Cloning and secretion expression of hepcidin in Pichia pastoris.
- Author:
Hui ZHANG
1
;
Qi-Peng YUAN
Author Information
1. Key Laboratory of Bioprocess of Beijing, Beijing University of Chemical Technology, Beijing 100029, China.
- Publication Type:Journal Article
- MeSH:
Amino Acid Sequence;
Anti-Bacterial Agents;
chemistry;
metabolism;
pharmacology;
Antimicrobial Cationic Peptides;
genetics;
metabolism;
pharmacology;
Bacillus subtilis;
drug effects;
growth & development;
Base Sequence;
Cloning, Molecular;
Electrophoresis, Polyacrylamide Gel;
Electroporation;
Escherichia coli;
drug effects;
growth & development;
Gene Expression;
Hepcidins;
Humans;
Molecular Sequence Data;
Molecular Weight;
Pichia;
genetics;
Plasmids;
genetics;
Polymerase Chain Reaction;
Recombinant Proteins;
chemistry;
metabolism;
pharmacology;
Transformation, Genetic
- From:
Chinese Journal of Biotechnology
2007;23(3):381-385
- CountryChina
- Language:Chinese
-
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).
