hasan abbas albuthabhak ( Assistant Lecturer )
Faculty of Science -
[email protected]
Recombinant production of hepcidin peptide in pichia pastoris
Introduction: Hepcidin is a small cystein-rich cationic peptide produced mainly by the liver. This hormone peptide is the key regulator of iron metabolism at the whole-body level and play roles in the inflammation, infection, hypoxia and anemia. This peptide is found in three forms; hepcidin 25, 22 and 20. Hepcidin 25 is the main form which is found in the bloodstream. Objective: the aim of this study was to recombinant production of hepcidin 25 in yeast Pichia pastoris. Materials and methods: the sequence of hepcidin 25 gene with its pre- and propeptides was synthesized according to the codon preference of Pichia pastoris and cloned in the pPICZA plasmid using EcoRI/KpnI restriction enzymes. The recombinant plasmid electroporated to the competent yeast and positive clones selected based on the zeocine antibiotic resistance (100 µg/ml). Some of positive clones were cultured in the YPD medium and grown cells were transformed to the YP medium containing 0.5% methanol. Induction with 1% methanol was continued for more next 3 days. The supernatant of culture media was analyzed for recombinant hepcidin using SDS-PAGE. Non-recombinant Pichia pastoris was used as the negative control. Results and conclusion: the bands of hepcidin peptide was observed in the supernatant of obtained positive recombinant clones. In conclusion, Pichia pastoris could be concerned as the appropriate host for recombinant production of hepcidin peptide. The obtained recombinant yeast producing hepcidin will be used for furthered studies.