Cloning and Secretion Expression of Hepcidin in Pichia pastoris

Hepcidin is a liver-expressed, small peptide rich in cysteine that acts as a regulator of systemic iron homeostasis. In this work, according to the partiality codon of Pichia pastoris, a DNA fragment containing the coding sequence of hepcidin was designed and synthesized, especially a Kex2 signal cleavage site was fused in the 5' end of the antibacterial peptide genes. The modified hepcidin gene was then inserted into the P. pastoris expression vector plasmid pPICZα-A. After electroporation of the resulting vector, pPICZα-A-Hepc, into the yeast host strain GS115, transformants with high copy inserts were selected by 1500 mg/L Zeocin™ selection. Under the control of the promoterAOX1 (alcohol oxidase 1), recombinant hepcidin secreted from P. pastoris had a molecular weight of 2.7 kD. After optimization of the flask-shaking culture fermentation, the yield of hepcidin reached 100 mg/L in the clarified broth. Through antibacterial assay, the recombinant hepcidin displayed obvious antibacterial activity against Bacillus subtilis. But it could not distinctly inhibit the growth of E. coliBL21(DE3).