Genetic Incorporation of Unnatural Amino Acids into Proteins in Yeast
Genetic Incorporation of Unnatural Amino Acids into Proteins in Yeast
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DOI：
10.1007/978-1-61779-331-8_12
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发表时间：
2012-01-01
期刊：
影响因子：
--
通讯作者：
Wang, Lei
中科院分区：
文献类型：
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作者：
Wang, Qian;Wang, Lei
通过使用一对正交的tRNA/氨酰-tRNA合成酶，非天然氨基酸可在活细胞中被基因编码整合到蛋白质中。在此我们描述了一种在酿酒酵母中高效表达正交tRNA和合成酶的方法，该方法能使非天然氨基酸高效地基因编码整合到酵母中的目标蛋白质中。我们还描述了使用一种无义介导的mRNA降解缺陷型酵母菌株，当使用终止密码子编码非天然氨基酸时，这种菌株可进一步提高非天然氨基酸的整合效率。这些策略将通过利用非天然氨基酸所提供的新特性，促进对酵母中蛋白质及其相关生物学过程的研究。
Unnatural amino acids can be genetically incorporated into proteins in live cells by using an orthogonal tRNA/aminoacyl-tRNA syntherase pair. Here we describe a method to efficiently express the orthogonal tRNA and synthetase in Saccharomyces cerevisiae, which enables unnatural amino acids to be genetically incorporated into target proteins in yeast with high efficiency. We also describe the use of a yeast strain deficient in the nonsense-mediated mRNA decay, which further increases the unnatural amino acid incorporation efficiency when a stop codon is used to encode the unnatural amino acid. These strategies will facilitate the investigation of proteins and their related biological processes in yeast by exploiting the novel properties afforded by unnatural amino acids.