title: Artificial synthesis of swine hepcidin gene and expression in Pichia pastoris.
abstract: In order to express swine hepcidin gene in Pichia pastoris, a DNA fragment coding hepcidin gene was synthesized with adaptation to yeast codon usage of highly expressed genes. A Kex2 signal cleavage site was fused in the 5' end of the DNA fragment for getting a peptide with the same N-end as native hepcidin. The 96-bp DNA fragment was ligated into the expression plasmid of pGAPZaA to construct pGAPZaA-hepcidin vector, which was transferred into P. pastoris (X33) to express hepcidin gene for extracellular secretion of protein at 86 µg/mL. A band of 2.76 kD molecular mass was detected by Tricine sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) analysis. Through antibacterial assay, the expressed hepcidin displayed obvious antibacterial activity. The minimal inhibitory concentration (MIC) was 5.38 and 2.69 µg/mL for Staphylococcus aureus and Bacillus subtilis prolification inhibitions, respectively.
authors.affiliation: a College of Animal Science and Veterinary Medicine , Henan Agricultural University , Zhengzhou , China.
authors.forename: Yuanran
authors.initials: Y
authors.lastname: Di
authors.forename: Wei
authors.initials: W
authors.lastname: Cheng
authors.forename: Juan
authors.initials: J
authors.lastname: Chang
authors.forename: Qingqiang
authors.initials: Q
authors.lastname: Yin
authors.forename: Min
authors.initials: M
authors.lastname: Lu
authors.forename: Lin
authors.initials: L
authors.lastname: Yuan
authors.forename: Xiaowei
authors.initials: X
authors.lastname: Dang
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology; D001412:Bacillus subtilis / Q000187:drug effects; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D003062:Codon / Q000235:genetics; D004247:DNA / Q000138:chemical synthesis* / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D064451:Hepcidins / Q000235:genetics* / Q000494:pharmacology; D006801:Humans; D010843:Pichia / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000494:pharmacology; D013203:Staphylococcal Infections / Q000188:drug therapy; D013211:Staphylococcus aureus / Q000187:drug effects; D013552:Swine / Q000235:genetics*
chemical_list: D000900:Anti-Bacterial Agents; D003062:Codon; D064451:Hepcidins; D011994:Recombinant Proteins; D004247:DNA
keywords: antibacterial activity; artificial syntheses; gene expression; swine hepcidin gene