Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 101, Issue 20, Pages 7566-7571Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0401517101
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With few exceptions the genetic codes of all known organisms encode the same 20 amino acids, yet all that is required to add a new building block are a unique tRNA/aminoacyl-tRNA synthetase pair, a source of the amino acid, and a unique codon that specifies the amino acid. For example, the amber nonsense codon, TAG, together with orthogonal Methanococcus jannaschii or Escherichia coli tRNA/synthetase pairs have been used to genetically encode a variety of unnatural amino acids in E. coli and yeast, respectively. However, the availability of noncoding triplet codons ultimately limits the number of amino acids encoded by any organism. Here, we report the design and generation of an orthogonal synthetase/ tRNA pair derived from archaeal tRNA(Lys) sequences that efficiently and selectively incorporates an unnatural amino acid into proteins in response to the quadruplet codon, AGGA. Frameshift suppression with L-homoglutamine (hGln) does not significantly affect protein yields or cell growth rates and is mutually orthogonal with amber suppression, permitting the simultaneous incorporation of two unnatural amino acids, hGln and O-methyl-L-tyrosine, at distinct positions within myoglobin. This work suggests that neither the number of available triplet codons nor the translational machinery itself represents a significant barrier to further expansion of the genetic code.
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