Department of Zoology, Biochemistry and Molecular Biology Laboratory, Centre of Advanced Study, Banaras Hindu University, Varanasi 221005, India.
Mol Biol Rep. 2011 Oct;38(7):4657-61. doi: 10.1007/s11033-010-0600-6. Epub 2010 Dec 12.
Nuclear magnetic resonance (NMR) spectroscopy is a useful biophysical technique to study the ligand-protein interaction. In this report, we have used bacterially produced ERβ and its domains for studying the functional analysis of ligand-protein interaction. Briefly, ERβ and its transactivation domain (TAD) and ligand binding domain (LBD) were subcloned and overexpressed using a prokaryotic expression system. The recombinant proteins were purified using Ni(+2)-IDA affinity chromatography and analyzed by NMR. Purified ERβ and TAD show similar conformation in the absence or presence of 17β-estradiol. However, LBD shows altered conformation in the presence of 17β-estradiol. These findings suggest that ERβ produced in bacteria exhibits a conformation such that its LBD remains masked and consequently it binds less to 17β-estradiol. Such study may help to develop the therapeutic approaches for controlling the estradiol-mediated gene expression in hormone dependent diseases.
核磁共振（NMR）光谱学是一种有用的生物物理技术，可用于研究配体-蛋白相互作用。在本报告中，我们使用细菌产生的 ERβ 及其结构域来研究配体-蛋白相互作用的功能分析。简而言之，使用原核表达系统亚克隆和过表达 ERβ 及其转录激活结构域（TAD）和配体结合结构域（LBD）。使用 Ni(+2)-IDA 亲和层析法纯化重组蛋白，并通过 NMR 进行分析。在缺乏或存在 17β-雌二醇的情况下，纯化的 ERβ 和 TAD 显示出相似的构象。然而，LBD 在存在 17β-雌二醇的情况下显示出改变的构象。这些发现表明，在细菌中产生的 ERβ 表现出一种构象，使得其 LBD 保持掩蔽，因此与 17β-雌二醇的结合减少。这样的研究可能有助于开发控制激素依赖性疾病中雌二醇介导的基因表达的治疗方法。
