(136c) High-Throughput Screening For Methionyl-Trna Synthetases That Enable Residue-Specific Incorporation Of Noncanonical Amino Acids Into Recombinant Proteins In Bacterial Cells

We report a high-throughput method of screening aminoacyl-tRNA synthetase (aaRS) libraries for global incorporation of noncanonical amino acids. The activity of aaRS toward amino acid analogs can be monitored by translation of model proteins in media depleted of the corresponding canonical amino acids. A variant of the green fluorescent protein was engineered to permit incorporation of methionine analogs without loss of fluorescence. Using the engineered GFP variant as a translational reporter, we screened a saturation mutagenesis library of Escherichia coli methionyl-tRNA synthetases (MetRS) for activity toward 6,6,6-trifluoronorleucine (Tfn), and identified a MetRS mutant that enabled high-yield expression of recombinant proteins containing Tfn. The screening method described here is simple and efficient, and directly applicable to Met analogs other than Tfn.