Expression, purification and structural characterization of recombinant hepcidin, an antimicrobial peptide identified in Japanese flounder, Paralichthys olivaceus

Par Conseil national de recherches du Canada

DOI	Trouver le DOI : https://doi.org/10.1016/j.pep.2008.05.012
Auteur	Rechercher : Srinivasulu, B.¹; Rechercher : Syvitski, R.; Rechercher : Seo, J- K.¹; Rechercher : Mattatall, N. R.¹; Rechercher : Knickle, L. C.²; Rechercher : Douglas, S. E.¹
Affiliation	Conseil national de recherches du Canada. Institut des biosciences marines du CNRC Conseil national de recherches du Canada. Développement des cultures et des ressources aquatiques
Format	Texte, Article
Résumé	The cysteine-rich peptide hepcidin is an antimicrobial peptide and iron transport regulator that has been found in vertebrates including birds, fish and mammals. To elucidate the structure and biological function of fish hepcidin, which is difficult to produce synthetically, we have cloned several plasmid constructs encoding hepcidin from Japanese flounder, Paralichthys olivaceus, and tested expression of recombinant peptides, each with an N-terminal hexahistidine (6×His) tag, in inclusion bodies or the periplasmic space of Escherichia coli. Hepcidin expressed in inclusion bodies was reduced, and subsequently refolded using a dilution technique with a cysteine redox system. The oxidized His-hepcidin monomer was separated from protein multimers and mass spectrometry analysis showed that the peptide was of the predicted size and contained four disulfide bonds. Removal of the 6×His tag was attempted using enzymatic cleavage by Factor Xa and tobacco etch virus (TEV) protease or chemical cleavage by hydroxylamine. The Factor Xa cleavage was unsuccessful and hydroxylamine cleavage resulted in aggregation of cleaved peptide. TEV protease cleavage was successful but immediately resulted in hexamer formation despite varying reaction conditions (redox, non-redox, pH, temperature, target protein concentration, type of buffer). However, the recombinant His-hepcidin fusion peptide monomer showed considerable antimicrobial activity. NMR-based studies showed that hepcidin contained a rare vicinal disulfide linkage at the top of a loop structure and a short β-sheet structure encompassing residues 7–13 and 19–25 that is stabilized by three disulfide bonds.
Date de publication	2008-09
Dans	Protein Expression and Purification 61, nº 1 (septembre 2008) : 36–44.
Langue	anglais
Publications évaluées par des pairs	Oui
Numéro du CNRC	NRCC 42780 1743
Numéro NPARC	3538402
Exporter la notice	Exporter en format RIS
Signaler une correction	Signaler une correction (s'ouvre dans un nouvel onglet)
Identificateur de l’enregistrement	c8c6b41b-dd4d-46de-bba1-618aea56c2dd
Enregistrement créé	2009-03-01
Enregistrement modifié	2020-04-15

Date de modification :: 2024-12-26