Expression Optimization And In-situ Characterization Of Seven-transmembrane Receptor

Membrane proteins make up about 20-30% of total protein in the organisms,receptor membrane proteins have always been a hot topic in the field of life sciences and drug discovery.Since phospholipid bilayer perform many important functions on membrane protein signal transduction,protein interaction and dynamic conformation,mimetics of native-like environment have been developed for primary structural methods after detergent-based purification of the protein.However it is still preferable to study membrane proteins in their natural membranes since membrane proteins perform their functions in the environment of a cell membrane.Seven-transmembrane receptor protein is the largest family of transmembrane proteins,and is divided into microbial seven-transmembrane photoreceptor proteins and animal seven-transmembrane receptor proteins on the basis of their source.In this thesis,we want to amend the current ambimbiguities of the applications of the seven-transmembrane receptor protein for in-situ characterization.Archaerhodopsin-4(aR4),a dual chromophore microbial seven-transmembrane receptor found in the claret membrane of Halobacterium species(H.sp.)XZ515,was used to demonstrate importance of in-situ expression for microbial seven-transmembrane receptor.The recombinant aR4 with only the Ret chromophore and endogenous lipids was expressed in Halobacterium salinarum L33,and the recombinant aR4 containing both the Ret and carotenoid chromophores together with the endogenous lipids was expressed in Halobacterium salinarum MPK409.Therefore,functions of bacterioruberin in aR4 can be investigated in a real in situ manner.Our results of stability measurement experiments,dynamic light scattering,FTIR,ATP generation rate determination,proton pump measurements,titration experiments,and photoinduced transient absorption spectroscopy showed that bacterioruberin not only plays an important role in maintaining the protein trimeric structure,but also mediates the conformation of retinal through the long-range correlation between them.For animal seven-transmembrane receptor protein,chemokine receptor CX3CR1 was used to construct a platform for optimizing the expression of GPCRs in mammalian cells.TRE-X 293 cell line and pcDNA4/TO tetracycline regulatory were used for expression,Rho33-rhodopsin N-terminal was constructed for fusion expression with CX3CR1,and eGFP combining at the C-terminal of CX3CR1 was used for fluorescence detection.Western Blot and fluorescence results showed that our expression and detection platform for CX3CR1 is successful.?3-AR was further expressed and detected with the same method,and confocal fluorescence microscope was used to detect the location of W64R/WT-?3-AR in cells.Our results showed that W64 R mutant was be expressed normally and localized on the plasma membrane,but its efficiency of the membrane insertion was lower than that of the wild type.Although crystal structure analysis of the membrane protein has developed rapidly in recent years,characterizing structure of the membrane protein in its native environment is a central goal of structural biologists due to the diversity,hydrophobicity,fluidity and instability of cell membrane.This work is not only important for understanding the importance of the in-situ expression and functional characterization of the seven-transmembrane receptor protein,but is also of great significance to understand the relationship between the structure and function of seven-transmembrane proteins.In addition,GPCRs are closely related to many diseases in humans,this work also provides some experimental date on the in-situ structure analysis of GPCRs and the structure-based drug design.