Discovery of Escherichia coli methionyl-tRNA synthetase mutants for efficient labeling of proteins with azidonorleucine in vivo.
Incorporation of noncanonical amino acids into cellular proteins often requires engineering new aminoacyl-tRNA synthetase activity into the cell. A screening strategy that relies on cell-surface display of reactive amino acid side-chains was used to identify a diverse set of methionyl-tRNA synthetase (MetRS) mutants that allow efficient incorporation of the methionine (Met) analog azidonorleucine (Anl). We demonstrate that the extent of cell-surface labeling in vivo is a good indicator of the rate of Anl activation by the MetRS variant harbored by the cell. By screening at low Anl concentrations in Met-supplemented media, MetRS variants with improved activities toward Anl and better discrimination against Met were identified.
Tanrikulu
I Caglar
IC
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
Schmitt
Emmanuelle
E
Mechulam
Yves
Y
Goddard
William A
WA
Tirrell
David A
DA
D001386:Azides / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D005434:Flow Cytometry; D006860:Hydrogen Bonding; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D008718:Methionine-tRNA Ligase / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation / Q000235:genetics; D009646:Norleucine / Q000031:analogs & derivatives* / Q000737:chemistry; D015202:Protein Engineering / Q000379:methods; D013194:Staining and Labeling / Q000379:methods
D001386:Azides; C000609218:azidonorleucine; D009646:Norleucine; D008715:Methionine; D008718:Methionine-tRNA Ligase
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