title: Expression, purification and antibacterial activity of the channel catfish hepcidin mature peptide.
abstract: 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 1mM 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.
authors.affiliation: College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China. Electronic address: ytao@shou.edu.cn.
authors.forename: Yan
authors.initials: Y
authors.lastname: Tao
authors.affiliation: College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China.
authors.forename: Dong-Mei
authors.initials: DM
authors.lastname: Zhao
authors.affiliation: College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China.
authors.forename: Ya
authors.initials: Y
authors.lastname: Wen
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000096:biosynthesis / Q000494:pharmacology; D023181:Antimicrobial Cationic Peptides / Q000096:biosynthesis / Q000235:genetics* / Q000494:pharmacology; D002397:Catfishes / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D006090:Gram-Negative Bacteria / Q000187:drug effects; D064451:Hepcidins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D010455:Peptides / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D064451:Hepcidins; D010455:Peptides; D011993:Recombinant Fusion Proteins
keywords: Antibacterial activity; Channel catfish; Enterokinase; Fusion protein; Hepcidin; Recombinant protein expression