Nature Methods
- 4, 239 - 244 (2007)
Published online: 25 February 2007; | doi:10.1038/nmeth1016
Genetic incorporation of unnatural amino acids into proteins in mammalian cellsWenshe Liu1, Ansgar Brock2, Shuo Chen1, 3, Shuibing Chen1 & Peter G Schultz1, 21
Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 Torrey Pines Road, La Jolla, California 92037, USA. 2
Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, USA. 3
Present address: Department of Biological Sciences, Ohio University, Athens, Ohio 45701, USA.
Correspondence should be addressed to Peter G Schultz schultz@scripps.edu We developed a general approach that allows unnatural amino acids with diverse physicochemical and biological properties to be genetically encoded in mammalian cells. A mutant Escherichia coli aminoacyl-tRNA synthetase (aaRS) is first evolved in yeast to selectively aminoacylate its tRNA with the unnatural amino acid of interest. This mutant aaRS together with an amber suppressor tRNA from Bacillus stearothermophilus is then used to site-specifically incorporate the unnatural amino acid into a protein in mammalian cells in response to an amber nonsense codon. We independently incorporated six unnatural amino acids into GFP expressed in CHO cells with efficiencies up to 1  g protein per 2  107 cells; mass spectrometry confirmed a high translational fidelity for the unnatural amino acid. This methodology should facilitate the introduction of biological probes into proteins for cellular studies and may ultimately facilitate the synthesis of therapeutic proteins containing unnatural amino acids in mammalian cells.
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