title: [Cloning and secretion expression of hepcidin in Pichia pastoris].
abstract: Hepcidin is a liver-expressed, small cysteine rich peptide that acts as a regulator of systemic iron homeostasis. In this work, according to the partiality codon of Pichia pastoris, a DNA fragment containing the coding sequence of hepcidin was designed and synthesized, especially a Kex2 signal cleavage site was fused in 5' end of the antibacterial peptide genes. Then the modified hepcidin gene was inserted into the Pichia pastoris expression vector plasmid pPICZalpha-A. After electroporation of the resulting vector, pPICZalpha-A-Hepc, into the yeast host strain GS115, transformants with high copy inserts were selected by 1500 mg/L Zeocin selection. Under the control of the promoter AOX1 (alcohol oxidase 1), recombinant hepcidin secreted from P. pastoris had a molecular weight of 2.7kD. After optimization of the flask-shaking culture fermentation, the yield of hepcidin reached 100 mg/L in the clarified broth. Through antibacterial assay, the recombinant hepcidin displayed obvious antibacterial activity against Bacillus subtilis. But it could not distinctly inhibit the growth of E. coli BL21 (DE3).
authors.affiliation: Key Laboratory of Bioprocess of Beijing, Beijing University of Chemical Technology, Beijing 100029, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
authors.forename: Qi-Peng
authors.initials: QP
authors.lastname: Yuan
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D023181:Antimicrobial Cationic Peptides / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D001412:Bacillus subtilis / Q000187:drug effects / Q000254:growth & development; D001483:Base Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D018274:Electroporation; D004926:Escherichia coli / Q000187:drug effects / Q000254:growth & development; D015870:Gene Expression; D064451:Hepcidins; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics*; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D014170:Transformation, Genetic
chemical_list: D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; C578702:HAMP protein, human; D064451:Hepcidins; D011994:Recombinant Proteins