Expression, purification and antibacterial activity of the channel catfish hepcidin mature peptide

• A channel catfish hepcidin was recombinantly expressed for the first time.
• pET-32a(+)-mCH expressed an enterokinase-cleavable thioredoxin–hepcidin fusion protein.
• The fusion protein strategy overcame previous insolubility problems in E. coli.
• The mature peptide released by enterokinase possessed antibacterial activity.

Hepcidins are small cysteine-rich cationic antimicrobial peptides. The channel catfish (Ictalurus punctatus) hepcidin cDNA has been characterized, but recombinant protein expression and purification was not reported. I. punctatus hepcidin is comprised of 96 residues, with eight functionally important conserved cysteine residues located in the C-terminal region of the mature peptide, suggesting that this region is responsible for the antibacterial activity. In this study, a cDNA fragment (mCH) encoding the 25 amino acid mature peptide was cloned from channel catfish liver, and inserted into vector pET-32a(+) to produce a construct that expressed a hexahistidine-tagged thioredoxin (trxA) fusion protein that was cleavable using enterokinase. The trxA–mCH fusion protein was expressed in Escherichia coli BL21 (DE3) at 25 °C, using 1 mM IPTG for induction. Greater than 80% of the fusion protein was expressed solubly, but was not biologically active. Removal of the trxA fusion partner by enterokinase resulted in mCH that exhibited antibacterial activity against two Gram-positive (Listeria monocytogenes and Staphylococcus aureus), and two Gram-negative (E. coli and Pseudomonas aeruginosa) bacteria.