Cloning And Secretion Expression Of Hepcidin In Pichia Pastoris

Hepcidin is a liver-expressed, small cysteine rich peptide that acts as a regulator of systemic iron homeostasis. Like other cysteine-rich antimicrobial peptides, hepcidin also exhibits obvious antibacterial and antifungal activity. In this work, we constructed the recombinant human hepcidin expression vector and secretion-expressed hepcidin in Pichia pastoris. Finally the method of isolation and purification of recombinant hepcidin is described.According to the partiality codon of Pichia pastoris, a DNA fragment containing the coding sequence of hepcidin was designed and synthesized, especially a Kex2signal cleavage site was fused in its5’end. Then the fragment was digested with NotI and XhoI and was inserted into the Pichia pastoris expression vector plasmid pPICZa-A. The resulting vector, pPICZa-A-Hepc, was linearized and transformed into the yeast host strain P. pastoris GS115by electroporation. The GS115strain with high copy inserts was obtained by1500μg/mL ZeocinTM selection. All Zeocin-resistant recombinants were tested to confirm the Mut phenotype.After methanol induction, recombinant hepcidin with a molecular weight of2.7kDa was secreted from the selected recombinants and was detected by Tricine-SDS-PAGE. After optimization of the flask-shaking culture fermentation, it was found that the greater expressed level of the recombinant hepcidin, as high as100mg/L, could be gained when the density of the seed yeast amounted to OD600value4-6, the yeast was cultured under28℃for48hours, and0.5%methanol was supplemented into the medium. Through antibacterial assay, the recombinant hepcidin displayed obvious antibacterial activity against Bacillus subtilis. But it could not distinctly inhibit the growth of E. coli BL21(DE3).Recombinant hepcidin was purified from culture supernatants after a24h induction by a process involving precipitation, untrafiltration and Sephadex G-25gel filtration chromatography. The peptide was first precipitated at the predicted pI of hepcidin (8.22). The precipitate which was dissolved in0.2M acetic acid, was then purified by gel-filtration chromatography using a Sephadex G-25column(1.0cm×100cm), the resulting protein was of at least90%purity.