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The new method to identify small molecules binding proteins by usingmutants and genome sequences

利用突变体和基因组序列识别小分子结合蛋白的新方法

基本信息

批准号：
19380066
负责人：
菅原二三男
金额：
$ 12.73万
依托单位：
Tokyo University of Science
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2007
资助国家：
日本
起止时间：
2007 至 2009
项目状态：
已结题

项目摘要

The present study aimed at the identification of the action new and in vivo target protein of the establishment of the new medicine receptivity evaluation method that used gene mutation Drosophila melanogaster and CPT that used it.CPT-20-B connecting peptide (NSASRGGSQRGRGEH) is identified by using the T7 phage display method with crystal departure pendulum microbalance (QCM) device. This peptide sequencing showed each array and the homology of human HshnRNP A1 and Drosophila melanogaster DmHrb87F. CPT united from the bead pull-down examination that used HeLa cell meltable picture with HshnRNP A1. CPT made HshnRNP A1-GST united changing protein, did the QCM interaction analysis, and was HshnRNP A1 and was dissociation constant 82.7 nM.As a result of the microbial sensitivity test, by gene loss Drosophila melanogaster, two Hrb87FKG02089 and Hrb87FBG02743 that was the DmHrb87F gene mutant showed CPT high receptivity compared with the wild strain. Topo I has working that cancels a super-coil of plasmid DNA. This topology change was observed under CPT and the HshnRNP A1 existence. CPT obstructed the topo I revitalization as reported. Moreover, hnRNP A1 also similarly obstructed the topo I revitalization. In addition, CPT has improved the topo I active inhibition to HshnRNP A1 under both existence.HshnRNP A1 or DmHrb87F united from the above-mentioned result with CPT, and the possibility of adjusting the topo I revitalization was suggested.

本研究旨在鉴定利用基因突变的黑腹果蝇和使用该方法的CPT建立新的药物感受性评价方法的新作用和体内靶蛋白。利用晶体离摆微天平(QCM)装置，利用T7噬菌体展示法对CPT-20-B连接肽(NSASRGGSQRGRGEH)进行鉴定。该肽测序显示了每个阵列以及人 HshnRNP A1 和果蝇 DmHrb87F 的同源性。 CPT 结合使用 HeLa 细胞可溶性图像的珠子下拉测试与 HshnRNP A1。 CPT制作HshnRNP A1-GST联合变化蛋白，进行QCM相互作用分析，为HshnRNP A1，解离常数为82.7nM。微生物敏感性试验的结果显示，由于基因缺失，果蝇中，DmHrb87F基因突变体的两个Hrb87FKG02089和Hrb87FBG02743 与野生株相比，喜树碱的感受性较高。 Topo I 正在研究消除质粒 DNA 的超级螺旋。在 CPT 和 HshnRNP A1 存在下观察到这种拓扑变化。据报道，CPT 阻碍了拓扑 I 的复兴。此外，hnRNP A1也同样阻碍了topo I的活化。另外，CPT在两者存在下均改善了对HshnRNP A1的topo I活性抑制。将HshnRNP A1或DmHrb87F与CPT联合，提示了调节topo I活化的可能性。