title: Tailoring the substrate specificity of yeast phenylalanyl-tRNA synthetase toward a phenylalanine analog using multiple-site-specific incorporation.
abstract: A yeast phenylalanyl-tRNA synthetase variant with T415G mutation (yPheRS (T415G)) was rationally designed to recognize various phenylalanine (Phe) analogs allowing site-specific incorporation into an amber site of a protein in E. coli. However, the relaxed substrate specificity of yPheRS (T415G) led to a significant tryptophan (Trp) misincorporation restricting the utility of yPheRS for biosynthesis of proteins containing a Phe analog. In order to obtain yPheRS variants with high substrate-specificity toward a Phe analog, we developed a general high-throughput screening method. This method uses fluorescence reduction of green fluorescence protein (GFP) upon efficient introduction of a Phe analog into multiple sites of GFP by breaking the degeneracy of the Phe codons. Combined use of positive and negative screenings of a yPheRS saturation library led to a yPheRS variant (yPheRS_naph) very selective toward 2-l-naphthylalanine (2Nal), a model Phe analog. The yPheRS_naph exhibited 6-fold higher relative activity toward 2Nal (vs Trp) in ATP-PPi exchange assays and led to high-fidelity incorporation of 2Nal into an amber site of murine dihydrofolate reductase in both minimal and rich media. These results successfully demonstrate that the high-throughput screening method developed can be used to evolve yPheRS to be very selective toward a Phe analog.
authors.affiliation: †School of Materials Science and Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712, Republic of Korea.|‡Department of Chemical Engineering, University of Virginia, Charlottesville, Virginia 22904-4741, United States.
authors.forename: Inchan
authors.initials: I
authors.lastname: Kwon
authors.affiliation: ‡Department of Chemical Engineering, University of Virginia, Charlottesville, Virginia 22904-4741, United States.
authors.forename: Sung In
authors.initials: SI
authors.lastname: Lim
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D051379:Mice; D010652:Phenylalanine-tRNA Ligase / Q000235:genetics*; D013379:Substrate Specificity / Q000235:genetics*; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D014364:Tryptophan / Q000235:genetics; D015003:Yeasts / Q000235:genetics*
chemical_list: D014364:Tryptophan; D013762:Tetrahydrofolate Dehydrogenase; D010652:Phenylalanine-tRNA Ligase
keywords: 2-l-naphthylalanine; high-throughput screening; multiple-site-specific incorporation,; saturation mutagenesis; substrate specificity; yeast phenylalanyl-tRNA synthetase