Optimized Secretory Expression, Purification, and Antibacterial Activity Evaluation of Disulfide-Rich Rainbow Trout β-Defensin 3 in Pichia pastoris

Rainbow trout β-defensin 3 ( rt Defb3) is a small cationic antimicrobial peptide identified at the gene level, but its recombinant expression and functional characterization have not been reported. In this study, the mature rt Defb3coding sequence was cloned from trout liver cDNA and heterologously expressed in Pichia pastoris GS115 using the pPIC9K vector with an α-factor secretion signal. Expression was optimized by adjusting methanol concentration (0.5–1.25%), induction temperature (26–30 °C), and induction duration (1–7 days). Optimized induction with 1.0% methanol at 30 ℃ for 96 h produced ~7 mg/mL of secreted peptide, which was purified to >90% purity using one-step Ni-IDA affinity chromatography. The identity and purity of the recombinant rt Defb3 were confirmed by SDS-PAGE, HPLC (~97% purity), and MALDI-TOF mass spectrometry. Functional assays revealed potent broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria. These findings demonstrate, for the first time, the successful production of active rt Defb3in P. pastoris , establishing a scalable expression platform for fish β-defensins. The recombinant rt Defb3peptide holds promise as a natural antimicrobial agent for aquaculture and potential therapeutic applications, addressing the urgent demand for alternatives to conventional antibiotics.