Production and purification of recombinant human hepcidin-25 with authentic N and C-termini

• Human hepcidin 25 (Hepc25) expression in Pichia pastoris using a modified version of the pPICZαA vector.
• Recombinant Hepc 25 is soluble, contains four disulfide bridges and native N- and C-termini.
• The purification process can be scaled-up.
• This is the first successful expression and purification of native human Hepc25 at a yield >1.5 mg/L of culture.

Hepcidin was first identified as an antimicrobial peptide present in human serum and urine. It was later demonstrated that hepcidin is the long-sought hormone that regulates iron homeostasis in mammals. Recombinant human Hepcidin-25 (Hepc25) was expressed in Pichia pastoris using a modified version of the pPICZαA vector. Hepc25 was then purified by a simple two-step chromatographic process to obtain 1.9 mg of soluble recombinant human Hepc25 per liter of culture at 96% purity. The sequence of Hepc25 and the presence of four disulfide bridges were confirmed by mass spectrometry analyses, and the recombinant Hepc25 exhibited antibacterial activity. This protocol of production and purification is the first step toward the production of human Hepc25 at a greater scale.