A eukaryotic expression strategy for producing the novel antimicrobial peptide PRW4

Area of Science:

• Biotechnology
• Microbiology
• Protein Engineering

Background:

• Antimicrobial peptides (AMPs) are crucial in innate immunity.
• PMAP-36, a porcine myeloid-derived AMP, was modified to create PRW4.
• PRW4 exhibits α-helical structure and broad-spectrum antimicrobial activity.

Purpose of the Study:

• To express and purify a recombinant form of the modified antimicrobial peptide PRW4.
• To evaluate the antimicrobial activity of the recombinant PRW4.
• To establish a production system for modified AMPs in Pichia pastoris.

Main Methods:

• PRW4 was fused to a 6× His-Trx tag for expression.
• Recombinant protein expression was carried out in Pichia pastoris GS115 using the pPICZαA vector.
• Maximal protein induction was achieved at 1% methanol, 96h, pH 6.0.
• Purification involved Ni-NTA resin chromatography and enterokinase digestion.

Main Results:

• Successfully expressed and purified recombinant PRW4 with 90% purity.
• Obtained 15 mg of recombinant PRW4 per liter of fermentation culture.
• Recombinant PRW4 demonstrated antimicrobial activity against a panel of Gram-negative and Gram-positive bacteria, similar to synthetic PRW4.

Conclusions:

• Pichia pastoris is an effective host for the production of recombinant PRW4.
• The recombinant production method provides a viable alternative to synthetic peptide production.
• This study serves as a reference for producing other modified antimicrobial peptides.