An orthogonal tRNA/aminoacyl-tRNA synthetase pair may genetically integrate unnatural amino acids into proteins in living cells. Here, we report a technique to effectively produce the orthogonal tRNA and synthetase in Saccharomyces cerevisiae, allowing for highly effective genetic incorporation of non-natural amino acids into target proteins in yeast. We also discuss the use of a yeast strain that is defective in nonsense-mediated mRNA decay, which improves the efficiency with which an unnatural amino acid is incorporated when a stop codon is used to encode it. By using the unique features provided by synthetic amino acids, these methods will make it easier to study proteins and the biological processes that are associated to them in yeast (Elmlinger et al., 2002).
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