title: Functional expression and purification of recombinant Hepcidin25 production in Escherichia coli using SUMO fusion technology.
abstract: Hepcidin25 is a small cysteine-rich peptide hormone known as a new class of antimicrobial peptides. The purpose of the present study was to express, purify and investigate the antibacterial properties of recombinant human hepcidin25 protein production in Escherichia coli. Human hepcidin25 gene was optimized and fused to a small ubiquitin-related modifier (SUMO) gene for higher expression. Then SUMO-hepcidin25 was cloned into the pET-32a (+) vector and expressed in E. coli Origami. The fusion protein with a molecular weight of approximately 35kDa was analyzed on SDS-PAGE gel. The highest expression was observed after 6h induction and the fusion protein consisted approximately 47% of the total cellular protein. The purified SUMO-hepcidin25 purity was determined to be higher than 95%, with a final yield of 3.9mgl-1of media. The recombinant hepcidin25 showed antibacterial activity against both Gram negative (Klebsiella pneumonia) and Gram positive (Staphylococcus aureus and Bacillus cereus) bacteria with minimum inhibitory concentrations (MICs) of 150μgml-1, 18.7μg/ml-1and 37.5μg/ml-1, respectively. These results indicated that thioredoxin and SUMO dual fusion system is an efficient production system for synthesis functional human hepcidin25.
authors.affiliation: Department of Biology, Faculty of Science, Shahrekord University, Shahrekord, Iran.
authors.forename: Vahideh
authors.initials: V
authors.lastname: Sadr
authors.affiliation: Department of Genetics, Faculty of Science, Shahrekord University, Shahrekord, Iran. Electronic address: Saffar_b@sci.sku.ac.ir.
authors.forename: Behnaz
authors.initials: B
authors.lastname: Saffar
authors.affiliation: Department of Biology, Faculty of Science, University of Isfahan, Isfahan, Iran.
authors.forename: Rahman
authors.initials: R
authors.lastname: Emamzadeh
mesh_terms: D001419:Bacteria / Q000145:classification / Q000187:drug effects; D003001:Cloning, Molecular; D003062:Codon; D004926:Escherichia coli / Q000235:genetics*; D064451:Hepcidins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D025841:Small Ubiquitin-Related Modifier Proteins / Q000235:genetics; D013879:Thioredoxins
chemical_list: D003062:Codon; D064451:Hepcidins; D011993:Recombinant Fusion Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; C545475:hepcidin 25, human; D013879:Thioredoxins
keywords: Antimicrobial peptides; Expression and purification; Fusion protein; Hepcidin25 gene; Small ubiquitin-related modifier (SUMO)