Expression and characterization of the antimicrobial peptide ABP-dHC-cecropin A in the methylotrophic yeast Pichia pastoris

- This is the first report of the use of Pichia pastoris for expression and purification of ABP-dHC-Cecropin A.
- The optimal conditions were 20 °C, 2% casamino acids, 0.5% methanol, which induced expression for 96 h.
- Approximately 48 mg of recombinant protein was secreted into 1L of medium.
- The antibacterial and antifungal activities of recombinant protein are similar to those of the synthetic protein.
- This method provides benefits of simple operation, high yield of the recombinant protein, and good activity.

ABP-dHC-cecropin A is a linear cationic peptide that exhibits antimicrobial properties. To explore a new approach for expression of ABP-dHC-cecropin A using the methylotrophic yeast Pichia pastoris, we cloned the ABP-dHC-cecropin A gene into the vector pPICZαA. The SacI-linearized plasmid pPICZαA-ABP-dHC-cecropin A was then transformed into P. pastoris GS115 by electroporation. Expression was induced after a 96-h incubation with 0.5% methanol at 20 °C in a culture supplied with 2% casamino acids to avoid proteolysis. Under these conditions, approximately 48 mg of ABP-dHC-cecropin A was secreted into 1L (4 × 250-mL)of medium. Recombinant ABP-dHC-cecropin A was purified using size-exclusion chromatography, and 21 mg of pure active ABP-dHC-cecropin A was obtained from 1L (4 × 250-mL)of culture. Electrophoresis on 4-20% gradient gels indicated that recombinant ABP-dHC-cecropin A was secreted as a protein approximately 4 kDa in size. Recombinant ABP-dHC-cecropin A was successfully expressed, as the product displayed antibacterial and antifungal activities (based on an antibacterial assay, scanning electron microscopy, and antifungal assay) indistinguishable from those of the synthesized protein.