INTRODUCTION OF NON-NATURAL AMINO ACIDS INTO PROTEINS

本提案的目的是制定一种通用方法， 将非天然氨基酸引入蛋白质。 位点特异性 诱变目前允许替换20种正常的 蛋白质的氨基酸成分与另一种。 尽管面临巨大 这种类型的操作在酶机制研究中的价值， 在其他领域，它受到严重限制，因为其他群体不能 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）应该允许 在一个指定的单一地点引入各种不同的群体， 蛋白质的位置。 例如，应该可以引入 特异性检测残基（例如，荧光标记氨基酸 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，对于特定的酶固定化， 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 肽递送至特定的细胞位点）。 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.