title: Recombinant overexpression of camel hepcidin cDNA in Pichia pastoris: purification and characterization of the polyHis-tagged peptide HepcD-His.
abstract: Hepcidin, a liver-expressed antimicrobial peptide, has been demonstrated to act as an iron regulatory hormone as well as to exert a wide spectrum of antimicrobial activity. The aim of this work was the expression, as secreted peptide, purification, and characterization of a new recombinant polyHis-tagged camel hepcidin (HepcD-His) in yeast Pichia pastoris. The use of this eukaryotic expression system, for the production of HepcD-His, having 6 histidine residues at its C terminus, was simpler and more efficient compared with the use of the prokaryotic system Escherichia coli. Indeed, a single purification step was required to isolate the soluble hepcidin with purity estimated more that 94% and a yield of 2.8 against 0.2 mg/L for the E coli system. Matrix-assisted laser desorption/ionization time-of-flight (TOF)/TOF mass spectrometry of the purified HepcD-His showed 2 major peaks at m/z 4524.64 and 4634.56 corresponding to camel hepcidin with 39 and 40 amino acids. Evaluation of disulfide bond connectivity with the Ellman method showed an absence of free thiol groups, testifying that the 8 cysteine residues in the peptide are displayed, forming 4 disulfide bridges. Circular dichroism spectroscopy showed that camel hepcidin structure was significantly modified at high temperature of 90°C and returns to its original structure when incubation temperature drops back to 20°C. Interestingly, this peptide showed also a greater bactericidal activity, at low concentration of 9.5μM, against E coli, than the synthetic analog DH3. Thus, the production, at a large scale, of the recombinant camel hepcidin, HepcD-His, may be helpful for future therapeutic applications including bacterial infection diseases.
authors: Laboratoire d'ingénierie des protéines et des molécules bioactives (LIP-MB), Institut National des Sciences Appliquées et de Technologie, Université de Carthage, BP 676, 1080, Tunis Cedex, Tunis, Tunisia.
authors: Mohamed
authors: M
authors: Boumaiza
authors: Laboratoire d'ingénierie des protéines et des molécules bioactives (LIP-MB), Institut National des Sciences Appliquées et de Technologie, Université de Carthage, BP 676, 1080, Tunis Cedex, Tunis, Tunisia.
authors: Haifa
authors: H
authors: Chahed
authors: Laboratoire d'ingénierie des protéines et des molécules bioactives (LIP-MB), Institut National des Sciences Appliquées et de Technologie, Université de Carthage, BP 676, 1080, Tunis Cedex, Tunis, Tunisia.
authors: Aymen
authors: 0000-0003-2074-6988
authors: A
authors: Ezzine
authors: Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601, Université Paris Descartes, CNRS, 45 rue des Saints Pères, 75270, Paris Cedex 06, Paris, France.
authors: Maryse
authors: M
authors: Jaouen
authors: Department of Molecular and Translational Medicine, DMMT, University of Brescia, Viale Europa 11, 25123, Brescia, Brescia, Italy.
authors: Alessandra
authors: A
authors: Gianoncelli
authors: Department of Molecular and Translational Medicine, DMMT, University of Brescia, Viale Europa 11, 25123, Brescia, Brescia, Italy.
authors: Giovanna
authors: G
authors: Longhi
authors: Department of Molecular and Translational Medicine, DMMT, University of Brescia, Viale Europa 11, 25123, Brescia, Brescia, Italy.
authors: Fernando
authors: F
authors: Carmona
authors: Department of Molecular and Translational Medicine, DMMT, University of Brescia, Viale Europa 11, 25123, Brescia, Brescia, Italy.
authors: Paolo
authors: P
authors: Arosio
authors: Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601, Université Paris Descartes, CNRS, 45 rue des Saints Pères, 75270, Paris Cedex 06, Paris, France.
authors: Marie-Agnès
authors: MA
authors: Sari
authors: Laboratoire d'ingénierie des protéines et des molécules bioactives (LIP-MB), Institut National des Sciences Appliquées et de Technologie, Université de Carthage, BP 676, 1080, Tunis Cedex, Tunis, Tunisia.
authors: Mohamed Nejib
authors: MN
authors: Marzouki
mesh_terms: D000818:Animals; D002162:Camelus; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000187:drug effects; D064451:Hepcidins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D006639:Histidine / Q000737:chemistry*; D008958:Models, Molecular; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013816:Thermodynamics
chemical_list: D004220:Disulfides; D064451:Hepcidins; D011994:Recombinant Proteins; D006639:Histidine
keywords: Pichia pastoris; affinity purification; antibacterial activity; camel hepcidin cDNA; circular dichroism; recombinant expression