High-level secretory expression of metchnikowin in Escherichia coli

• We developed a method to produce secretory metchnikowin in recombinant E. coli.
• The recombinant plasmid contained a fusion gene coding for metchnikowin and Cheery.
• The fusion protein was successfully expressed and secreted with a yield of 300 μg/ml.
• Active metchnikowin was released by cleaving the Asp-Pro bond between fused proteins.
• Recombinant metchnikowin had antibacterial activities against B. subtilis and E. coli.

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 300 μg/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.