Solid-phase extraction with the functionalization of calcium-sensing receptors onto magnetic microspheres as an affinity probe can capture ligands selectively from herbal extract.

Abstract

Magnetic solid phase extraction with the functionalization of protein onto micro- or nano-particles as a probe is favorable for the discovery of new drugs from complicated natural products. Herein, we aimed to develop a rapid method by immobilizing halogenated alkane dehalogenase (Halo)-tagged calcium-sensing receptor (CaSR) directly out of crude cell lysates onto the surface of magnetic microspheres (MM) with no need to purify protein. Thereby we achieved CaSR-functionalized MM for revealing adsorption characteristics of agonist neomycin and screening ligands from herbal medicine Radix Astragali (RA). About 43.87mg CaSR could be immobilized per 1g MM within 30min, and the acquired CaSR-functionalized MM showed good stability and activity for 4weeks. The maximum adsorption capacity of neomycin on CaSR-functionalized MM was determined as 4.70 × 10-4 ~ 3.96 × 10-4mol/g within 277 ~ 310K, and its adsorption isotherm characteristics described best by the Temkin model were further validated using isothermal titration calorimetry. It was inferred that CaSR's affinity for neomycin was driven by electrostatic forces in a spontaneous process when the system reached an equilibrium state. Moreover, the ligands from the RA extract were screened, three of which were assigned as astragaloside IV, ononin, and calycosin based on HPLC-MS. Our findings demonstrated that the functionalization of a receptor onto magnetic materials designed as an affinity probe has the capability to recognize its agonist and capture the ligands selectively from complex matrices like herbs.