INTRODUCTION OF NON-NATURAL AMINO ACIDS INTO PROTEINS

这项建议的目标是开发一种通用的方法 将非天然氨基酸引入蛋白质。站点特定 突变目前允许替换20个正常的 蛋白质的氨基酸组成与另一种。尽管有巨大的 这类操作在酶机制研究中的价值，以及 在其他地区，它受到严重限制，因为其他群体不能 通过这种方法引入的。合成琥珀抑制剂系统的使用 (UAG终止密码子和从头合成的酰化t-RNA)应该允许 介绍了各种不同的团体在单一指定 蛋白质中的位置。例如，应该可以引入 特定检测残留物(例如，荧光标记的氨基酸 酶代谢研究)、催化残留物(例如，金属络合 活性)、连接残基(例如，能够形成共价键的基团 激活后，对于特定的酶进行固定化，加强加热 稳定性，或翻译后修饰)，裂解残基(用于 酶原-酶转化)和受体/细胞特异性残基(用于 多肽递送到特定的细胞位置)。圆满完成 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。

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.