CUL5 E3 Ligase Establish In Vitro Ubiquitination System

Human cells exist a variety of protein degradation pathway, by26S will beubiquitin protein markers protein substrate degradation in the way of cellularhomeostasis and with many such as virus infection, tumor, cancer and other disease hasimportant connection.Including HIV-1virus infection is hot research topic for manyyears, however, the treatment is still used today all kinds of defects. Vif is based inimportant cofactor, HIV can APOBEC3G raised CUL5E3ligase complex, make A3G isubiquitin, and through the ubiquitin-proteasome pathway to its degradation.Currentlyin view of the ubiquitin proteasome system of new cures for diseases quickly becamethe research hot spot.So the purpose of this study is designed to establish CUL5E3ligase ubiquitin vitro system, for the study of the ubiquitin system functions and theinteractions among various components, further explore the mechanism of action ofHIV/AIDS treatment, development of targeted drugs have great practicalsignificance.And, more importantly, a successful build CUL5E3ligase ubiquitin vitrosystem class as a tool, but there is an interaction by changing the substrate protein orprotein components, study other protein ubiquitin between mechanism.Theestablishment of this system may also be a variety of disease there is no relativeeffective treatments involving protein interaction mechanism, provides an effectiveapproach.This article mainly on two aspects of research:(1) the expression and purificationCUL5ubiquitin E3ligase vitro all related proteins in the system.Protein within thesystem of the construction of expression vector, gene transformation after get positivemonoclonal, for the expression of recombinant proteins, and purification bycorresponding affinity chromatography.Further through the FPLC separation proteinwith high purity.Experiments and optimize the protein purification methods within thesystem, and make the original of protein expression, to express was successfullypurified through together.(2) to detect CUL5ubiquitin E3ligase in vitro activity of thesystem.By Western blotting to establish the detection of protein ubiquitin system activity.Research system within the protein ubiquitin situation, through the controlvariable method further verification to explore2Vif is based and A3G of ubiquitinsystem.Inside each protein proportion, and through regulating system for subsequentoptimization of the system into a more mature practical stronger in vitro ubiquitinresearch tools to lay the foundation.