Prokaryotic Expression, Purification And Biological Activity Of Human Lysozyme

Objective：To express and purification human lysozyme (hLyz) gene in Escherichia coli（E.coli）, and determine the biological activity of the protein. It will provide an experimentalbasis for the industrial application of hLyz.Methords：The gene sequence of hLyz. was prepared by RT-PCR from PC3cell, andcloned into prokaryotic expression vector pET-15b with His fusion labeling. The ligationproduct pET-15b-hlysozyme,was transformed to competent cell DH5α. Then the constructedrecombinant plasmid was extracted form the cell and transformed to E. coli gami forexpression. After induction with IPTG, the product was identified by SDS-PAGE. Afteroptimization of the expression conditions, the target protein were expressed in a lager scale.The insoluble protein was solubilized in5M guanidine-HCl and refolded in the presence ofoxido-shuZing agent (GSH/GSSG). Through the refolding process the rhLyz wassolubilized and purified. The purified human lysozyme was identified by Western blot andmass spectrometry, and then enzyme activity was determined by Micrococcus lysodeikticus.Results：Both agarose electrophoresis and nucleotide sequencing proved that recombinantplasmid pET-15b-hlysozyme was constructed correctly. SDS-PAGE showed a target proteinband with relative molecular mass of about17kDa. Western blot and mass spectrometryshowed that the target protein was expressed in E. coli gami. The recombinant Protein washighly expressed，and reached maximum level of about49.6%of total somatic Proteins afterinduction with0.3mmol/L IPTG at25℃for6h. Through the refolding process, the rhLyzwas solubilized and purified with an purity over95%. The rhLyz was found to show strongenzyme activity and antibacterial activity. At30μg/mL, the rhLyz showed47.59%,58.02%and35.43%, respectively against the bacteria E. coli, Pseudomonas aeruginosa,Staphylococcus aureus.Conclusion：The human hlysozume was successfully expressed in E. coli and showed goodbiological activity.