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Genetic Incorporation of Unnatural Amino Acids into Proteins in Yeast

  • Qian Wang
  • Lei WangEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 794)

Abstract

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 words

Unnatural amino acid Yeast Orthogonal tRNA Orthogonal synthetase Amber suppression Polymerase III promoter Nonsense-mediated mRNA decay Green fluorescent protein 

Notes

Acknowledgments

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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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Tianjin Institute of Industrial BiotechnologyChinese Academy of SciencesYianjinChina
  2. 2.The Jack H. Skirball Center for Chemical Biology & Proteomics, The Salk Institute for Biological StudiesLa JollaUSA

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