[Cloning and secretion expression of hepcidin in Pichia pastoris].
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).
Zhang
Hui
H
Key Laboratory of Bioprocess of Beijing, Beijing University of Chemical Technology, Beijing 100029, China.
Yuan
Qi-Peng
QP
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
D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; C578702:HAMP protein, human; D064451:Hepcidins; D011994:Recombinant Proteins