人降钙素受体胞外域的复性及新的体外活性测定方法

人降钙素受体胞外域是降钙素受体的药物靶点区域.本研究截取降钙素受体的N端胞外域22~140氨基酸残基区域,依据大肠杆菌偏爱密码子优化并人工合成基因,克隆至pET22b(+)载体,构建的表达质粒转入大肠杆菌BL21(DE3)中表达.表达产物经复性、纯化后进行受体-配体结合实验.结果表明,目的蛋白质绝大部分以包涵体形式存在.本研究探索出了一套高效的复性方法,经SDS-PAGE鉴定,纯化蛋白质为单一条带,质谱测定蛋白质分子量与理论分子量一致.复性后的蛋白质采用新的体外活性测定方法证实,有较强的结合鲑鱼降钙素(salmon calcitonin,sCT)的能力.这为其进一步的结构与功能研究奠定了基础.

Method of in vitro Bioactivity Assay for Refolded Human Ectodomain of Calcitonin Receptor

The human ectodomain of calcitonin receptor (eCTR) is considered as a drug target region of calcitonin receptors. The DNA encoding the N-terminus truncated 22～140 amino acid residues of eCTR gene was synthesized, codon optimized for E.coli and inserted into a pET-22b (+) vector to transform BL21(DE3) for expression. The expressed proteins were refolded, purified, and tested for ligand binding ability. The results showed that eCTR was expressed mainly in inclusion bodies. Efficient refolding allowed the purification of protein as a single band in SDS PAGE, which confirmed by mass spectrometry. The purified protein exerted high binding affinity to salmon calcitonin by in vitro assay. This study might be helpful for the further investigation structure -function research of calcitonin receptor.