Expression And Purification Of Human Chemokine Receptor CCR4 Protein

Chemokine receptors play essential roles in human development,homeostasis,immune surveillance,inflammation,and other physiological processes.The chemokine receptor CCR4 has attracted much attention as a critical drug target for treating allergic diseases and solid tumors,but the structure study of human CCR4 has not been reported.The ultimate purpose of this research is to analyze the inactive and active state structures of CCR4 protein.The analysis of the CCR4 system will clarify the molecular mechanism of the binding of small molecule drugs to CCR4 targets and deepen the understanding of the activation mechanism of chemokine receptors,providing a structural basis for the development of CCR4 target drugs.To analyze the structures of the CCR4 receptor in the inactive state and the activated state,the premise is to obtain high-purity and stable CCR4 receptor protein and CCR4-CCL22-Gi complex protein,respectively.In this study,human CCR4 protein was expressed in eukaryotic insect cells,and the protein was extracted,purified,and identified.The CCR4 protein was obtained through the optimization of clones,the screening of small molecule inhibitors,and the improvement of experimental conditions.The complex protein was obtained by screening the baculovirus ratio and optimizing the experimental conditions.In this project,CCR4 and its complex were successfully expressed in Sf9 cells,and purified,stable,and uniform CCR4 receptor protein and CCR4-CCL22-Gi complex protein were obtained after protein purification.These results lay a good foundation for subsequent structural studies.