Production And Biological Activity Characterization Of Human Chemokine Receptor CCR3

G protein-coupled receptor(GPCR) comprises the largest family of integral membrane protein in human body. They function as signal messengers to sense extracellular signals and amplify them inside the cells. They play crucial roles in various physiological processes, so that they are important drug targets for diseases. In order to develop drugs, we must know the GPCR structure, biophysical and biochemical function. Because less physiological expression makes it difficult to carry on the system study, it is very important to produce much GPCR with biological activity by heterologous expression. GPCR has seven hydrophobic transmembrane α-helical structures, which leads to not easily purified as soluble protein. Therefore, it is crucial to choose proper surfactant to effectively solubilize GPCR. In addition, how to maintain GPCR biological activity is also a problem that must be solved. The human chemokine receptor CCR3 is a member of GPCR family. It plays a key role in allergic diseases, and is also co-receptor of some isolates of HIV-1, so it is important drug target and is hot spot of research. CCR3 realizes its function by binding with its chemokine ligands, such as CCL11, CCL24. In our research, CCR3 was produced by heterologous expression, which provided the material foundation for the in-depth study of CCR3 structure, biophysical and biochemistry function. CCR3 binding ligand biological activity was also studied, and the effect of surfactant on the biological activity of CCR3 was further understood.CCR3 was produced in tetracycline inducible mammalian cells. After the screening of high expression cell line, the concentration of tetracycline and sodium butyrate was optimized. After the screening more than ten common surfactants solubilization of CCR3, Fos choline-14(FC-14) was finally selected for the production of CCR3. After protein purification, milligram quantity of CCR3 was produced with high purity. To our knowledge, the human chemokine receptor CCR3 was successfully produced for the first time. The circular dichroism(CD) spectrum showed that the produced CCR3 had the typical characteristics of α-helix, indicating the proper folding of CCR3. The distribution of CCR3 fused to green fluorescent protein was observed by laser scanning confocal microscope. CCR3 was mainly found in the cytoplasm, but not on the cell membrane.CCR3 was produced in Escherichia coli(E.coli). In order to avoid the misfolding of CCR3 and toxicity in E.coli, CCR3 fused to maltose binding protein(MBP) was expressed. After the screening of host bacteria, E.coli TB1 was finally selected. The culture medium, induction phase and induction concentration were optimized. FC-14 was found to be an optimum surfactant for solubilization and stabilization of MBP-CCR3. MBP-CCR3 could be purified at milligram yield with two-step chromatography. It had a typical ?-helix structure in the circular dichroism spectrum.CCR3 is activated by binding its ligands, and then the activated CCR3 transmits signals into cell. In the signal transduction, chemokine binding with CCR3 is an important step. The surface plasmon resonance(SPR) is a typical method to study protein biological activity. CCR3 produced in lab could bind with bioactive CCL11 and CCL24 by SPR, which indicated that CCR3 had biological activity. The bioactive CCR3 could also bind CCL11 and CCL24 produced in lab, which indicated that the chemokines made in lab also had biological activity. The dissociation equilibrium constant(KD) between CCR3 and CCL11 or CCL24 was 7.0?10-8 M and 6.2?10-8 M, when N-dodecyl-b-D-maltoside(DDM) was used in production and SPR experiments; the KD values were 3.7×10-7 M and 3.0×10-7 M, when Triton X-100(Trx-100) was used in production and SPR experiments; while the KD values were 1.3?10-6 M and 1.6?10-6 M, when FC-14 was used in production and SPR experiments. The experiment rasults indicated that the surfactant could significantly influence the biological activity of CCR3 binding with its ligand. It was noticed that the change of surfactant from DDM to FC-14 resulted in a variation in binding affinity by more than one order of magnitude, indicating a strong environmental dependence of CCR3 activity. When FC-14 was changed into DDM, the biological activity of CCR3 binding with its ligand was increased, which further indicated the great influence of surfactant on the biological activity of CCR3. In addition, CCR3 fused to MBP also had biological activity. The KD values were 2.9×10-6 M and 1.8×10-6 M when DDM was used in SPR experiments.The liposome was prepared. MBP-CCR3 was recombined into liposome, and then MBP-CCR3 proteoliposome was produced. The exprements of liposomes and FC-14 interaction indicated that the Rsat and Rsol values were 1 mM and 6 mM, respectively. When the molar rate of POPC and MBP-CCR3 was 2000:1 under 3 mM FC-14, MBP-CCR3 proteoliposome could be made. MBP-CCR3 proteoliposome provided the experimental foundation for the study of CCR3 in liposome on the activity and structure.