title: Discovery of aminoacyl-tRNA synthetase activity through cell-surface display of noncanonical amino acids.
abstract: The incorporation of noncanonical amino acids into recombinant proteins in Escherichia coli can be facilitated by the introduction of new aminoacyl-tRNA synthetase activity into the expression host. We describe here a screening procedure for the identification of new aminoacyl-tRNA synthetase activity based on the cell surface display of noncanonical amino acids. Screening of a saturation mutagenesis library of the E. coli methionyl-tRNA synthetase (MetRS) led to the discovery of three MetRS mutants capable of incorporating the long-chain amino acid azidonorleucine into recombinant proteins with modest efficiency. The Leu-13 --> Gly (L13G) mutation is found in each of the three MetRS mutants, and the MetRS variant containing this single mutation is highly efficient in producing recombinant proteins that contain azidonorleucine.
authors.affiliation: *Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125.
authors.forename: A James
authors.initials: AJ
authors.lastname: Link
authors.affiliation: *Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125.
authors.forename: Mandy K S
authors.initials: MKS
authors.lastname: Vink
authors.affiliation: Departments of Chemistry and Molecular and Cell Biology and Howard Hughes Medical Institute, University of California, Berkeley, CA 94720; and.|Materials Science Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Agard
authors.affiliation: Departments of Chemistry and Molecular and Cell Biology and Howard Hughes Medical Institute, University of California, Berkeley, CA 94720; and.|Materials Science Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Prescher
authors.affiliation: Departments of Chemistry and Molecular and Cell Biology and Howard Hughes Medical Institute, University of California, Berkeley, CA 94720; and.|Materials Science Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720.
authors.forename: Carolyn R
authors.initials: CR
authors.lastname: Bertozzi
authors.affiliation: *Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125; tirrell@caltech.edu.
authors.forename: David A
authors.initials: DA
authors.lastname: Tirrell
mesh_terms: D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism*; D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D003300:Copper / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D015394:Molecular Structure; D009154:Mutation / Q000235:genetics; D019151:Peptide Library; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D000596:Amino Acids; D019151:Peptide Library; D011994:Recombinant Proteins; D001710:Biotin; D003300:Copper; D000604:Amino Acyl-tRNA Synthetases