Recombinant Preparation And Functional Characterization Of Human Chemokines CCL5 And CCL8

Chemokines CCL5 and CCL8 are a kind of cytokines with cell chemotaxis.They and their receptors play an important role in the process of cellular immune inflammatory response and are important drug design templates or target.Despite its relatively small molecular weight and relatively simple structure,the study of chemokines and their receptors is limited by two factors:1)the content of natural chemokines is very low,and chemokines in Escherichia coli recombinant expression is easy to form inclusion bodies.Thus,the need for chemokines in E.coli to achieve mg-class activity-soluble expression,and applied to structural and functional studies and drug screening is also increasingly urgent.2)Due to the complexity of the cellular immune chemotaxis system,the same receptor?CCR3?can interact with multiple chemokines such as CCL5,CCL8,CCL11 and CCL24.Therefore,the detailed characterization of the interaction of different chemokines with their receptor and their functional analysis are important for more specific and efficient drug design.In view of the above problems,CCL8 and CCL5 were used as the research object to explore the excessive and soluble expression of chemokines in Escherichia coli.The soluble overexpression of CCL8 in Escherichia coli was achieved by optimizing the vector construction mode and induction culture conditions,and a simple and effective method for the purification of CCL8 was established.Using this method,CCL8 with a purity greater than 98%was obtained,the yield was about 1.5 mg/L.The circular dichroism spectroscopy analysis showed that the purified CCL8 was similar to other typical chemokines,indicating that it had been substantially folded correctly.The interactions of CCL8 with CCR3 by quartz crystal microbalance?QCM?show that CCL8 can be combined with CCR3 in vitro,and the k_D is1.22×10^-7 by fitting.Using the same method,we have studied the CCL5 recombinant preparation method.The results showed that CCL5 fusion protein could achieve excessive soluble expression in Escherichia coli,and the fusion protein with purity of more than 90%could be obtained,the yield was about 0.4 mg/L.The fusion proteins were characterized by SDS-PAGE,Western-Blot and circular dichroism spectroscopy.However,after digestion of the fusion protein,CCL5 showed a large amount of precipitate,and it was difficult to obtain a large amount of recombinant CCL5 protein after cleavage.On the basis of the above work,we discussed the molecular chaperones GroEL and GroES to reduce the expression of inclusion bodies in Escherichia coli by using CCL5 as the main research object.Using molecular chaperone GroEL,GroES and CCL5 plasmid co-transformation,we found that molecular chaperone GroEL and GroES can significantly increase the soluble expression of CCL5.The yield of CCL5 reached 0.47 mg/L,which was increased by 17.5%and the formation of inclusion bodies was significantly decreased by further optimizing the co-transformation conditions.It was proved that the chaperones GroEL and GroES had an auxiliary folding effect on CCL5 fusion protein,can effectively reduce the production of E.coli inclusion bodies.Similar to several other chemokines,we demonstrate that the chemokine CCL8 prepared in the laboratory can cause the internalization of CCR3-EGFP transiently expressed on the cell membrane,it also has cell chemotaxis activity,such as at chemokine concentration of 200 nM,the chemotaxis index was 4.76,but the chemotactic activity was concentration dependent.Chemotaxis experiments also showed that the four chemokines had significant chemotactic activity,but the chemotactic ability of different chemokines was significantly different,and CCL5 and CCL8 was slightly lower than CCL11 and CCL24.In addition,we also explored the phenomenon of calcium flow caused by chemokines.This study provides a certain method and experimental basis for clarifying the preference activation of different chemokines on the same receptor.