Expression,Purification And Functional Study Of Gonadotropin-releasing Hormone Receptor Protein

Gonadotropin-releasing hormone receptor(Gn RHR)is a member of the G protein-coupled receptor family and is characterized by a seven-pass transmembrane spiral structure,which is expressed on the surface of pituitary gonadotropin cells and breast,ovary and prostate.After the gonadotropin-releasing hormone(Gn RH)binds to it,the receptor binds to the second messenger G protein,and the activation of the receptor eventually leads to the release of gonadotropin-producing hormone and follicle-stimulating hormone.The study of the structure of this receptor can pave the way for breaking through the structural analysis bottleneck of this receptor,and understand its specific downstream mechanism of action.It can provide a certain the oretical basis for drug development with this receptor as the target.When it is a drug target,it provides new ideas and strategies for the treatment of prostate cancer,endometriosis,precocious puberty and other related diseases.This subject intends to conduct expression purification and functional research on gonadotropin-releasing hormone receptors.Cloning design and cloning optimization of the target gene of the receptor protein,selecting different frameworks to construct molecular clones,and performing expression purification experiments on the target protein to detect its protein expression.Then conduct a functional research experiment,perform molecular mutations on the gene plasmid,and want to construct a protein receptor plasmid that responds well to the ligand,and test its vitality through the reporter gene experiment.If the receptor protein is of good nature,high thermal stability,strong activity,single conformation,etc,then conduct protein expression purification experiments to check its expression.The subject designed several frameworks for the target gene and completed the construction of the p Fast Bac-HA-Bril-h Gn RHR-tev-OMBP-MBP-H8,pc DNA3-HA-Flag-H10-tev-Bril-h Gn RHR,p Fast Bac-HA-h Gn RHR-tev-OMBP-MBP plasmid.The downstream pathway of the recept or protein was verified as NFAT,and the protein receptor under the mutation of delta K191,(S186G,delta K191),(L112F/Q208E/L300V/D302 E,delta K191),(delta K191,F130W),(delta K191,F272Y),(delta K191,F130 W,F272Y)and P303 A were found to have enhanced responsiveness to ligand.The pure expression of the protein is not ideal,and the large amount of protein aggregation is not good.The yield is low,so it is necessary to further explore in the future to find more suitable mutations that are more suitable for the framework,so that its yield is higher and its properties are more stable.