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Published July 2007 | Supplemental Material
Journal Article Open

Evolved orthogonal ribosomes enhance the efficiency of synthetic genetic code expansion

Abstract

In vivo incorporation of unnatural amino acids by amber codon suppression is limited by release factor-1–mediated peptide chain termination. Orthogonal ribosome-mRNA pairs function in parallel with, but independent of, natural ribosomes and mRNAs. Here we show that an evolved orthogonal ribosome (ribo-X) improves tRNA_(CUA)-dependent decoding of amber codons placed in orthogonal mRNA. By combining ribo-X, orthogonal mRNAs and orthogonal aminoacyl-tRNA synthetase/tRNA pairs in Escherichia coli, we increase the efficiency of site-specific unnatural amino acid incorporation from ∼ 20% to >60% on a single amber codon and from <1% to >20% on two amber codons. We hypothesize that these increases result from a decreased functional interaction of the orthogonal ribosome with release factor-1. This technology should minimize the functional and phenotypic effects of truncated proteins in experiments that use unnatural amino acid incorporation to probe protein function in vivo.

Additional Information

© 2007 Macmillan Publishers Limited. Received 20 February; accepted 23 May; published online 24 June 2007. J.W.C. is an EMBO Young Investigator. K.W. is grateful for a Medical Research Council-Laboratory of Molecular Biology (MRC-LMB) Cambridge Scholarship, an Honorary External Research Studentship from Trinity College, Cambridge, and an Overseas Research Studentship Award. H.N. is an MRC Career Development Fellow. We are grateful to O. Barrett and W. An for sharing unpublished materials and assisting in early stages of this project. We are grateful to M. Babu for extracting E. coli amber codon usage, P.G. Schultz (TSRI) for the pSUP Bpa vector. This work was funded by The Medical Research Council. The authors declare no competing financial interests.

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Created:
August 19, 2023
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October 18, 2023