Genetic incorporation of unnatural amino acids into proteins in mammalian cells
Genetic incorporation of unnatural amino acids into proteins in mammalian cells
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DOI：
10.1038/nmeth1016
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发表时间：
2007-03-01
期刊：
影响因子：
48
通讯作者：
Schultz, Peter G.
中科院分区：
文献类型：
--
作者：
Liu, Wenshe;Brock, Ansgar;Schultz, Peter G.
我们开发了一种通用的方法，允许具有不同理化和生物学特性的非天然氨基酸在哺乳动物细胞中进行遗传编码。突变型大肠杆菌氨酰-tRNA合成酶（阿尔斯）首先在酵母中进化，以用感兴趣的非天然氨基酸选择性地氨酰化其tRNA。然后将该突变体阿尔斯与来自嗜热脂肪芽孢杆菌的琥珀抑制tRNA一起用于响应琥珀无义密码子将非天然氨基酸位点特异性地掺入哺乳动物细胞中的蛋白质中。我们独立地将六种非天然氨基酸掺入CHO细胞中表达的GFP中，效率高达1 μ g蛋白/2 × 10（7）细胞;质谱法证实了非天然氨基酸的高翻译保真度。这种方法应有助于将生物探针引入蛋白质中进行细胞研究，并最终有助于在哺乳动物细胞中合成含有非天然氨基酸的治疗性蛋白质。
We developed a general approach that allows unnatural amino acids with diverse physicochemical and biological properties to be genetically encoded in mammalian cells. A mutant Escherichia coli aminoacyl-tRNA synthetase (aaRS) is first evolved in yeast to selectively aminoacylate its tRNA with the unnatural amino acid of interest. This mutant aaRS together with an amber suppressor tRNA from Bacillus stearothermophilus is then used to site-specifically incorporate the unnatural amino acid into a protein in mammalian cells in response to an amber nonsense codon. We independently incorporated six unnatural amino acids into GFP expressed in CHO cells with efficiencies up to 1 mu g protein per 2 x 10(7) cells; mass spectrometry confirmed a high translational fidelity for the unnatural amino acid. This methodology should facilitate the introduction of biological probes into proteins for cellular studies and may ultimately facilitate the synthesis of therapeutic proteins containing unnatural amino acids in mammalian cells.