Genetic Incorporation of Unnatural Amino Acids into Proteins in Yeast
Unnatural amino acids can be genetically incorporated into proteins in live cells by using an orthogonal tRNA/aminoacyl-tRNA synthetase pair. Here we describe a method to efficiently express the orthogonal tRNA and synthetase in Saccharomyces cerevisiae, which enables unnatural amino acids to be genetically incorporated into target proteins in yeast with high efficiency. We also describe the use of a yeast strain deficient in the nonsense-mediated mRNA decay, which further increases the unnatural amino acid incorporation efficiency when a stop codon is used to encode the unnatural amino acid. These strategies will facilitate the investigation of proteins and their related biological processes in yeast by exploiting the novel properties afforded by unnatural amino acids.
Key wordsUnnatural amino acid Yeast Orthogonal tRNA Orthogonal synthetase Amber suppression Polymerase III promoter Nonsense-mediated mRNA decay Green fluorescent protein
We thank Dr. Vicki Lundblad and members of the Lundblad lab for providing reagents and advice on yeast protocols. This work was supported by CIRM (RN1-00577-1) and NIH (1DP2OD004744).
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