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Evolved orthogonal ribosomes enhance the efficiency of synthetic genetic code expansion

Wang, Kaihang and Neumann, Heinz and Peak-Chew, Sew Y. and Chin, Jason W. (2007) Evolved orthogonal ribosomes enhance the efficiency of synthetic genetic code expansion. Nature Biotechnology, 25 (7). pp. 770-777. ISSN 1087-0156. doi:10.1038/nbt1314. https://resolver.caltech.edu/CaltechAUTHORS:20180627-090110712

[img] PDF (Supplementary Fig. 1 - The anticodon stem loops of the tRNAs used) - Supplemental Material
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[img] PDF (Supplementary Fig. 2 - The context of UAGA and UAG selector codons in cat reporter genes) - Supplemental Material
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[img] PDF (Supplementary Fig. 3 - The linker region of the gst-malE expression construct) - Supplemental Material
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[img] PDF (Supplementary Fig. 4 - 35S misincorporation in GST-MBP) - Supplemental Material
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[img] PDF (Supplementary Fig. 5 - The ribosome dependence of O-DLR derived Renilla luciferase (O-R-luc) activity) - Supplemental Material
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[img] PDF (Supplementary Fig. 6 - The Ribo-X mediated enhanced unnatural amino acid incorporation efficiency is robust in minimal medium) - Supplemental Material
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[img] PDF (Supplementary Table 1 - 528/36 16S rDNA Libraries Construction) - Supplemental Material
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[img] PDF (Supplementary Table 2 - Primers Used) - Supplemental Material
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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.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1038/nbt1314DOIArticle
https://rdcu.be/11pnPublisherFree ReadCube access
ORCID:
AuthorORCID
Wang, Kaihang0000-0001-7657-8755
Chin, Jason W.0000-0003-1219-4757
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.
Funders:
Funding AgencyGrant Number
European Molecular Biology Organization (EMBO)UNSPECIFIED
Medical Research Council (UK)UNSPECIFIED
Trinity CollegeUNSPECIFIED
Overseas Research Studentship AwardUNSPECIFIED
Issue or Number:7
DOI:10.1038/nbt1314
Record Number:CaltechAUTHORS:20180627-090110712
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20180627-090110712
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:87376
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:27 Jun 2018 16:15
Last Modified:15 Nov 2021 20:47

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