Expression,Purification And Characterization Of Recombinant Human PLC-zeta Protein

Background:PLC?is a new isoenzyme of the PLC family,which plays an extremely important role in the activation of mammalian oocytes.Mounting evidence implies dysfunctional isoforms or significantly reduced levels of PLC?in sperm underlie certain types of male factor infertility.However,while active recombinant mouse PLC?protein has been purified,the successful production of active recombinant human PLC?protein has not yet been described in the literature.It would,therefore,be necessary to obtain an active,purified version of recombinant human PLC?protein and it is essential that this is obtained for the viable therapeutic use of PLC?within the clinic.Objective:In this study,the recombinant human PLC?protein was expressed and purified in vitro by constructing different expression vectors,and the protein activity was determined,which laid a foundation for exploring its feasibility of using it as artificially assisted oocyte to activate biological agents.Methods:The PLC?amino acid sequence was submitted to the proteomics server,and the basic composition and physicochemical properties of the PLC?protein were predicted using Pro Param software.The full length of human PLC?gene was cloned into the p Fast Bac-HTA plasmid to form the recombinant donor plasmid which was further transformed into DH10Bac Escherichia coli cells to construct the recombined bacmid by the site-specific transposition,which was screened by resistance and blue-white spots.Then the bacmid was transfected to Sf9 insect cells via cellfectin for package of the recombinant baculovirus.After the amplification of the recombinant baculovirous,the recombinant protein was expressed from the cells transduced by the recombinant baculovirus and was purified by Ni-NTA resin.Purified protein was identified by Western blot and time-of-flight mass spectrometry and the enzyme activity was determined.The optimized human PLC?gene was cloned into p ET-43.1a plasmid,and the PLC?recombinant plasmid expression vector p ET43a-PLC?containing the Nus A fusion tag was constructed.The recombinant plasmid was transformed into competent Escherichia coli BL-21?DE3?for prokaryotic expression of the protein.The soluble expression of the fusion protein Nus A-PLC?was induced by IPTG concentration and culture temperature.The fusion protein was purified by Ni-NTA resin and identified by Western blot and time-of-flight mass spectrometry and the enzyme activity was determined.The absorbance at 410 nm wavelength before and after the reaction of yellow p-nitrophenol by p-NPPC from PLC was determined,and the activity of PLC?enzyme was calculated according to the standard curve method to study the proteolytic properties of recombinant protein.Results:The human PLC?gene was cloned into p Fast Bac-HTA plasmid,and the shuttle plasmid Bacmid-PLC?was successfully constructed.The recombinant bacmid Bacmid-PLC?was transfected into Sf9 insect cells.The P3 generation baculovirus was determined to have a virus titer of 5.0×10⁸ PFU/m recombinant PLC?protein in the Sf9 cells was achieved at 72 hours after baculovirus infection and expressed in secreted form in cell culture medium.The recombinant protein purified by Ni²⁺affinity column was identified as PLC?by Western blot and ionization time-of-flight mass spectrometry and the enzyme activity was up to 656.64 U/m L.The recombinant expression vector p ET-43.1a?+?-PLC?carrying human PLC?gene fragment was successfully constructed,and the soluble expression of recombinant human PLC?protein in Escherichia coli was achieved by Nus A fusion tag.The expressed product was subjected to Ni²⁺affinity chromatography and gel filtration chromatography to obtain an active recombinant human Nus A-PLC?fusion protein,which was identified as PLC?and the enzyme activity was up to 150.52 U/m L.The optimal temperature of the two proteins was,the optimum p H was 8.0,and the stability was good between p H 7.0 and 8.5.Conclusion:The recombinant human PLC?protein was successfully expressed and purified by using Bac-to-Bac baculovirus expression system,and the soluble expression of recombinant human Nus A-PLC?fusion protein in Escherichia coli was achieved by using Nus A fusion tag.It has laid a good foundation for the in-depth study of large-scale production of recombinant human prion protein and biomedical applications.