Production of biologically active forms of recombinant hepcidin, the iron-regulatory hormone.
Hepcidin is a liver produced cysteine-rich peptide hormone that acts as the central regulator of body iron metabolism. Hepcidin is synthesized under the form of a precursor, prohepcidin, which is processed to produce the biologically active mature 25 amino acid peptide. This peptide is secreted and acts by controlling the concentration of the membrane iron exporter ferroportin on intestinal enterocytes and macrophages. Hepcidin binds to ferroportin, inducing its internalization and degradation, thus regulating the export of iron from cells to plasma. The aim of the present study was to develop a novel method to produce human and mouse recombinant hepcidins, and to compare their biological activity towards their natural receptor ferroportin. Hepcidins were expressed in Escherichia coli as thioredoxin fusion proteins. The corresponding peptides, purified after cleavage from thioredoxin, were properly folded and contained the expected four-disulfide bridges without the need of any renaturation or oxidation steps. Human and mouse hepcidins were found to be biologically active, promoting ferroportin degradation in macrophages. Importantly, biologically inactive aggregated forms of hepcidin were observed depending on purification and storage conditions, but such forms were unrelated to disulfide bridge formation.
Gagliardo
Bruno
B
CNRS (UMR 8601), Université Paris Descartes, France.
Faye
Audrey
A
Jaouen
Maryse
M
Deschemin
Jean-Christophe
JC
Canonne-Hergaux
François
F
Vaulont
Sophie
S
Sari
Marie-Agnès
MA
D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000096:biosynthesis* / Q000302:isolation & purification / Q000502:physiology; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D064451:Hepcidins; D006801:Humans; D035925:Iron-Regulatory Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000502:physiology; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism
D023181:Antimicrobial Cationic Peptides; D017931:DNA Primers; C578702:HAMP protein, human; C579599:Hamp protein, mouse; D064451:Hepcidins; D035925:Iron-Regulatory Proteins; D011994:Recombinant Proteins