INTRODUCTION OF NON-NATURAL AMINO ACIDS INTO PROTEINS

将非天然氨基酸引入蛋白质中

• 批准号: 3301523
• 负责人: A RICHARD CHAMBERLIN
• 金额: $ 11.53万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-04-01 至 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3301523
• 关键词: RNA biosynthesis; aminoacid analog; aminoacid tRNA ligase; aminoacyl tRNA; biomarker; chemical binding; chemical synthesis; genetic translation; nuclear runoff assay; point mutation; protein biosynthesis; protein engineering; protein sequence; ribosomes; site directed mutagenesis; suppressor mutations; synthetic nucleic acid; synthetic peptide; transfer RNA

The objective of this proposal is to develop a general method of introducing non-natural amino acids into proteins. Site specific mutagenesis currently allows replacement of any one of the twenty normal amino acids constituents of proteins with another. Despite the enormous value of this type of manipulation in the study of enzyme mechanisms, and other areas, it is severely limited because other groups cannot be introduced by this method. The use of a synthetic amber suppressor system (a UAG stop codon and de novo synthesized acylated t-RNAs) should allow the introduction of a variety of different groups at a single designated position in proteins. For example, it should be possible to introduce specific detection residues (e.g., fluorescence-labeled amino acids for enzyme metabolism studies), catalytic residues (e.g., metal chelating activity), linking residues (e.g., groups capable of forming covalent bonds after activation, for specific enzyme immobilization, enhanced thermal stability, or post-translational modification), cleaving residues (for proenzyme -> enzyme conversion), and receptor/cell-specific residues (for peptide delivery to specific cellular sites). The successful completion of this project would provide significant new tools for genetic engineering, enzymologists, biochemists, and bioorganic chemists.

本提案的目的是开发一种通用方法 将非天然氨基酸引入蛋白质中。 特定地点 诱变目前允许替换二十种正常物质中的任何一种 蛋白质的氨基酸成分与另一种不同。 尽管规模巨大 这种类型的操作在酶机制研究中的价值，以及 在其他领域，它受到严重限制，因为其他群体无法 通过这种方法引入。 使用合成琥珀抑制系统 （UAG 终止密码子和从头合成的酰化 t-RNA）应允许 在一个指定的地方引入各种不同的群体 在蛋白质中的位置。 例如，应该可以引入 特定的检测残基（例如，荧光标记的氨基酸 酶代谢研究）、催化残留物（例如金属螯合 活性），连接残基（例如，能够形成共价键的基团 活化后，用于特定酶固定化，增强热 稳定性或翻译后修饰），切割残基（用于 酶原 -> 酶转化）和受体/细胞特异性残基（用于 肽递送到特定的细胞位点）。 顺利完成 该项目将为基因工程提供重要的新工具， 酶学家、生物化学家和生物有机化学家。