title: Molecular cloning, recombinant expression, and antimicrobial activity of EC-hepcidin3, a new four-cysteine hepcidin isoform from Epinephelus coioides.
abstract: 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.
authors.affiliation: State Key Laboratory of Marine Environmental Science, College of Oceanography and Environmental Science, Xiamen University, Fujian, PR China.
authors.forename: HaiDong
authors.initials: H
authors.lastname: Qu
authors.forename: Bei
authors.initials: B
authors.lastname: Chen
authors.forename: Hui
authors.initials: H
authors.lastname: Peng
authors.forename: KeJian
authors.initials: K
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000096:biosynthesis / Q000276:immunology* / Q000494:pharmacology; D023181:Antimicrobial Cationic Peptides / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology; D001492:Bass / Q000276:immunology* / Q000378:metabolism; D003001:Cloning, Molecular; D003545:Cysteine / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005091:Exons; D015870:Gene Expression; D064451:Hepcidins; D007113:Immunity, Innate; D007438:Introns; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010802:Phylogeny; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D044223:Pseudomonas stutzeri / Q000187:drug effects* / Q000254:growth & development; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D016415:Sequence Alignment; D013211:Staphylococcus aureus / Q000187:drug effects* / Q000254:growth & development
chemical_list: D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D064451:Hepcidins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D003545:Cysteine