Production of cyclic peptides and proteins in vivo
Production of cyclic peptides and proteins in vivo
复制标题
DOI：
10.1073/pnas.96.24.13638
复制
发表时间：
1999-11-23
影响因子：
11.1
通讯作者：
Benkovic, SJ
中科院分区：
文献类型：
--
作者：
Scott, CP;Abel-Santos, E;Benkovic, SJ
合成和天然产物的组合文库是研究生物靶标的分子信息的重要来源。通过将小分子固有的发现潜力与通过细胞内方法实现的大文库大小相结合，细胞内产生稳定小分子文库的方法将是对现有文库技术的有价值的补充。我们探索了使用分裂内含肽（内部蛋白质）进行肽骨架环化的细胞内催化，作为生成稳定对抗细胞分解代谢的蛋白质和小肽的方法。来自集胞藻属 (Synechocystis sp.) 的 DnaE 分裂内含肽。 PCC6803 用于环化大肠杆菌酶二氢叶酸还原酶，并在细菌中产生环状八氨基酸酪氨酸酶抑制剂pseudostellarin F。环状二氢叶酸还原酶表现出改善的体外热稳定性，并且通过抑制重组抗生素链霉菌酪氨酸酶催化的黑色素产生，体内的假星菌素 F 的产生是显而易见的。体内生成和筛选主链环状产物的能力是实现生成细胞内环肽和蛋白质库的目标的重要里程碑。
Combinatorial libraries of synthetic and natural products are an important source of molecular information for the interrogation of biological targets. Methods for the intracellular production of libraries of small, stable molecules would be a valuable addition to existing library technologies by combining the discovery potential inherent in small molecules with the large library sizes that can be realized by intracellular methods. We have explored the use of split inteins (internal proteins) for the intracellular catalysis of peptide backbone cyclization as a method for generating proteins and small peptides that are stabilized against cellular catabolism. The DnaE split intein from Synechocystis sp. PCC6803 was used to cyclize the Escherichia coli enzyme dihydrofolate reductase and to produce the cyclic, eight-amino acid tyrosinase inhibitor pseudostellarin F in bacteria. Cyclic dihydrofolate reductase displayed improved in vitro thermostability, and pseudostellarin F production was readily apparent in vivo through its inhibition of melanin production catalyzed by recombinant Streptomyces antibioticus tyrosinase. The ability to generate and screen for backbone cyclic products in vivo is an important milestone toward the goal of generating intracellular cyclic peptide and protein libraries.