Exploration Of NIRF In Vitro Ubiquitination Towards HBV Core Protein

Objective: NIRF is an ubiquitination E3 ligase which can bind to E2-Ub recombinant and substrate, fascinating polyubiquity transfer from E2 to substrate. Construct various expression plasmids, including eukaryotic expression plasmid pFLAG-NIRF and prokaryotic expression pGST-NIRF and pGST-HBc, and then to express target proteins and to purify GST-NIRF and GST-HBc by GST 4B gel. Protein-protein interaction between NIRF and HBc was indentified by GST pull down. Develop an ubiquitination system to explore whether NIRF can ubiquitylize HBc in vitro by ubiquitination analysis.Methods:(1) Primers were designed according to the sequences of NIRF, and NIRF genes were amplified by PCR and then inserted into prokaryotic expression plasmid pGEX-4T-1 to construct pGST-NIRF. pGST-NIRF was transformed into E.coil BL21 (DE3) to express GST-NIRF proteins and culture conditions were optimized to produce high concentration and full-length target proteins. Purification process was optimized to obtain high-purity GST-NIRF proteins. Eukaryotic plasmid pcDNA3-HBc was transfected into HEK293 cells to produce HBc proteins. Purified GST-NIRF and cells lysate with HBc in which were incubated together to allow the NIRF capture HBc and then products were purified by GST 4B gel and subjected to SDS-PAGE analysis.(2) Primers were designed according to the sequences of HBc, and HBc genes were amplified by PCR and then inserted into prokaryotic expression plasmid pGEX-4T-1 to construct pGST-HBc. pGST-HBc was transformed into E.coil BL21 (DE3) to express GST-HBc proteins and culture conditions were optimized to produce high concentration and full-length target proteins. NIRF genes was amplified by the methods above mentioned and then inserted into eukaryotic plasmid p3*FLAG-CMV-10 to get pFLAG-NIRF. pFLAG-NIRF was transfected into HEK293 cells to produce FLAG-NIRF fusion proteins. GST-pull down was processed through the methods above to prove the protein-protein interaction between HBc and NIRF.(3) Purified GST-NIRF and GST-HBc were added into ubiqutination system including E1, E2 (Ubc5b), ubiquity and so on. Products via ubiquitination reactivates were separated by SDS-PAGE and then subjected to western blot to identify whether NIRF can ubiquity HBc in vitro.Results: Construct eukaryotic plasmid pFLAG-NIRF successfully, two prokaryotic plasmids pGST-NIRF and pGST-HBc too. The eukaryotic plasmid was well expressed in HEK293T cells, so did the two prokaryotic plasmids in BL21 (DE3). GST-NIRF and GST-HBc proteins were purified by GST 4B gel. Combination between NIRF and HBc was confirmed through GST pull down, whether GST-NIRF pulls down HBc or GST-HBc pulls down NIRF. We have a clue that NIRF can ubiquity HBc in vitro.Conclusion: Protein-protein interaction between NIRF and HBc was confirmed by GST pull down analysis. Ubiquitination experiment showed that NIRF may ubiquinate HBc in vitro, which is not confirmed because of the lack of proofs.