Directed evolution of O6-alkylguanine-DNA alkyltransferase for applications in protein labeling.
The specific reaction of O6-alkylguanine-DNA alkyltransferase (AGT) with O6-benzylguanine (BG) derivatives allows for a specific labeling of AGT fusion proteins with chemically diverse compounds in living cells and in vitro. The efficiency of the labeling depends on a number of factors, most importantly on the reactivity, selectivity and stability of AGT. Here, we report the use of directed evolution and two different selection systems to further increase the activity of AGT towards BG derivatives by a factor of 17 and demonstrate the advantages of this mutant for the specific labeling of AGT fusion proteins displayed on the surface of mammalian cells. The results furthermore identify two regions of the protein outside the active site that influence the activity of the protein towards BG derivatives.
Gronemeyer
Thomas
T
Ecole Polytechnique Fédérale de Lausanne (EPFL), Institute of Chemical Sciences and Engineering CH-1015 Lausanne, Switzerland.
Chidley
Christopher
C
Juillerat
Alexandre
A
Heinis
Christian
C
Johnsson
Kai
K
D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D019143:Evolution, Molecular; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D008958:Models, Molecular; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019151:Peptide Library; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*
D019151:Peptide Library; D011993:Recombinant Fusion Proteins; C064976:O(6)-benzylguanine; D006147:Guanine; D019853:O(6)-Methylguanine-DNA Methyltransferase