Purification and partial characterisation of recombinant human hepcidin.
Hepcidin is a liver-expressed antimicrobial and iron regulatory peptide. A number of studies have indicated that hepcidin is important for the correct regulation of body iron homeostasis. The aims of this study were to analyse the expression, trafficking and regulation of human hepcidin in an in vitro cell culture system. Human hepcidin was transfected into human embryonic kidney cells. Immunofluorescence and confocal microscopy analysis revealed that recombinant hepcidin localised to the Golgi complex. Recombinant hepcidin is secreted from the cell within 1 h of its synthesis. Recombinant hepcidin was purified from the cell culture medium using ion-exchange and metal-affinity chromatography and was active in antimicrobial assays. Amino-terminal sequence analysis of the secreted peptide revealed that it was the mature 25 amino acid form of hepcidin. Our results show that recombinant myc-His tagged human hepcidin was expressed, processed and secreted correctly and biologically active in antimicrobial assays.
Wallace
Daniel F
DF
Membrane Transport Laboratory, Cancer and Cell Biology Division, The Queensland Institute of Medical Research, PO Royal Brisbane Hospital, Qld. 4029, Brisbane, Queensland, Australia.
Jones
Marc D
MD
Pedersen
Palle
P
Rivas
Lucy
L
Sly
Lindsay I
LI
Subramaniam
V Nathan
VN
D023181:Antimicrobial Cationic Peptides / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D001410:Bacillus megaterium / Q000187:drug effects; D002478:Cells, Cultured; D019084:Fluorescent Antibody Technique, Indirect; D006056:Golgi Apparatus / Q000378:metabolism; D064451:Hepcidins; D006801:Humans; D007668:Kidney / Q000196:embryology / Q000378:metabolism; D016982:Micrococcus luteus / Q000187:drug effects; D011994:Recombinant Proteins / Q000302:isolation & purification
D023181:Antimicrobial Cationic Peptides; C578702:HAMP protein, human; D064451:Hepcidins; D011994:Recombinant Proteins