Scalable recombinant production of bioactive human neutrophil peptide-1 in Komagataella phaffii.

The escalating crisis of antimicrobial resistance demands novel therapeutics that overcome the limitations of conventional antibiotics. Human α-defensins, such as Human Neutrophil Peptide-1 (HNP-1), represent compelling candidates due to their potent, broad-spectrum antimicrobial activity and membrane-disrupting mechanism. However, clinical translation has been hindered by the absence of scalable production systems capable of delivering high yields of functional peptide. To address this challenge, we developed an efficient expression platform in the yeast Komagataella phaffii. In this system, a codon-optimized HNP-1 sequence was fused to a His6-SUMO tag downstream of the α-factor secretion signal to enhance solubility, folding, and recovery. Initial shake-flask optimization identified pH 6.0 with 96 h of induction as optimal fermentation conditions, yielding 19.75 ± 1.1 mg/L of recombinant protein. Subsequent scale-up to a controlled 5 L bioreactor significantly enhanced production, achieving 122 mg/L of the fusion protein. Following purification and precise cleavage, this process delivered 15.25 mg/L of pure, mature HNP-1 representing, to our knowledge, the highest yield of recombinant HNP-1 reported. The final product demonstrated potent antibacterial activity against Staphylococcus aureus and Escherichia coli while exhibiting excellent hemocompatibility, confirming preservation of native structure and function. This work establishes a scalable production platform for HNP-1 and provides an adaptable framework for expressing other structurally complex antimicrobial peptides with therapeutic potential.