Recombinant Expression,Purification And Characterization Of Antimicrobial Peptide VpDef In Pichia Pastoris

With the development of economy, people's living standard improvement, we pay more attention to our health and want to use non-drug, non-toxic, no side effects and non-pharmaceutical products to achieve good health effects. Antimicrobial peptides with broad-spectrum antibacterial, not only have a higher resistance to bacteria, but also have a good killing effect on viruses, tumors, fungi and protozoa. Antibacterial peptides also play an important role in immune response and wound healing. Therefore, antimicrobial peptides as a great potential of functional factors, will cause more and more attention.The antimicrobial peptide in this study is derived from the clam Venerupis philippinarum, VpDef was expressed in hemocytes and mantles, as well as in other major tissues. This peptide consisted 49 amino acids, VpDef possessed combined helix and beta structure, and four potential disulfide bonds. VpDef gene was cloned in vector pPICZaA,then we used electroporation transformed into Pichia pastor is GS115. When the concentration of methanol was 1.5% and the induction time was 144 h, the highest protein expression was 128.98 ?g/mL. In addition to target protein, there are other proteins.Through cation exchange column purification method, when the starting buffer was 50 mmol/L acetic acid (pH = 4.89) and the elution buffer was 50 mmol/L acetic acid,100 mmol/L NaCl (pH = 4.89) ,the target protein was successful elution, and the yield was 2.92% and the purity of the target protein was 98%. The minimal inhibitory concentration(MIC) assay showed that the recombinant protein VpDef had broad-spectrum antimicrobial activity. The MICs of L. monocytogenes, E. coli 0157(ATCC 35150) and Escherichia coli(ATCC 10305) were 75 ?g/mL,the MIC of Bacillus subtilis Lzz-133 was 95 ?g/mL and the MIC of Staphylococcus aureus was 120 ?g/mL.Finally, the properties of antimicrobial peptide VpDef were studied. VpDef was treated for 1 h with different temperature (4, 25, 37, 60, 80, 100?). There was no significant difference in inhibition zone (p>0.05). VpDef was treated for 4 h with different pH, the results showed that the activity of VpDef was not significantly affected at pH 2.0,4.0, 6.0 and 8.0, but decreased in alkaline. In addition, the protease stability of VpDef was studied by protease treatment for 2 h, and the stability of pepsin, trypsin, protease K and papain were also good.In summary, we have successfully expressed VpDef in P. pastor is , which has good tolerance of temperature, protease and pH.