A GAL4-based yeast three-hybrid system for the identification of small molecule-target protein interactions
A GAL4-based yeast three-hybrid system for the identification of small molecule-target protein interactions
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DOI：
10.1016/s0006-2952(02)00884-5
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发表时间：
2002-05-01
影响因子：
5.8
通讯作者：
Meldrum, E
中科院分区：
文献类型：
--
作者：
Henthorn, DC;Jaxa-Chamiec, AA;Meldrum, E
我们报告的开发酵母菌株设计用于测定化合物-蛋白质的相互作用，通过激活报告基因的表达。已经证明，在FK 506和FK 506结合蛋白12（FKBP 12）之间以及甲氨蝶呤（MTX）和二氢叶酸还原酶（DHFR）之间的化合物-蛋白相互作用中，由GAL 4启动子驱动的lacZ表达激活。报告基因的表达完全废除在竞争性的方式存在过量的FK 506或MTX，分别。此外，表达突变DHFR克隆的菌株与MTX结合亲和力降低，不能激活报告基因表达。虽然菌株敏感性具有化合物依赖性，但对于FK 506-FKBP 12相互作用，驱动报告基因表达所需的最小化合物浓度为20 nM。该菌株作为鉴定未知化合物结合蛋白的工具的实用性已通过筛选小鼠cDNA文库中编码能够结合MTX的蛋白的克隆而得到证实。在筛选5 X IO 6个克隆后，鉴定了小鼠DHFR的四个文库克隆。筛选背景较低，假阳性很容易识别，使得该酵母系统特别适合用于筛选新型化合物-蛋白质相互作用。(C)2002年由Elsevier Science Inc.出版
We report the development of a yeast strain designed for assaying compound-protein interactions through activation of reporter gene expression. Activation of lacZ expression, driven by the GAL4 promoter, has been demonstrated for precedented compound-protein interactions between FK506 and FK506 binding protein 12 (FKBP12) and also between methotrexate (MTX) and dihydrofolate reductase (DHFR). Reporter gene expression was completely abrogated in a competitive manner by the presence of excess FK506 or MTX, respectively. In addition, a strain expressing a mutated DHFR clone with decreased binding affinity for MTX was not capable of activating reporter gene expression. While strain sensitivity is compound-dependent, the minimum compound concentration necessary to drive reporter gene expression was 20 nM for the FK506-FKBP12 interaction. The utility of this strain as a tool for identifying unknown compound-binding proteins has been demonstrated by screening a mouse cDNA library for clones that encode proteins capable of binding MTX. Four library clones of mouse DHFR were identified after screening 5 x 106 clones. The screen background was low and false positives were easily identified, making this yeast system particularly amenable for use in a screening context for novel compound-protein interactions. (C) 2002 Published by Elsevier Science Inc.