Engineering of an orthogonal aminoacyl-tRNA synthetase for efficient incorporation of the non-natural amino acid O-methyl-L-tyrosine using fluorescence-based bacterial cell sorting.

@article{Kuhn2010EngineeringOA,
  title={Engineering of an orthogonal aminoacyl-tRNA synthetase for efficient incorporation of the non-natural amino acid O-methyl-L-tyrosine using fluorescence-based bacterial cell sorting.},
  author={Sebastian M Kuhn and Marina Rubini and Markus Fuhrmann and Ina Theobald and Arne Skerra},
  journal={Journal of molecular biology},
  year={2010},
  volume={404 1},
  pages={70-87}
}
We describe a strategy for the rapid selection of mutant aminoacyl-tRNA synthetases (aaRS) with specificity for a novel amino acid based on fluorescence-activated cell sorting of transformed Escherichia coli using as reporter the enhanced green fluorescent protein (eGFP) whose gene carries an amber stop codon (TAG) at a permissive site upstream of the fluorophore. To this end, a one-plasmid expression system was developed encoding an inducible modified Methanocaldococcus jannaschii (Mj) tyrosyl… CONTINUE READING
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