Purification And Structure Of The REG?-20s Proteasome And HCV Core Protein

Protein Degradation Pathway is very important for the maintenance of life activities.There are two main types of Protein Degradation Systems: Proteasome System and Lysosome System.Most proteins in cells are completed by the Proteasome Degradation System.In addition,most proteins are degraded through the Ubiquitin-proteasome Pathway,which is the 26 S proteasome degradation process.However,some proteins can also be degraded through other proteasome pathways,namely the 11 S proteasome activating factor REG?-mediated degradation process,which is independent of ubiquitination and ATP.As a member of the REG family,more and more research results show that REG? has abnormal expression in tumors which are closely related to tumor development.REG? can activate 20 S proteasome to degrade different target proteins and regulate the related signaling pathway.In the REG?-mediated protein degradation pathway,How does REG? control the gate of20 S core particles? How does REG? recognize its target protein? None of these have been reported.Purification of the REG?,20 S core particle and His-HCV Core protein(1-151)proteins in vitro and observation of them under a Cryo-Electron Microscope to construct a Three-Dimensional Model to further analyze the structure of the REG?-20 S proteasome complex and REG?-His HCV Core protein complex were investigated in this research with results as follows.1.REG? protein was purified by Profinity e Xact Affinity Chromatography.Induction conditions: BL21 competent cells,OD600 is 0.6,0.2 m M IPTG,induced at16 ? for 12 h;2.The 20 S core particle was purified by newborn bovine blood as the raw material.It was purified through a Hitrap Q ion exchange column,gel filtration chromatography column,and CHT-1 column to obtain 20 S core particles with better quality and concentration;3.The purification of His-HCV Core protein adopted inclusion body purification.Induction conditions: Rosetta competent cells,OD600 is 1.0,1 m M IPTG,induced at 25 ? for 5h;4.The structural analysis of the REG?-20 S proteasome complex showed that C-terminal amino acid of REG? at 254 site played an important role.The C-terminus of REG? would be inserted into the ?3-?4 pocket,?5-?6 pocket and ?6-?7 pocket,which may form a hydrogen bond with the amino acid of the ?-loop subunit of the20 S core particle and play a role in controlling the switch.