| Membrane proteins are abundant in organisms and participate in various physiological processes.If they are abnormal,they will affect life activities.Therefore,membrane proteins have become a hot research object in recent years.However,there are three problems in the study of membrane proteins : it is difficult to obtain proteins,the lipid bilayer environment in which proteins are located is complex,and proteins are insoluble in aqueous solution.Liposomes are vesicles composed of phospholipids,and in aqueous solution are spheres composed of lipid bilayers.Membrane protein and liposome are reconstituted to proteoliposome,which can more realistically simulate the lipid bilayer environment of membrane protein in cells,and then study the protein,so liposome is a good tool in membrane protein research.The energy coupling factor(ECF)transporter is a membrane protein that exists in prokaryotes and mainly transports trace elements required by organisms.It is mainly composed of two ATP-hydrolyzing,nucleotide-binding domains(ECF-A and ECF-A’)and a transmembrane scaffold protein(ECF-T)and a substrate binding protein S component.The nucleotide binding domain and the transmembrane scaffold protein together constitute the ECF module.In recent years,it has been studied mainly in detergents.The main reason is that the ECF transporter is too dynamic and protein liposome reconstitution is difficult.In this study,ECF transporter was selected as the experimental object to explore a set of protein liposome reconstitution methods that may be universal for membrane proteins,which provided a basis for the study of the structure and function of ECF transporter on lipid bilayer,and provided a reference for the preparation of liposomes of other membrane proteins.In this study,the genes of BtuM,LhaS and ECF-Pan T complex were synthesized,and their expression and purification were optimized,the following results were obtained:(1)The expression vectors of BtuM,LhaS and ECF-Pan T complex were successfully constructed.By optimizing strain conditions,protein tag and position,and expression mode,the expression conditions of ECF transporters were successfully obtained,which have higher expression levels.(2)Both BtuM and LhaS were able to obtain high purity proteins by selecting detergent and adding substrate,and the ECF-Pan T complex was able to obtain high purity and homogeneous protein by purification selection.(3)Proteoliposomes can be successfully reconstituted by screening the type and concentration of detergent,the type of phospholipids,and whether to add substrates when BtuM and LhaS are used to construct liposomes.The protein was in a normal state and had secondary structure as determined by SDS-PAGE and CD detection.In summary,this study explored a set of optimized experimental conditions from protein expression and purification to successful reconstitution of proteoliposomes by taking ECF transporter as the experimental object,and successfully reconstitution of proteoliposomes.This study provides a basis for further study of the structure and transport mechanism of ECF transporter,and also provides experimental reference for proteoliposomes reconstitution of other membrane proteins. |
