Evolved orthogonal ribosomes enhance the efficiency of synthetic genetic code expansion
Evolved orthogonal ribosomes enhance the efficiency of synthetic genetic code expansion
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DOI：
10.1038/nbt1314
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发表时间：
2007-07-01
影响因子：
46.9
通讯作者：
Chin, Jason W.
中科院分区：
文献类型：
--
作者：
Wang, Kaihang;Neumann, Heinz;Chin, Jason W.
通过琥珀密码子抑制体内掺入非天然氨基酸受到释放因子-1介导的肽链终止的限制。正交核糖体- mRNA对与天然核糖体和mRNA平行但独立地起作用。在这里，我们表明，一个进化的正交核糖体（ribo-X）提高tRNA（CUA）依赖的琥珀密码子放置在正交mRNA的解码。通过在大肠杆菌中组合ribo-X、正交mRNA和正交氨酰-tRNA合成酶/tRNA对，我们将位点特异性非天然氨基酸掺入的效率从单个琥珀密码子上的20%提高到> 60%，并且从两< 1% to >个琥珀密码子上的20%提高。我们推测这些增加是由于正交核糖体与释放因子-1的功能性相互作用降低所致。该技术应在使用非天然氨基酸掺入来探测体内蛋白质功能的实验中最小化截短蛋白质的功能和表型效应。
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.