期刊
NATURE BIOTECHNOLOGY
卷 25, 期 7, 页码 770-777出版社
NATURE RESEARCH
DOI: 10.1038/nbt1314
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资金
- MRC [MC_U105181009] Funding Source: UKRI
- Medical Research Council [MC_U105181009] Funding Source: researchfish
- Medical Research Council [MC_U105181009] Funding Source: Medline
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 similar to 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.
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