Purified human vitamin D receptor overexpressed in E. coli and baculovirus systems does not bind 1,25-dihydroxyvitamin D3 hormone efficiently unless supplemented with a rat liver nuclear extract.
Purified human vitamin D receptor overexpressed in E. coli and baculovirus systems does not bind 1,25-dihydroxyvitamin D3 hormone efficiently unless supplemented with a rat liver nuclear extract.
复制标题
在大肠杆菌和杆状病毒系统中过表达的纯化人维生素 D 受体不能有效结合 1,25-二羟基维生素 D3 激素，除非补充大鼠肝核提取物。
DOI：
10.1006/bbrc.1993.2504
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发表时间：
1993
影响因子：
3.1
通讯作者：
Haussler,MR
中科院分区：
文献类型：
--
作者：
Nakajima,S;Hsieh,JC;MacDonald,PN;Haussler,CA;Galligan,MA;Jurutka,PW;Haussler,MR
我们在此报道了从me中提取的高纯度人维生素D受体（hVDR）。当单独分析这些制剂时，彩色杆状病毒表达系统不表现出可饱和的、高亲和力的1,25-二羟基维生素D3（1,25(OH)2D3）配体结合。大鼠肝核提取物本身不具有可检测到的1,25(OH) 2d3结合活性，因此需要将其纳入细菌或昆虫细胞分离的hVDR中，使其具有高亲和力的激素结合特性（Kd0.13-0.22 nM）。这一观察结果将有助于在过表达hVDR样品中有效测定125 (OH) 2d3结合活性和动力学。此外，由于大鼠肝核提取物含有类视黄醇X受体和其他可能与hVDR形成异源二聚体的辅助因子，进而与维生素D响应元件相关联，我们假设，与DNA结合一样，1,25(OH) 2d3与hVDR的结合需要一个共受体或一些未表征的受体激活/稳定因子的合作。
We report here that highly purified human vitamin D receptor (hVDR) derived fromE. colior baculovirus expression systems does not exhibit saturable, high affinity 1,25-dihydroxyvitamin D3(1,25(OH)2D3) ligand binding when these preparations alone are analyzed. Inclusion of rat liver nuclear extract, which does not itself contain detectable 1,25(OH)2D3binding activity, is required to endow hVDR isolated from bacterial or insect cells with the property of high affinity hormone binding (Kd0.13-0.22 nM). This observation should facilitate the valid assay of 1,25(OH)2D3binding activity and kinetics in samples of overexpressed hVDR. Moreover, since rat liver nuclear extract contains retinoid X receptors and possibly other auxiliary factors capable of forming heterodimers with hVDR that in turn associate with vitamin D responsive elements, we hypothesize that like DNA binding, 1,25(OH)2D3binding to hVDR requires the cooperation of a co-receptor or some uncharacterized receptor activating/stabilizing factor.