Recombinant expression and purification of a hybrid antimicrobial peptide Aur-Defb3 in Komagataellaphaffii

Antimicrobial peptides (AMPs) are promising alternatives to conventional antibiotics against multidrug-resistant pathogens, but their application is limited by low yield, structural instability, and potential toxicity. In this study, we designed a hybrid peptide, Aur-Defb3, by fusing a bioinformatically optimized rainbow trout β-defensin-derived sequence with a conserved aurein motif. To improve predicted physicochemical properties, we introduced a Leucine-to-Phenylalanine (L-to-F) substitution into the defensin-derived segment, hypothesizing that the aromatic side chain may contribute to hydrophobic-core stability and membrane interaction. Recombinant Aur-Defb3 was expressed in Komagataella phaffii GS115 and purified by Ni-IDA affinity chromatography. HPLC analysis showed that the major purified peak accounted for 97.08% of the integrated peak area, and the concentration of the purified fraction was estimated by A₂₈₀ measurement. Antimicrobial assays revealed antibacterial activity against both Gram-positive and Gram-negative bacteria, with MIC values ranging from 31.25 to 250 μg/mL (average 97.7 μg/mL). Hemolysis assays showed low hemolytic activity toward rainbow trout erythrocytes under the tested conditions, with approximately 10% hemolysis observed at 256 μg/mL. Scanning electron microscopy (SEM) showed concentration-dependent morphological damage to bacterial cells, including surface roughening, deformation, and disruption, suggesting that membrane damage may contribute to the antibacterial activity of Aur-Defb3. These results demonstrate the successful expression of Aur-Defb3, supporting its potential for further structural characterization, precise yield quantification, and in vivo mechanistic evaluation.