title: High-level secretory expression of metchnikowin in Escherichia coli.
abstract: Metchnikowin is a proline-rich peptide from Drosophila with antibacterial and antifungal activities. Its commercial application is limited due to the low immune-inducible expression in vivo. Here we present a method to produce high-yield metchnikowin in recombinant Escherichia coli. The genes coding for metchnikowin and the fusion partner Cherry tag were subcloned into the pET22b vector to construct a recombinant expression plasmid and transformed into E. coli BL21 (DE3). The fusion protein was successfully expressed and secreted with a yield of 300μg/ml after 18-h induction. Active metchnikowin was released by cleavage of the Asp-Pro bond between fused proteins by 72-h formic acid hydrolysis at 50°C. After 24-h dialysis, metchnikowin was purified to electrophoretic homogeneity and showed significant antibacterial activities against both Bacillus subtilis and E. coli DH5α. It is the first report on efficient production of metchnikowin in recombinant E. coli.
authors.affiliation: School of Life Sciences, Anhui University, Hefei, Anhui 230601, PR China.
authors.forename: Di
authors.initials: D
authors.lastname: Wu
authors.forename: Yinghu
authors.initials: Y
authors.lastname: Lu
authors.forename: Huoqing
authors.initials: H
authors.lastname: Huang
authors.forename: Lijuan
authors.initials: L
authors.lastname: Ma
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Che
authors.forename: Xiangdong
authors.initials: X
authors.lastname: Zha
authors.forename: Bin
authors.initials: B
authors.lastname: Yao
authors.forename: Peilong
authors.initials: P
authors.lastname: Yang
mesh_terms: D000595:Amino Acid Sequence; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D005561:Formates / Q000737:chemistry; D006868:Hydrolysis; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D023181:Antimicrobial Cationic Peptides; D029721:Drosophila Proteins; D005561:Formates; D011993:Recombinant Fusion Proteins; C030544:formic acid; C096696:Mtk protein, Drosophila
keywords: Antimicrobial peptide; Asp-Pro cleavage; Metchnikowin; Secretory expression