Directed evolution of O6-alkylguanine-DNA alkyltransferase for efficient labeling of fusion proteins with small molecules in vivo.
We report here the generation of mutants of the human O(6)-alkylguanine-DNA alkyltransferase (hAGT) for the efficient in vivo labeling of fusion proteins with synthetic reporter molecules. Libraries of hAGT were displayed on phage, and mutants capable of efficiently reacting with the inhibitor O(6)-benzylguanine were selected based on their ability to irreversibly transfer the benzyl group to a reactive cysteine residue. Using synthetic O(6)-benzylguanine derivatives, the selected mutant proteins allow for a highly efficient covalent labeling of hAGT fusion proteins in vivo and in vitro with small molecules and therefore should become important tools for studying protein function in living cells. In addition to various applications in proteomics, the selected mutants also yield insight into the interaction of the DNA repair protein hAGT with its inhibitor O(6)-benzylguanine.
Institute of Molecular and Biological Chemistry, Swiss Federal Institute of Technology, CH-1015 Lausanne, Switzerland.
Alexandre
A
Juillerat
Thomas
T
Gronemeyer
Antje
A
Keppler
Susanne
S
Gendreizig
Horst
H
Pick
Horst
H
Vogel
Kai
K
Johnsson
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D017931:DNA Primers; D019020:Directed Molecular Evolution; D005456:Fluorescent Dyes; D017930:Genes, Reporter; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry; D007202:Indicators and Reagents; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000737:chemistry / Q000235:genetics*; D019151:Peptide Library; D040901:Proteomics; D011994:Recombinant Proteins / Q000737:chemistry*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D008024:Ligands; D019151:Peptide Library; D011994:Recombinant Proteins; C064976:O(6)-benzylguanine; D006147:Guanine; D019853:O(6)-Methylguanine-DNA Methyltransferase