Evolved orthogonal ribosomes enhance the efficiency of synthetic genetic code expansion.
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 approximately 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.
Wang
Kaihang
K
Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, England, UK.
Neumann
Heinz
H
Peak-Chew
Sew Y
SY
Chin
Jason W
JW
D001483:Base Sequence; D001709:Biotechnology / Q000379:methods*; D003062:Codon; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D013058:Mass Spectrometry; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012333:RNA, Messenger / Q000378:metabolism; D012343:RNA, Transfer / Q000737:chemistry; D012270:Ribosomes / Q000737:chemistry* / Q000378:metabolism
D003062:Codon; D012333:RNA, Messenger; D012343:RNA, Transfer; D005982:Glutathione Transferase; D003545:Cysteine