Engineering Substrate Specificity of O super(6)-Alkylguanine-DNA Alkyltransferase for Specific Protein Labeling in Living Cells

Fusion proteins of human O super(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 (wtAG...

Full description

Saved in:
Bibliographic Details
Published in:Chembiochem : a European journal of chemical biology 2005-07, Vol.6 (7), p.1263-1269
Main Authors: Juillerat, Alexandre, Heinis, Christian, Sielaff, India, Barnikow, Jan, Jaccard, Hugues, Kunz, Beatrice, Terskikh, Alexey, Johnsson, Kai
Format: Article
Language:English
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Fusion proteins of human O super(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.
ISSN:1439-4227
1439-4227
DOI:10.1002/cbic.200400431