Research abstract
Article abstract
Nature Biotechnology 25, 770 - 777 (2007)
Published online: 24 June 2007 | doi:10.1038/nbt1314
Evolved orthogonal ribosomes enhance the efficiency of synthetic genetic code expansion
Kaihang Wang1,2, Heinz Neumann1,2, Sew Y Peak-Chew1 & Jason W Chin1
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 tRNACUA-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.
- Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, England, UK.
- These authors contributed equally to this work.
Correspondence to: Jason W Chin1 e-mail: chin@mrc-lmb.cam.ac.uk
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