Synthesis and purification of soluble ligand binding domain of the human vitamin D3 receptor.
Synthesis and purification of soluble ligand binding domain of the human vitamin D3 receptor.
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人维生素 D3 受体可溶性配体结合域的合成和纯化。
DOI：
10.1006/bbrc.1996.0160
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Kumar,R
中科院分区：
文献类型：
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作者：
Craig,TA;Kumar,R
我们利用谷胱甘肽-S-转移酶融合蛋白表达系统表达和纯化了毫克量的人1，25-二羟基维生素D3受体的配体结合域。表达了105-427个氨基酸。Colias是一种GST融合蛋白，在降低(20°C)的温度下，通过谷胱甘肽琼脂糖凝胶纯化。将融合蛋白吸附在谷胱甘肽琼脂糖凝胶上，与凝血酶切割，得到可溶性的105-427人1，25-二羟基维生素D3受体。用Mono Q离子交换层析进一步纯化了105-427人1，25-二羟基维生素D3受体，经SDS-聚丙烯酰胺凝胶电泳法鉴定为单一条带。人1，25-二羟基维生素D3受体与1，25-二羟基维生素D3高亲和力结合(Kd值约为10−~9M)，结合容量为47pmoles/nmole蛋白。105-427人1，25-二羟基维生素D3受体的大规模表达将提供适合于结构研究的人1，25-二羟基维生素D3受体配体结合域。
We expressed and purified milligram quantities of the ligand binding domain of the human 1,25-dihydroxyvitamin D3receptor using a glutathione-S-transferase (GST) fusion protein expression system. Amino acids 105–427 were expressed inE. colias a GST fusion protein at a reduced (20°C) temperature and purified on glutathione sepharose. The fusion protein adsorbed to glutathione sepharose was cleaved with thrombin to yield soluble 105–427 human 1,25-dihydroxyvitamin D3receptor. The 105–427 human 1,25-dihydroxyvitamin D3receptor was further purified by Mono Q ion exchange chromatography and was characterized as a single band on SDS–polyacrylamide gel electrophoresis. The 105–427 human 1,25-dihydroxyvitamin D3receptor bound 1,25-dihydroxyvitamin D3with high affinity (Kdapproximately 10−9M) and with a binding capacity of 47 pmoles/nmole protein. Large scale expression of 105–427 human 1,25-dihydroxyvitamin D3receptor will provide human 1,25-dihydroxyvitamin D3receptor ligand binding domain suitable for structural studies.