Abstract
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 μg 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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Acknowledgements
This work was funded by a US National Institutes of Health grant GM62159. This is manuscript 18455 of the Scripps Research Institute.
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W.L. developed and evaluated the method, A.B. carried out the MS analysis, Shou C. and Shuibing C. helped with protein characterization, and W.L. and P.G.S. designed the project, analyzed the data and prepared the manuscript.
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Supplementary information
Supplementary Fig. 1
Amber suppression is dependent upon both the EcTyrRS and BstRNATyrCUA genes in both T-rex CHO and T-rex 293 cells. (PDF 51 kb)
Supplementary Fig. 2
Western blot analysis of expression of six EcTyrRS variants in T-rexTM CHO and 293 cells. (PDF 46 kb)
Supplementary Fig. 3
ESI-TOF MS spectrum of affinity purified wild-type GFP. (PDF 14 kb)
Supplementary Fig. 4
Annotated tandem MS spectra of the peptide FSVSGEGEGDATY*GK from mutant GFP containing pAzpa. (PDF 56 kb)
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Liu, W., Brock, A., Chen, S. et al. Genetic incorporation of unnatural amino acids into proteins in mammalian cells. Nat Methods 4, 239–244 (2007). https://doi.org/10.1038/nmeth1016
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DOI: https://doi.org/10.1038/nmeth1016
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