Expression,Purification,Identification And Crystallization Of LMP1 Intracellular Soluble Proteins And Complex Proteins

Epstein-Barr virus(EBV)is a human ?-herpes virus which has an infection rate up to 90% in population.It stays latent in host cells life-long,until transforms into lytic,which may trigger genesis of several types of malignant tumors.Latent membrane protein 1,the important latent membrane proteins encoded by EBV,is anchored in the lipid raft region of the host cell membrane,interacting with signaling molecules,such as families of TRAF,Caspase and STAT.LMP1,involved in many signaling pathways,is considered closely related to lymphoproliferative diseases and tumorigenesis through cell migration and apoptosis.However,its role of neoplastic processes is ambiguity.Therefore,to research the structure and the molecular mechanism of the signal transduction pathwayit is of great significance.In this study,was used biological chemical methods to explore the effective plasmid transfection method of mammalian cells,and then the recombinant plasmid was transfected into HEK293 f cells to express LMP1 protein successfully with this method.While,homogeneous,stable and high concentration of LMP1-8(LMP1-Cterminal soluble protein)were expressed and purified in prokaryotic expression system.The stable complex protein of LMP1-8 and TRAF3-258(TRAF domain of TRAF3)was assembled in vitro and the complex protein crystals with a resolution of 3.2 ? were obtained by means of gradient screening of crystallization conditions.In addition,this study used analytical ultracentrifuge to detect the molecular weight and protein status of LMP1-8 and complex protein,and found that LMP1-8 and TRAF3-258 were all dimer proteins and the form of LMP1-8 interact with TRAF3-258 was 1:3.The results of this study provides a more solid theoretical basis for the analysis of the structure of LMP1 protein and its pathogenesis.