Expression And Purification Of G Protein-Coupled Receptors As Drug Targets

G protein-coupled receptor(GPCR)is an important membrane protein on the cell surface in human body.It participates in many physiological and pathological functions and is the largest class of drug targets.Determination of the three-dimensional structure of the GPCR-drug complex will contribute to the drug discovery targeting GPCR.However,as a kind of membrane protein,GPCR has low expression,poor stability and is difficult to analyze the structure.Therefore,extensive expression and purification of GPCR are needed to obtain the GPCR proteins with high expression leveles,high purity,good homogeneity and good thermal stability,so as to lay an important foundation for the structural determination of GPCR protein.Histamine is an important active substance in human body.It mediates different physiological and pathological reactions through four histamine GPCR subtypes(H1R,H2 R,H3R and H4R).Among them,H2 R is mainly expressed in the stomach,which is related to gastric acid secretion.Bradykinin is an important part of kallikrein-kinin system,which contains two bradykinin GPCR subtypes B1 R and B2 R.B1R is abundantly expressed in tumor tissues and cells,and B1 R antagonists have the potentials to treat tumors.However,the protein structures of histamine receptors H2 R and H4 R as well as bradykinin receptor B1 R have not been resolved,which limits the discovery of drugs targeting these GPCRs.The purpose of this research is to express and purify the proteins of histamine receptors H2 R and bradykinin receptor B1 R for protein three-dimensional structure determination,so as to clarify the protein structure of these important GPCR drug targets,reveal the molecular mechanism of drugs acting on these GPCRs,and provide important scientific basis for drug discovery targeting these GPCRs.Through a series of GPCR protein expression and purification,including N/C terminal truncations screening,fusion protein types and fusion sites screening,site directed mutations screening,protein expression and purification conditions screening,we successfully obtained the proteins of histamine receptor H2 R and bradykinin receptor B1 R with high expression,high purity,good homogeneity and good thermal stability,which lays an important foundation for the structural analysis of GPCR protein.