IN VIVO INCORPORATION OF UNNATURAL AMINO ACIDS

描述：为特定地点的 将非天然氨基酸在体内掺入蛋白质中，直接从 生长培养基，将大大提高非天然氨基酸的力量 诱变例如，合成大量 含有重原子的蛋白质会促进蛋白质结构 测定，以及位点选择性取代荧光团或 在活细胞中将可光裂解的基团转化为蛋白质， 研究体内蛋白质功能的工具。或者，我们可以 通过为蛋白质提供新的 官能团，例如含酮基的氨基酸。为此中央 Schultz集团已开发出一种通用的多阶段方法， 将非天然氨基酸掺入体内蛋白质中。总体战略 由四个要素组成：（1）非天然氨基的设计和合成 酸性底物;（2）正交琥珀抑制tRNA的构建 （O-tRNACUA），其不是任何天然存在的 氨酰tRNA合成酶，并最终将用于提供 在一些实施方案中，所述非天然氨基酸是响应于编码所述非天然氨基酸的mRNA中的UAG密码子的非天然氨基酸。 目的蛋白质;（3）从目的蛋白质中选择氨酰tRNA合酶; 识别O-tRNACUA但不识别任何 内源性tRNA;和（4）筛选该tRNA的突变体文库 氨酰合成酶的能力，使O-tRNACUA与氨酰 非天然氨基酸，但不是普通氨基酸。

DESCRIPTION: The development of a general approach for the site-specific incorporation of unnatural amino acids into proteins in vivo, directly from the growth media, would greatly enhance the power of unnatural amino acid mutagenesis. For example, the ability to synthesize large quantities of proteins containing heavy atoms would facilitate protein structure determination, and the ability to site-selectively substitute fluorophores or photocleavable groups into proteins in living cells would provide powerful tools for studying protein function in vivo. Alternatively, one might be able to enhance the properties of proteins by providing building blocks with new functional groups, such as a keto-containing amino acid. To this end, the Schultz group has developed a general multistage approach for site specifically incorporating unnatural amino acids into proteins in vivo. The overall strategy consists of four elements: (1) The design and synthesis of the unnatural amino acid substrate; (2) The construction of an orthogonal amber suppressor tRNA (O-tRNACUA) that is not the substrate for any of the naturally occurring aminoacyl tRNA synthetases and which will eventually be used to deliver the unnatural amino acid in response to a UAG codon in the mRNA encoding the protein of interest; (3) the selection of aminoacyl tRNA synthase from a library of mutants that recognizes the O-tRNACUA but does not recognize any endogenous tRNAs; and (4) the screening of a library of mutants of this tRNA aminoacyl synthetase for the ability to acylate the O-tRNACUA with the unnatural amino acid but not a common amino acid.