Human calcium-sensing receptor (CaSR), a member of the G-protein-coupled receptor superfamily (GPCR), has been a therapeutic target for developing new drugs against calciotropic disorders and non-calciotropic diseases. The highly efficient methodologies for pursuing novel ligands/drugs remained a challenge due to the redundant purification processes of membrane protein in some widely-used methods including NMR, X-ray crystallography, Fluorescence Titration Spectroscopy, and Circular Dichroism. Herein, extracellular domain (ECD) of CaSR as its functional fragment was used to develop a rapid chromatographic method, which involved the synthesis of stationary phase material based on the site-specific covalent reaction of Halogenated alkane dehalogenase (Halo)-tagged ECD of CaSR in cell lysate with 6-chlorocaproic acid modified silica beads, the use of the immobilized CaSR column for revealing the interaction of three known agonists with CaSR and further screening ligands from complex matrix like Chinese herb medicine ‘Shuangdan’. The immobilized CaSR column was prepared rapidly without the protein purification and retained a good stability and specificity for at least 35 days. It was revealed that one type of binding sites occurred on CaSR with the binding affinity of neomycin > gentamicin-C / kanamycin, presumably which related to the number of structural amino groups attached. This method allowed for recognizing specifically novel ligands from ’Shuangdan’, demonstrating one type of binding sites on CaSR with the binding affinity of gallic acid > caffeic acid > paeonol. These results indicated that, the immobilization of a representative extracellular domain of CaSR to silica beads as biomaterial is feasible to develop a new rapid method, which can be successfully applied in screening novel ligands efficiently from complex matrices.

人类钙敏感受体 (CaSR) 是 G 蛋白偶联受体超家族 (GPCR) 的成员，一直是开发抗骨性紊乱和非骨性疾病新药的治疗靶点。由于在核磁共振、X 射线晶体学、荧光滴定光谱和圆二色谱等一些广泛使用的方法中膜蛋白的重复纯化过程，追求新型配体/药物的高效方法仍然是一个挑战。在此，以 CaSR 的细胞外结构域 (ECD) 作为其功能片段，开发了一种快速色谱方法，该方法涉及基于 CaSR 的卤代烷脱卤酶 (Halo) 标记的 ECD 的位点特异性共价反应合成固定相材料在含有 6-氯己酸修饰的二氧化硅珠的细胞裂解物中，使用固定化 CaSR 柱揭示三种已知激动剂与 CaSR 的相互作用，并进一步从复杂基质（如中草药“双丹”）中筛选配体。固定化 CaSR 柱无需蛋白质纯化即可快速制备，并保持良好的稳定性和特异性至少 35 天。结果表明，CaSR上出现了一类结合位点，其结合亲和力为新霉素>庆大霉素-C/卡那霉素，可能与连接的结构氨基数有关。这种方法可以识别来自“双丹”的特异性新配体，证明了 CaSR 上的一种结合位点具有没食子酸 > 咖啡酸 > 丹皮酚的结合亲和力。这些结果表明，