Xu Yan, Li Pengju, Nie Jiaojiao, Zhao Qi, Guan Shanshan, Kuai Ziyu, Qiao Yongbo, Jiang Xiaoyu, Li Ying, Li Wei, Shi Yuhua, Kong Wei, Shan Yaming
National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University Changchun Jilin China
Faculty of Health Sciences, University of Macau Macau China.
RSC Adv. 2018 May 10;8(31):17218-17223. doi: 10.1039/c8ra02798f. eCollection 2018 May 9.
According to the binding site structure and the catalytic mechanism of the native glutathione peroxidase (GPX), three glutathione derivatives, GSH-S-DNP butyl ester (hapten Be), GSH-S-DNP hexyl ester (hapten He) and GSH-S-DNP hexamethylene ester (hapten Hme) were synthesized. By a four-round panning with a human synthetic phage library against three haptens, the enrichment of the phage particles with specific binding activity could be determined. Three phage particles were selected binding to each glutathione derivative, respectively. After a two-step chemical mutation to convert the serine residues of the phage particles into selenocysteine residues, GPX activity could be observed and determined upto 3000 U μmol in the selenium-containing phage abzyme which was isolated by affinity capture against the hapten Be. Also the phage abzymes elicited by different antigens displayed different catalytic activities. After a directed evolution by DNA shuffling to improve the affinity to the hapten Be, a secondary library with GPX activity was created in which the catalytic activity of the selenium-containing phage abzyme could be increased 17%. This study might be helpful for new haptens or antigens design to optimize the abzymes with high binding activities and might also provide a novel scheme for GPX mimic candidates for drug development.
根据天然谷胱甘肽过氧化物酶（GPX）的结合位点结构和催化机制，合成了三种谷胱甘肽衍生物，即谷胱甘肽-S-二硝基苯基丁酯（半抗原Be）、谷胱甘肽-S-二硝基苯基己酯（半抗原He）和谷胱甘肽-S-二硝基苯基己二醇酯（半抗原Hme）。通过用人源合成噬菌体文库针对三种半抗原进行四轮淘选，可以确定具有特异性结合活性的噬菌体颗粒的富集情况。分别选择了三种与每种谷胱甘肽衍生物结合的噬菌体颗粒。经过两步化学突变将噬菌体颗粒中的丝氨酸残基转化为硒代半胱氨酸残基后，在通过针对半抗原Be进行亲和捕获分离得到的含硒噬菌体抗体酶中可以观察到并测定出高达3000 U/μmol的GPX活性。而且由不同抗原引发的噬菌体抗体酶表现出不同的催化活性。通过DNA改组进行定向进化以提高对半抗原Be的亲和力后，创建了一个具有GPX活性的二级文库，其中含硒噬菌体抗体酶的催化活性提高了17%。该研究可能有助于设计新的半抗原或抗原以优化具有高结合活性的抗体酶，也可能为药物开发的GPX模拟候选物提供一种新方案。
