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.
Keywords: Calcium-Sensing Receptor (CaSR); Halogenated alkane dehalogenase (Halo); extracellular domain (ECD); ‘Shuangdan’.
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