Labeling of fusion proteins of O6-alkylguanine-DNA alkyltransferase with small molecules in vivo and in vitro.
The in vivo and in vitro labeling of fusion proteins with synthetic molecules capable of probing and controlling protein function has the potential to become an important method in functional genomics and proteomics. We have recently introduced an approach for the specific labeling of fusion proteins, which is based on the generation of fusion proteins with the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (hAGT) and the irreversible reaction of hAGT with O6-benzylguanine derivatives. Here, we report optimized protocols for the synthesis of O6-benzylguanine derivatives and the use of such derivatives for the labeling of different hAGT fusion proteins in vivo and in vitro.
Institute of Molecular and Biological Chemistry, Ecole Polytechnique Fédérale de Lausanne, Lausanne CH-1015, Switzerland.
Antje
A
Keppler
Maik
M
Kindermann
Susanne
S
Gendreizig
Horst
H
Pick
Horst
H
Vogel
Kai
K
Johnsson
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D047628:Estrogen Receptor alpha / Q000235:genetics / Q000378:metabolism; D019793:Fluorescein / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008727:Methotrexate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D008856:Microscopy, Fluorescence; D015394:Molecular Structure; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*; D013347:Subcellular Fractions / Q000378:metabolism; D013629:Tamoxifen / Q000031:analogs & derivatives* / Q000494:pharmacology; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D047628:Estrogen Receptor alpha; D019793:Fluorescein; D005456:Fluorescent Dyes; D005656:Fungal Proteins; D006147:Guanine; D008727:Methotrexate; D019853:O(6)-Methylguanine-DNA Methyltransferase; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D013629:Tamoxifen; D000099160:LexA Repressor Protein; C064976:O(6)-benzylguanine; C016601:afimoxifene