Purification And Structure Of The REGγ-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 REGγ-mediated degradation process,which is independent of ubiquitination and ATP.As a member of the REG family,more and more research results show that REGγ has abnormal expression in tumors which are closely related to tumor development.REGγ can activate 20 S proteasome to degrade different target proteins and regulate the related signaling pathway.In the REGγ-mediated protein degradation pathway,How does REGγ control the gate of20 S core particles? How does REGγ recognize its target protein? None of these have been reported.Purification of the REGγ,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 REGγ-20 S proteasome complex and REGγ-His HCV Core protein complex were investigated in this research with results as follows.1.REGγ 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 REGγ-20 S proteasome complex showed that C-terminal amino acid of REGγ at 254 site played an important role.The C-terminus of REGγ would be inserted into the α3-α4 pocket,α5-α6 pocket and α6-α7 pocket,which may form a hydrogen bond with the amino acid of the α-loop subunit of the20 S core particle and play a role in controlling the switch.