A yeast sensor of ligand binding
A yeast sensor of ligand binding
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DOI：
10.1038/nbt1101-1042
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发表时间：
2001-11-01
影响因子：
46.9
通讯作者：
Fields, S
中科院分区：
文献类型：
--
作者：
Tucker, CL;Fields, S
我们描述了一种生物传感器，它将小分子配体与蛋白质的结合报告为温度敏感型酵母生长的变化。酵母菌株缺乏二氢叶酸还原酶(DHFR)，并得到含有插入柔性环中的配体结合结构域的小鼠DHFR的补充。表达两个配体结合域融合的酵母菌株，FKBP12-DHFR和雌激素受体-α(ERα)-DHFR，在相应配体的存在下显示出更快的生长。我们使用这个传感器来识别ERα残基中对配体结合重要的突变，以及通常影响蛋白质活性或表达的突变。我们还针对化学阵列测试了传感器，以确定与FKBP12或ERα结合的配体。ERA传感器能够区分雌激素类似物，显示与其相对结合亲和力(RBAs)相关的类似物不同程度的生长。这种生长实验提供了一种简单、廉价的方法来选择新的配体和配体结合域。
We describe a biosensor that reports the binding of small-molecule ligands to proteins as changes in growth of temperature-sensitive yeast. The yeast strains lack dihydrofolate reductase (DHFR) and are complemented by mouse DHFR containing a ligand-binding domain inserted in a flexible loop. Yeast strains expressing two ligand-binding domain fusions, FKBP12-DHFR and estrogen receptor-alpha (ER alpha)-DHFR, show increased growth in the presence of their corresponding ligands. We used this sensor to identify mutations in residues of ER alpha important for ligand binding, as well as mutations generally affecting protein activity or expression. We also, tested the sensor against a chemical array to identify ligands that bind to FKBP12 or ER alpha. The ERa sensor was able to discriminate among estrogen analogs, showing different degrees of growth for the analogs that correlated with their relative binding affinities (RBAs). This growth assay provides a simple and inexpensive method to select novel ligands and ligand-binding domains.