 1015 Lausanne, Switzerland.
Heinis
Christian
C
Sielaff
India
I
Barnikow
Jan
J
Jaccard
Hugues
H
Kunz
Beatrice
B
Terskikh
Alexey
A
Johnsson
Kai
K
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D008958:Models, Molecular; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015202:Protein Engineering; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity; D014162:Transfection
D005456:Fluorescent Dyes; D011506:Proteins; D011993:Recombinant Fusion Proteins; D019853:O(6)-Methylguanine-DNA Methyltransferase