固定化分选酶介导的多肽环化  

Enzymatic cyclization of peptides using immobilized sortase A

作　　者：张书香 刘忞之 杨燕 程克棣 孔建强 王伟 

机构地区：[1]中国医学科学院、北京协和医学院药物研究所,天然药物活性物质与功能国家重点实验室,国家卫生和计划生育委员会天然药物生物合成重点实验室,北京100050

出　　处：《药学学报》2015年第5期627-632,共6页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金资助项目(30772677,81072562);国家“重大新药创制”科技重大专项资助项目(2012ZX09301002);中央高校基本科研业务费(2012N06)

摘　　要：多肽环化修饰是提高其稳定性和生物活性的重要手段之一,本研究建立了含有适于亲和色谱纯化的6个组氨酸和几丁质结合域两个融合标签的分选酶表达系统,纯化获得了目的蛋白;酶促动力学实验结果表明融合标签对其催化活性没有影响;以N-端含有3个甘氨酸和C-端含有分选酶识别序列的线性肽为底物研究了固定化酶催化线性肽的环化,该技术操作简单,易于酶促反应产物的分离纯化和固定化酶再利用,可直接用于环肽类药物的化学-酶法合成。Peptide cyclization, a pivotal approach to modifying linear precursors of proteins and peptides, has been used to enhance their biological activities and serum stabilities. Recently, sortase A （SrtA） from Staphyloccus aureus becomes a promising new technology for efficiently incorporating site specific modifications into proteins, conjugating the cell surface and cyclizing the linear peptides. In this study, we constructed two recombinant expression systems, one with chitin binding domain and the other with six-histidine tag and chitin binding domain on the N-terminal of SrtA, separately. The results of enzymatic kinetics indicate that the two recombinant tags do not impair the transpeptidase activity of SrtA compared with the standard reaction reported under the same reaction condition. The two synthesized peptides with N-ternimal three glycines and C-terminal penta-amino acid motif, LPETG, were cyclized using immobilized and recycled SrtA. The SrtA-based cyclization promises to represent a simple method for easy and efficient enzymatic synthesis of large cyclic peptides.

关 键 词：分选酶 固定化酶 环肽 环化 

分 类 号：R931[医药卫生—生药学]