Discovery of Escherichia coli methionyl-tRNA synthetase mutants for efficient labeling of proteins with azidonorleucine in vivo.

I Caglar Tanrikulu (1) , Emmanuelle Schmitt (2) , Yves Mechulam (2) , William A Goddard (1) , David A Tirrell (1)

1 DCCE-Caltech - Division of Chemistry and Chemical Engineering
2 BIOC - Laboratoire de Biochimie de l'Ecole polytechnique

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.

Domaines: Biochimie, Biologie Moléculaire

I Caglar Tanrikulu, Emmanuelle Schmitt, Yves Mechulam, William A Goddard, David A Tirrell. Discovery of Escherichia coli methionyl-tRNA synthetase mutants for efficient labeling of proteins with azidonorleucine in vivo.. Proceedings of the National Academy of Sciences of the United States of America, 2009, 106 (36), pp.15285-90. ⟨10.1073/pnas.0905735106⟩. ⟨hal-00502034⟩