title: Iron regulatory and bactericidal properties of human recombinant hepcidin expressed in Pichia pastoris.
abstract: Hepcidin is a circulating cysteine-rich peptide with antimicrobial properties. It functions as a hormonal regulator of iron homeostasis by controlling iron efflux from target cells via ferroportin (FPN1), which is internalized and degraded upon hepcidin binding. Because of its profound biomedical significance, hepcidin has become the target of intense biochemical studies. The aim of this study was to produce functional recombinant hepcidin in sufficient quantities for advanced research or potential clinical use, as the native hepcidin can be isolated from urine in very low yield. We report the expression, purification and functional characterization of hepcidin variants in yeast P. pastoris. The yield of untagged hepcidin 20- and 25-mer peptides was too low for complete functional characterization. By contrast, Hep20 and Hep25 tagged with either single 6xHis or double Myc-6xHis epitopes were expressed at high quantities (5-7mg/l of culture), yet mostly in oligomeric forms. Purification of monomeric tagged hepcidins was achieved by size exclusion chromatography, with a yield of 0.5-1mg/l of culture. All recombinant hepcidins exhibited bacteriostatic activity and the ability to control cellular iron homeostasis, with Hep25-His being the most potent. Thus, Hep25-His promoted an increase in the levels of the labile iron pool (LIP) in macrophages and consistently bound to ferroportin (FPN1) causing its internalization and the subsequent downregulation of transferrin receptor 1 (TfR1) expression. Analysis by mass-spectrometry suggested that all eight cysteines participated in disulfide bond formation. Our results suggest that only the recombinant Hep25-His monomer was a fully active peptide. As Hep25-His faithfully recapitulates the functional properties of native Hep25, it represents a powerful tool for biochemical studies and potential diagnostic and therapeutic applications.
authors.affiliation: Department of Biochemistry, Laboratory of Molecular Biology and Immunobiotechnology, Hellenic Pasteur Institute, 127 Vas. Sofias Ave, 11521 Athens, Greece.
authors.forename: Vasiliki
authors.initials: V
authors.lastname: Koliaraki
authors.forename: Martha
authors.initials: M
authors.lastname: Marinou
authors.forename: Martina
authors.initials: M
authors.lastname: Samiotaki
authors.forename: George
authors.initials: G
authors.lastname: Panayotou
authors.forename: Kostas
authors.initials: K
authors.lastname: Pantopoulos
authors.forename: Avgi
authors.initials: A
authors.lastname: Mamalaki
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry / Q000494:pharmacology*; D002460:Cell Line; D064451:Hepcidins; D006801:Humans; D007501:Iron / Q000378:metabolism*; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid
chemical_list: D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; C578702:HAMP protein, human; C579599:Hamp protein, mouse; D064451:Hepcidins; D011994:Recombinant Proteins; D007501:Iron