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A unique transfer RNA (tRNA)/aminoacyl-tRNA synthetase pair has been generated that expands the number of genetically encoded amino acids in Escherichia coli. When introduced into E. coli, this pair leads to the in vivo incorporation of the synthetic amino acid O-methyl-L-tyrosine into protein in response to an amber nonsense codon. The fidelity of translation is greater than 99%, as determined by analysis of dihydrofolate reductase containing the unnatural amino acid. This approach should provide a general method for increasing the genetic repertoire of living cells to include a variety of amino acids with novel structural, chemical, and physical properties not found in the common 20 amino acids.
The genetic code of all organisms encodes the same 20 common amino acids. These amino acids can be modified by posttranslational modification, e.g., phosphorylation or oxidation, and in rarer instances, augmented by selenocysteine (1). Nonetheless, it is remarkable that polypeptides synthesized from 20 simple building blocks carry out all of the complex processes of life. Is it possible that the properties of proteins, or possibly an entire organism, could be enhanced by expanding the genetic code to include additional amino acids with novel biological, chemical, or physical properties? To begin to address this question, we developed a strategy that makes it possible to site-specifically incorporate unnatural amino acids directly into proteins in living cells. This methodology should also provide a powerful tool for analyzing protein function both in vitro and in vivo.
Unnatural amino acids can be site-specifically incorporated into proteins in vitro by the addition of chemically aminoacylated suppressor tRNAs to protein synthesis reactions programmed with a gene containing a desired amber nonsense mutation (2-6). One can also substitute a number of the common 20 amino acids with close structural homologs using auxotrophic strains (7-9). However, the addition of a new amino acid to the genetic repertoire in vivo requires additional components for the biosynthetic machinery. A new tRNA must be constructed that is not recognized by existing E. coli aminoacy1-tRNA synthetases, but functions efficiently in translation (an orthogonal tRNA). This tRNA must deliver the novel amino acid in response to a codon that does not encode any of the common 20 amino acids, e.g., nonsense or four base codons. The former have been used together...