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Reprogramming the amino-acid substrate specificity of orthogonal aminoacyl-tRNA synthetases to expand the genetic code of eukaryotic cells

Nature Protocols volume 2, pages 2590–2600 (2007)Cite this article

Abstract

The genetic code of living organisms has been expanded to allow the site-specific incorporation of unnatural amino acids into proteins in response to the amber stop codon UAG. Numerous amino acids have been incorporated including photo-crosslinkers, chemical handles, heavy atoms and post-translational modifications, and this has created new methods for studying biology and developing protein therapeutics and other biotechnological applications. Here we describe a protocol for reprogramming the amino-acid substrate specificity of aminoacyl-tRNA synthetase enzymes that are orthogonal in eukaryotic cells. The resulting aminoacyl-tRNA synthetases aminoacylate an amber suppressor tRNA with a desired unnatural amino acid, but no natural amino acids, in eukaryotic cells. To achieve this change of enzyme specificity, a library of orthogonal aminoacyl-tRNA synthetase is generated and genetic selections are performed on the library in Saccharomyces cerevisiae. The entire protocol, including characterization of the evolved aminoacyl-tRNA synthetase in S. cerevisiae, can be completed in approximately 1 month.

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Figure 1
Figure 2: Plasmid maps and EcYRS structure.
Figure 3: Examples of screened phenotypes of the O-methyltyrosine selection.
Figure 4

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Acknowledgements

T.A.C. is grateful to the University of Maryland for financial support. J.W.C. is an EMBO Young Investigator and is supported by the Medical Research Council. We are grateful to P.G.S. for supporting this work.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland, 20742-2021, USA

    T Ashton Cropp

  2. Department of Bioengineering, University of California, 717 Potter Street, Berkeley, 94720, California, USA

    J Christopher Anderson

  3. Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK

    Jason W Chin

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  1. T Ashton Cropp
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Correspondence to T Ashton Cropp or Jason W Chin.

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Cropp, T., Anderson, J. & Chin, J. Reprogramming the amino-acid substrate specificity of orthogonal aminoacyl-tRNA synthetases to expand the genetic code of eukaryotic cells. Nat Protoc 2, 2590–2600 (2007). https://doi.org/10.1038/nprot.2007.378

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