人视黄醇结合蛋白在大肠杆菌中的高效表达及其活性测定

视黄醇结合蛋白 (retinol- binding protein,RBP)是体内结合、转运维生素 A的重要载体蛋白 ,对生长、繁殖、视觉及维持上皮细胞分化状态至关重要 ,不但是临床营养支持蛋白质营养评价的最灵敏指标 ,而且可作为慢性肾病、严重肝病、甲亢等相关疾病的辅助诊断指标 .同时 ,RBP可作为疏水小分子结合蛋白家族构效关系研究的模型 ,具有重要的理论与应用研究价值 .我们已克隆出了它的 c D-NA1 ] ,本文报道了人 RBP在大肠杆菌中的高效表达及其产物的初步纯化和活性测定 .1　材料与方法1 .1　材料含目的片段的克隆质粒 p GEM- RBP为本室构建 …

Expression of Human Retinol-Binding Protein and Its Characteristics

Retinol\|binding protein(RBP)is the transport protein of vitamin A and is one of the members in the family of proteins which may all bind small hydrophobic molecules.In order to study the structure\|function relationship of RBP,the plasmid pET3a\|RBP was constructed,and RBP was expressed in E.coli Bl21(DE3)successfully.The expressed protein,with molecular weight of about 21 kD,was about 36.95 percent of the total bacteria protein as detected by SDS\|PAGE and densitometry analysis.Then the production was confirmed by probing Western blot of E.coli lysate separated on SDS\|PAGE with antisera against hRBP.In succession,the fluorescence enhancement properties and shift in absorption spectrum had also confirmed the production's functional characteristics.It could bind ROH in ratio of 1∶1 and K d=1.5×10 -7 mol/L.