Evolved orthogonal ribosomes enhance the efficiency of synthetic genetic code expansion

被引：240

作者：

Wang, Kaihang ^{[1
]}

Neumann, Heinz ^{[1
]}

Peak-Chew, Sew Y. ^{[1
]}

Chin, Jason W. ^{[1
]}

机构：

[1] MRC, Mol Biol Lab, Cambridge CB2 2QH, England

来源：

NATURE BIOTECHNOLOGY | 2007年 / 25卷 / 07期

基金：

英国医学研究理事会;

关键词：

D O I：

10.1038/nbt1314

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 ; 0836 ; 090102 ; 100705 ;

摘要：

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.

引用

收藏

页码：770 / 777

页数：8

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