胸苷酸合酶的表达、纯化和抑制剂筛选体系的建立

doi:10.5246/jcps.2013.02.024

胸苷酸合酶的表达、纯化和抑制剂筛选体系的建立

李超, 郭莹, 田超, 周受辛, 闫汝峰, 张志丽, 王孝伟, 刘俊义^*

北京大学医学部药学院化学生物学系, 北京 100191

收稿日期:2012-12-11 修回日期:2013-02-03 出版日期:2013-03-18 发布日期:2013-03-18
通讯作者: 刘俊义*

Expression, purification and activity assay of recombinant human thymidylate synthase

Chao Li, Ying Guo, Chao Tian, Shouxin Zhou, Rufeng Yan, Zhili Zhang, Xiaowei Wang, Junyi Liu^*

Department of Chemical Biology, School of Pharmaceutical Science, Peking University Health Science Center, Beijing 100191, China

Received:2012-12-11 Revised:2013-02-03 Online:2013-03-18 Published:2013-03-18
Contact: Junyi Liu*

摘要/Abstract

摘要：

胸苷酸合酶(TS)催化人体内脱氧胸苷酸唯一的从头合成反应, 是抗叶酸代谢类药物的重要靶点。为方便地筛选化合物对TS的抑制活性, 本论文建立了重组人TS为基础的表达、纯化和活性筛选体系, 并以其为基础进行了新化合物的TS抑制活性筛选, 其中4个化合物显示出了较好的抑制活性, 阳性对照药物雷替曲塞的IC50值为3.4 μM。实验结果表明该方法具有方便、快速和稳定的特点。

关键词: 胸苷酸合成酶抑制剂, 重组蛋白质表达, 镍亲和色谱, 筛选模型

Abstract:

Thymidylate synthase (TS, E.C.2.1.1.45) catalyzes a critical reaction in the only pathway of de novo synthesis of thymidylate (dTMP) in human cells, and is an important target of chemotherapy. To evaluate the inhibitory activities of novel compounds to TS, a convenient method of activity assay using 6x His-tagged recombinant human TS (rhTS) was established and 49 novel synthetic folate analogues were screened to discover potential TS inhibitors. During the process, 4 novel compounds were found to effectively inhibit TS, while the IC50 of a positive control raltitrexed was 3.4 μM in this assay.

Key words: Thymidylate synthase inhibitors, Recombinant protein expression, Ni-resin column, Screening model

中图分类号:

R916.41

Supporting: Foundation items: National Natural Science Foundation of China (Grant No. 20972011, 21042009, 21172014).
^*Corresponding author. Tel.: 86-10-82801706

李超, 郭莹, 田超, 周受辛, 闫汝峰, 张志丽, 王孝伟, 刘俊义^*. 胸苷酸合酶的表达、纯化和抑制剂筛选体系的建立[J]. , DOI: 10.5246/jcps.2013.02.024.

Chao Li, Ying Guo, Chao Tian, Shouxin Zhou, Rufeng Yan, Zhili Zhang, Xiaowei Wang, Junyi Liu^*. Expression, purification and activity assay of recombinant human thymidylate synthase[J]. , DOI: 10.5246/jcps.2013.02.024.

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