Expression and preparation of recombinant hepcidin in Escherichia coli.
Hepcidin is a low-molecular-weight, highly disulfide bonded peptide relevant to small intestine iron absorption and body iron homeostasis. In this work, hepcidin was expressed in Escherichia coli as a 10.5 kDa fusion protein (His-hepcidin) with a N-terminal hexahistidine tag. The expressed His-hepcidin existed in the form of inclusion bodies and was purified by IMAC under denaturation condition. Since the fusion partner for hepcidin did not contain other cysteine residues, the formation of disulfide bonds was performed before the His-tag was removed. Then, the oxidized His-hepcidin monomer was separated from protein multimers through gel filtration. Following monomer refolding, hepcidin was cleaved from fusion protein by enterokinase and purified with reverse-phase chromatography. The recombinant hepcidin exhibited obvious antibacterial activity against Bacillus subtilis.
Zhang
Huai
H
Department of Pharmaceutical Engineering, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, PR China.
Yuan
Qipeng
Q
Zhu
Yaping
Y
Ma
Runyu
R
D000595:Amino Acid Sequence; D023181:Antimicrobial Cationic Peptides / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D001412:Bacillus subtilis / Q000187:drug effects; D003001:Cloning, Molecular; D004765:Enteropeptidase / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D064451:Hepcidins; D007093:Imidazoles / Q000737:chemistry; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D023181:Antimicrobial Cationic Peptides; D064451:Hepcidins; D007093:Imidazoles; D011993:Recombinant Fusion Proteins; C029899:imidazole; D004765:Enteropeptidase