IN VIVO INCORPORATION OF UNNATURAL AMINO ACIDS

描述：制定针对特定站点的通用方法 在体内将非天然氨基酸掺入蛋白质中，直接从 生长介质，将大大提高非天然氨基酸的能力 诱变。例如，能够合成大量的 含有重原子的蛋白质有助于蛋白质结构 测定，以及有选择性地取代荧光团或 在活细胞中可光裂解成蛋白质的基团将提供强大的 研究体内蛋白质功能的工具。或者，一个人也许能够 通过提供新的构建块来增强蛋白质的性质 官能团，如含酮的氨基酸。为此， Schultz小组针对特定的站点开发了一个通用的多阶段方法 在体内将非天然氨基酸加入蛋白质中。总体战略 由四个元素组成：(1)非天然氨基的设计和合成 酸性底物；(2)正交琥珀抑制因子tRNA的构建 (O-tRNACUA)不是任何自然发生的 氨酰tRNA合成酶，最终将被用来传递 非天然氨基酸对编码基因中的UAG密码子的反应 目的蛋白的筛选；(3)从 识别O-tRNACUA但不识别任何 内源tRNA；以及(4)该tRNA突变体文库的筛选 氨酰合成酶与O-tRNACUA的酰化能力 非天然氨基酸，但不是常见的氨基酸。

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.