title: Forced Ambiguity of the Leucine Codons for Multiple-Site-Specific Incorporation of a Noncanonical Amino Acid.
abstract: Multiple-site-specific incorporation of a noncanonical amino acid into a recombinant protein would be a very useful technique to generate multiple chemical handles for bioconjugation and multivalent binding sites for the enhanced interaction. Previously combination of a mutant yeast phenylalanyl-tRNA synthetase variant and the yeast phenylalanyl-tRNA containing the AAA anticodon was used to incorporate a noncanonical amino acid into multiple UUU phenylalanine (Phe) codons in a site-specific manner. However, due to the less selective codon recognition of the AAA anticodon, there was significant misincorporation of a noncanonical amino acid into unwanted UUC Phe codons. To enhance codon selectivity, we explored degenerate leucine (Leu) codons instead of Phe degenerate codons. Combined use of the mutant yeast phenylalanyl-tRNA containing the CAA anticodon and the yPheRS_naph variant allowed incorporation of a phenylalanine analog, 2-naphthylalanine, into murine dihydrofolate reductase in response to multiple UUG Leu codons, but not to other Leu codon sites. Despite the moderate UUG codon occupancy by 2-naphthylalaine, these results successfully demonstrated that the concept of forced ambiguity of the genetic code can be achieved for the Leu codons, available for multiple-site-specific incorporation.
authors.affiliation: School of Materials Science and Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju, Republic of Korea.|Department of Chemical Engineering, University of Virginia, Charlottesville, Virginia, United States of America.
authors.forename: Inchan
authors.initials: I
authors.lastname: Kwon
authors.affiliation: School of Materials Science and Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju, Republic of Korea.|Department of Biological Sciences, College of Natural Sciences, Chonnam National University, Gwangju, Republic of Korea.
authors.forename: Eun Sil
authors.initials: ES
authors.lastname: Choi
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism; D000926:Anticodon / Q000235:genetics / Q000378:metabolism; D003062:Codon / Q000235:genetics / Q000378:metabolism*; D007930:Leucine / Q000235:genetics / Q000378:metabolism*; D009281:Naphthalenes / Q000378:metabolism*; D010652:Phenylalanine-tRNA Ligase / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D000926:Anticodon; D003062:Codon; D009281:Naphthalenes; D029701:Saccharomyces cerevisiae Proteins; D010652:Phenylalanine-tRNA Ligase; D007930:Leucine; D000409:Alanine; C513002:2-naphthylalanine