In Vivo Optimized Expression Of Recombinant Hepcidin Using Pichia Pastoris

The hepatic peptide hormone hepcidin, which has recently been isolated from human plasma and urine, is thought to be a central regulator of iron homeostasis. Hepcidin is a low molecular weight cysteine rich circulating antimicrobial and antifungal peptide expressed in liver. It can act as signaling molecule involved in the maintenance of iron homeostasis. The chemical synthesis and purification of hepcidin from the natural sources is difficult and expensive. To date the expression and preparation of small peptide like hepcidin in eukaryotes has not been well reported.The present study is therefore designed to get a high yield of active recombinant hepcidin in yeast because the yeast can be grown just as easily as bacteria in continuous culture and will express a cloned gene and process the resulting protein, in a manner more related to that occurring in higher animals. In the present study we expressed human hepcidin in Pichia pastoris (P. pastoris) as recombinant hepc 20. We aimed to express and purify a small peptide (without any tag), which is indeed a very small peptide (2.2 kD) and optimize the conditions for its expression.First of all according to codon preference in yeast and amino acid sequence of hepcidin, the full length synthesized gene of the hepcidin (20 amino acids) protein was cloned in the expression vector pPIC9K and transformed into P. pastoris strain GS115 as pPIC9K-H. The hepcidin fragment was amplified and then verified by restriction enzymes analysis and DNA sequencing.After transformation the geneticin-resistant (G418~R) recombinant colonies were cultivated on MD and MM plates and were found as His+Mut+. Twenty transformants were selected each had the alcohol oxidase gene and Hepc gene. The hepcidin protein coding sequence was correctly integrated into the P. pastoris genome. In order to optimize the conditions for fermentation, the influence of different factors on biomass and hepcidin protein production during induction phase was subsequently studied by growing a single recombinant colony (resistant to G418,1mg/ml) in shake flask. Tricine-SDS-PAGE showed that hepcidin peptide was successfully expressed as secretory protein in P. pastoris and shown as 2.2 kDa band. The peptone, yeast extracts, steady pH and temperature had prominent effects on the growth and expression of recombinant P. pastoris. These are the most important factors which influenced the expression level of hepcidin and P. pastoris growth. After 48 hrs induction with 0.5% methanol at 28°'C, the BMMY medium was found optimal for hepcidin expression as compared with various other media. The magnitude of expression level reached 3 mg/L. The optimal harvesting time of cells was determined as 48-60 h after induction. Peptide expression level was confirmed by western blotting. Studies revealed that active recombinant hepcidin peptide can be successfully expressed in methylotrophic yeast.Recombinant peptide was purified from culture supernatants using Sephadex G-25 gel filtration chromatography. The fractions containing hepcidin were further purified by RP-HPLC. The elution time for the peak by RP-HPLC column (11th min) was the same as that of native hepcidin. In addition, the measured molecular weight accorded with the calculated molecular weight (2191.77Da) of hepcidin.Each of the fractions was analyzed by ELISA and confirmed that the material was indeed hepcidin. Furthermore the peak having a molecular weight of 2192.0 Da by LC-ESI—MS analysis was determined as recombinant hepcidin. Finally the recombinant hepcidin demonstrated obvious antimicrobial activity against Staphyloccocus aureus and Bacillus subtilis.