Molecular Cloning, Recombinant Expression, and Antimicrobial Activity of EC-hepcidin3, a New Four-Cysteine Hepcidin Isoform from Epinephelus coioides

Hepcidin, a cysteine-rich antimicrobial peptide, is widespread in fish and shows multiple activities, including antimicrobial, antivirus, and antitumor. Here, a new four-cysteine hepcidin isoform gene, EC-hepcidin3, was cloned from the marine-cultured orange-spotted grouper (Epinephelus coioides). The complete cDNA sequence consisted of 603 bases with an open reading frame (ORF) of 270 bases. The genomic DNA sequence was composed of two introns and three exons, and its 312-bp upstream region had multiple putative transcription factor binding sites. Soluble recombinant protein EC-proHep3 containing a His-tag at the C-terminus was obtained from expression plasmid pET-28a/EC-proHep3 in Escherichia coli Rosetta. It was purified by immobilized metal affinity chromatography (IMAC), and it showed antibacterial activity in vitro. Kinetic studies indicated that recombinant EC-proHep3 has strong, rapid activity against Staphylococcus aureus and Pseudomonas stutzeri. The results indicate that EC-hepcidin3 might be an effective component in the innate immune system of groupers.