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An orthogonalized platform for genetic code expansion in both bacteria and eukaryotes

Nature Chemical Biology volume 13, pages 446–450 (2017)Cite this article

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Abstract

In this study, we demonstrate the feasibility of expanding the genetic code of Escherichia coli using its own tryptophanyl–tRNA synthetase and tRNA (TrpRS–tRNATrp) pair. This was made possible by first functionally replacing this endogenous pair with an E. coli–optimized counterpart from Saccharomyces cerevisiae, and then reintroducing the liberated E. coli TrpRS–tRNATrp pair into the resulting strain as a nonsense suppressor, which was then followed by its directed evolution to genetically encode several new unnatural amino acids (UAAs). These engineered TrpRS–tRNATrp variants were also able to drive efficient UAA mutagenesis in mammalian cells. Since bacteria-derived aminoacyl–tRNA synthetase (aaRS)–tRNA pairs are typically orthogonal in eukaryotes, our work provides a general strategy to develop additional aaRS–tRNA pairs that can be used for UAA mutagenesis of proteins expressed in both E. coli and eukaryotes.

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Figure 1: Overview of the strategy to create an aaRS–tRNA pair that can drive genetic code expansion in both eukaryotes and E. coli.
Figure 2: Establishing the EcTrpRS–tRNAEcTrp pair as an orthogonal nonsense suppressor in an engineered E. coli strain.
Figure 3: Evolution of EcTrpRS to develop mutants charging 5HTP (1) with high fidelity and efficiency.
Figure 4: Site-specific incorporation of tryptophan analogs into proteins expressed in both ATMW1 E. coli and mammalian cells using polyspecific EcTrpRS variants.
Figure 5: 5AzW and 5PrW residues facilitate site-selective bioconjugation to proteins.

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Acknowledgements

The EcNR1 strain was a kind gift from G.M. Church (Harvard). A.C. is thankful for support from the Richard and Susan Smith Family Foundation, Newton, MA.

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Authors and Affiliations

  1. Department of Chemistry, Boston College, Chestnut Hill, Massachusetts, USA

    James S Italia, Partha Sarathi Addy, Chester J J Wrobel, Lisa A Crawford, Yunan Zheng & Abhishek Chatterjee

  2. Department of Biochemistry, University of Washington, Seattle, Washington, USA

    Marc J Lajoie

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Contributions

J.S.I. designed the experiments, performed strain development/characterization, cloning/mutagenesis, directed evolution, and protein expression, and wrote the manuscript; P.S.A. synthesized 5AzW and 5PrW; C.J.J.W. performed cloning/mutagenesis, directed evolution, and protein expression; L.A.C. performed the digestion/MS analysis of reporter proteins; M.J.L. assisted with the design of the recombination experiments; Y.Z. assisted with the protein expression in mammalian cells; A.C. conceived the idea, designed the experiments, and wrote the manuscript.

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Correspondence to Abhishek Chatterjee.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–13 and Supplementary Tables 1–3. (PDF 2035 kb)

Supplementary Note 1

Synthesis of 5PrW and associated characterization data. (PDF 727 kb)

Supplementary Note 2

Sequences for plasmids and other gene cassettes. (PDF 1690 kb)

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Italia, J., Addy, P., Wrobel, C. et al. An orthogonalized platform for genetic code expansion in both bacteria and eukaryotes. Nat Chem Biol 13, 446–450 (2017). https://doi.org/10.1038/nchembio.2312

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