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

Optimized Secretory Expression, Purification, and Antibacterial Activity Evaluation of Disulfide-Rich Rainbow Trout β-Defensin 3 in Pichia pastoris | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Optimized Secretory Expression, Purification, and Antibacterial Activity Evaluation of Disulfide-Rich Rainbow Trout β-Defensin 3 in Pichia pastoris Jiali Cai, Haimei Wang, Shengfeng He, Meiqi Li, Dandan Peng, Bo Yao This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7618669/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Apr, 2026 Read the published version in Applied Microbiology and Biotechnology → Version 1 posted You are reading this latest preprint version Abstract 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. β-Defensin Rainbow trout (Oncorhynchus mykiss) Pichia pastoris pPIC9K Expression optimization Antimicrobial activity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Key points First successful recombinant production of rainbow trout β-defensin 3 in Pichia pastoris via optimized induction strategy. High-yield secretory expression (~7 mg/mL) with >90% purity confirmed by HPLC and biochemical analysis. Recombinant rtDefb3 displayed potent broad-spectrum antibacterial activity, offering a repeatable and scalable system and functional validation. Introduction The rapid rise of antimicrobial-resistant (AMR) pathogens has become a critical global threat to human health, creating an urgent need for alternative strategies to traditional antibiotics (Zheng et al. 2025 ). In 2021 alone, more than one million deaths were attributed to antibiotic-resistant bacterial infections, and this burden continues to increase (Charani et al. 2022 ). Antimicrobial peptides (AMPs), as key effectors of the innate immune system, have attracted considerable attention due to their broad-spectrum antimicrobial activity, immunomodulatory properties, and lower propensity to induce resistance. These features make AMPs promising candidates for novel antimicrobial therapeutics (Mahlapuu et al. 2020 ; Le et al. 2022 ; Mwangi et al. 2019 ). Numerous recent reviews have summarized AMP biological functions, mechanisms of action, and the challenges and opportunities in clinical translation (Magana et al. 2020 ; Mookherjee et al. 2020 ; Yang et al. 2025 ). β-Defensins are a conserved class of small cationic AMPs characterized by six cysteine residues forming three intramolecular disulfide bonds (Fig. 1 ). They are widely distributed in vertebrates and function in both direct antimicrobial defense and immunomodulation (Ganz 2003 ; Das et al. 2022 ). In teleost, β-defensins contribute to mucosal immunity; for example, certain homologues from zebrafish and tilapia exhibit measurable antimicrobial activity (Dong et al. 2015 ). In rainbow trout ( Oncorhynchus mykiss ), four β-defensin genes ( om BD-1 to om BD-4) have been identified (Casadei et al. 2009 ). Previous studies characterized their cDNA sequences and tissue expression profiles, with om BD-1 showing antiviral activity against viral hemorrhagic septicemia virus (VHSV) (Falco et al. 2008 ). However, om BD-2, om BD-3, and om BD-4 have mainly been investigated at the sequence and expression level (Casadei et al. 2009 ; Falco et al. 2008 ). To date, there has been no direct experimental evidence confirming the antimicrobial activity of rainbow trout β-defensin 3 ( rt Defb3, also known as om BD-3). Producing bioactive disulfide-rich polypeptides in heterologous systems remains challenging. Bacterial expression often results in misfolded products or inclusion bodies due to the reductive cytoplasm, whereas chemical synthesis of cysteine-rich polypeptides is costly and impractical at scale. Eukaryotic secretory expression systems such as Pichia pastoris (recently reclassified as Komagataella phaffii ) provide advantages for these polypeptides, as they allow proper oxidative folding, post-translational modifications, and efficient secretion into the culture medium, thereby simplifying purification. However, high-yield production of correctly folded polypeptides in P. pastoris requires careful optimization of the expression construct and culture conditions (Lv and Cai 2025 ). Critical factors include the choice of signal peptide, methanol induction level, temperature, and induction duration, which together determine the yield of soluble and bioactive product. In this study, we cloned the cDNA of mature rt Defb3 and expressed it in P. pastoris GS115 using the pPIC9K vector containing the yeast α-mating factor signal peptide to direct secretion. Methanol concentration, induction temperature, and induction time were systematically optimized to obtain maximum production of correctly folded rt Defb3. The secreted recombinant polypeptide was purified and subjected to antimicrobial assays against a panel of Gram-positive and Gram-negative bacteria. We report, for the first time, the successful recombinant production of rt Defb3 and demonstrate its potent broad-spectrum antimicrobial activity. This provides critical functional evidence for a fish β-defensin. Furthermore, our strategy-combining a secretion signal with systematic induction optimization-proved effective for producing an active, disulfide-rich polypeptide in a eukaryotic system. This approach offers a reproducible framework for scalable production of cysteine-rich AMPs (Karbalaei et al. 2020 ) and is consistent with recent advances in P. pastoris secretion engineering that enhance yield and economic feasibility (Lv and Cai 2025 ). Materials and Methods Experimental Animals, Strains, and Vectors Total RNA was isolated from the liver of rainbow trout (maintained in our laboratory) and subsequently reverse-transcribed into cDNA. Pichia pastoris GS115 (Shanghai RuiChu Biotechnology Co., Ltd.) was employed as the expression host, and the vector pPIC9K (Chongqing Tsingke Biotechnology Co., Ltd., China) was used for construction of the recombinant plasmid. The antimicrobial activity of the purified peptide was evaluated against the following test strains: Staphylococcus aureus ATCC 25923, Bacillus subtilis CMCC (B) 63501, Bacillus cereus CMCC (B) 63303, Escherichia coli ATCC 25923, Klebsiella pneumoniae CMCC (B) 46117, Pseudomonas aeruginosa ATCC 27853, Pseudomonas fluorescens ATCC 7966, Salmonella ATCC 12022, and Trichoderma reesei RUT-C30 (maintained in our laboratory). Reagents and Equipment The mRNA RT SuperMix reverse transcription kit (SynScript® III) was purchased from Tsingke Biotechnology Co., Ltd. (Beijing, China). The DNA gel extraction kit, yeast RNA extraction kit, and DNA marker A (25–500 bp) were obtained from Sangon Biotech Co., Ltd. (Shanghai, China). Restriction enzymes EcoR I, Not I, and Sac I-HF were purchased from New England Biolabs (NEB, USA). The DL2000 DNA marker and DNA ligation kit were purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China). Low molecular weight pre-stained protein marker was obtained from Thermo Fisher Scientific (USA). All other reagents were of analytical grade and sourced domestically in China. The A200 thermal cycler was provided by Hangzhou Long Gene Scientific Instrument Co., Ltd. (Hangzhou, China). The UV-Vis spectrophotometer (Model 6132) was supplied by Eppendorf (Germany). The protein electrophoresis system was provided by Bio-Rad (USA). The gel imaging system (Model 1100/26M) was purchased from Quantum ST4 (USA). The incubator shaker (Model ZQTY-70N) was supplied by Zhichu Instrument Co., Ltd. (Shanghai, China). The high-performance liquid chromatography (HPLC) system (Model LC5090) was provided by Fuli Analytical Instruments Co., Ltd. (Zhejiang, China). Construction of the Expression Vector Synthesis of the First-Strand cDNA Total mRNA was extracted from the liver of rainbow trout and reverse-transcribed into cDNA using the SynScript® III miRNA RT SuperMix reverse transcription kit (Tsingke Biotechnology Co., Ltd., Beijing, China). The reverse transcription reaction was performed with 10 ng –1 µg of mRNA as the template, 2 µL of RTase Mix, 10 µL of 2× Reaction Buffer, and RNase-free water to a final volume of 20 µL. The mixture was incubated at 37°C for 60 minutes and then treated at 85°C for 5 minutes. The resulting first-strand cDNA was stored at -20°C. Cloning of the cDNA Encoding the D4K and 6×His Tagged Target Gene To express the antimicrobial peptide with a native N-terminal, a D4K protease cleavage site was added at the N-terminus, and a 6×His tag was introduced at the C-terminus for purification purposes. The first-strand cDNA was used as the template, and specific primers Def-1 (forward) and Def-2 (reverse) (Table 1 ) were designed for PCR amplification of the rt Defb3 cDNA fragment. The PCR reaction mixture and conditions were as follows: 5 µL of 10× PCR Buffer, 4 µL of dNTPs (2.5 mmol/L), 1 µL each of forward and reverse primers, 0.2 µL of first-strand cDNA, 0.5 µL of Pfu DNA Polymerase, and RNase-free water to a total volume of 50 µL. The PCR conditions were: 94°C for 4 minutes, followed by 38 cycles of 94°C for 30 seconds, 55°C for 40 seconds, and 72°C for 20 seconds, with a final extension at 72°C for 10 minutes. The amplified product was cloned into the pMD19-T simple vector and transformed into Escherichia coli DH5α. Positive clones were selected and sequenced by Shanghai Sangon Biotechnology Co., Ltd. The cDNA fragment was further amplified using primers Def-F (forward) and Def-R (reverse) (Table 1 ), introducing an EcoR I site at the 5′ end and a 6×His tag, stop codon, and Not I site at the 3′ end. The PCR reaction was carried out under the same conditions, and the amplified target gene was confirmed by sequencing at Tsingke Biotechnology Co., Ltd. The molecular weight and isoelectric point of the encoded protein were predicted using ExPASy software ( http://web.expasy.org/compute.pi/ ). Table 1 Primer sequences Primer name Primer sequence (5’-3’) Size (bp) Def-1 GCGAATTCATGTCTCTACACTTATGTTTCATTAGTGG 37 Def-2 ATTCGCGGCCGCCATCATCACCATCACCACTCATTTACAGCATACATTCGGCCATG 56 Def-F CGGAATTCGACGACGATGACAAGTCTCTACACTTATGTTTCATTAGTGGGGG 52 Def-R ATTCGCGGCCGCTTAGTGATGGTGATGGTGGTGTTTACAGCATACATTCGGCCATGTAC 59 3'AOX1 GCAAATGGCATTCTGACATCC 21 5'AOX1 GACTGGTTCCAATTGACAAGC 21 Construction of the Recombinant Expression Vector and Transformation into Pichia pastoris GS115 The pMD19-T-Defensin plasmid was double-digested with EcoR I and Not I to obtain the target gene fragment. The fragment was then ligated into the pPIC9K vector (pre-digested with the same restriction enzymes) using 5 µL of ligation mix and incubated at 16°C for 30 minutes. The ligation reaction product was transformed into Escherichia coli DH5α competent cells, and positive clones were confirmed by colony PCR and sequencing. The verified pPIC9K-Defensin plasmid was digested with Sac I-HF and transformed into Pichia pastoris GS115 competent cells using the Yeast Transformation Kit (Biyuntian, China), with a 1:6 (v/v) transformation ratio. An empty pPIC9K vector was also transformed into Pichia pastoris GS115 as a negative control. Screening of High-Copy Pichia pastoris Transformants The transformed yeast solution was plated on MD plates (15 g/L agar, 13.4 g/L YNB, 22 g/L glucose, 0.07 g/L biotin) and incubated at 30°C for 3–4 days. Single colonies were selected, resuspended in sterile water, and plated on YPD plates containing geneticin at final concentrations of 0.5, 0.75, 1, 2, and 3 mg/mL. The plates were incubated for 2–5 days, and high-copy transformants utilizing methanol were screened. Genomic DNA was extracted from the selected yeast transformants, and PCR was performed using the universal primers 5′AOX1 and 3′AOX1 (Table 1 ) for confirmation. Expression of the Target Protein in Pichia pastoris GS115 and Optimization of Expression Conditions The PCR-confirmed yeast transformants were inoculated into 5 mL YPD medium and cultured overnight at 30°C, 250 rpm, until the OD 600 reached 0.6–0.8. One milliliter of the culture was then transferred into 100 mL BMGY medium (10 g/L yeast extract, 20 g/L peptone, 13.4 g/L YNB, 2 mL/L 500× biotin, 2 mL/L 50% glycerol, 100 mmol/L potassium phosphate buffer, pH 6.0) and cultured for 24 hours under the same conditions. The culture was then shifted to BMMY medium (same formulation as BMGY, except for 5 mL/L methanol replacing glycerol) and cultured at 30°C, 250 rpm for 72 hours. Methanol was added every 24 hours to a final concentration of 0.5%. The culture supernatant was collected, concentrated by TCA precipitation, and subsequently analyzed by Tricine-SDS-PAGE. Electrophoresis was carried out using polyacrylamide gels with concentrations of 4%, 10%, and 16% for the stacking, intermediate, and separating layers, respectively. In parallel, the methanol concentration in BMMY medium was adjusted to 0.5%, 0.75%, 1.0%, and 1.25%, while the cultivation temperature was set to 26°C, 28°C, and 30°C. Yeast transformants carrying pPIC9K-Defensin were cultured under these conditions to determine the optimal methanol concentration and expression temperature. Based on the obtained results, the same transformants were further incubated for different induction periods to identify the optimal expression time. Purification and Characterization of the Recombinant Protein The selected pPIC9K-Defensin transformants were cultured in 500 mL of the optimized medium. After centrifugation, the supernatant was mixed with an equal volume of Binding Buffer (5 mmol/L imidazole) and applied to a column. The column was washed with 15 column volumes of Binding Buffer to remove unbound proteins, followed by elution with Elution Buffer (25 mmol/L imidazole) until the absorbance returned to the baseline of 280 nm. The eluted protein was analyzed by HPLC. The HPLC system used a C 18 column (4.6×250 mm, 5 µm) with pure acetonitrile (Pump B) and ultrapure water (Pump C) as mobile phases, at a flow rate of 1 mL/min and a column temperature of 32°C. The injection volume was 10 µL, and detection was performed at 214 nm. The purified product was analyzed by Tricine–SDS–PAGE, and the target band was excised for subsequent MALDI-TOF/TOF mass spectrometric analysis. Antimicrobial Activity Evaluation of the Recombinant Protein The antimicrobial activity and spectrum of recombinant rt Defb3 were evaluated using agar diffusion method and microdilution assays. Laboratory-maintained Gram-positive, Gram-negative bacteria, and fungi were selected as test strains: Staphylococcus aureus (ATCC 25923), Bacillus subtilis (CMCC(B) 63501), Bacillus cereus (CMCC(B) 63303), Escherichia coli (ATCC 25923), Klebsiella pneumoniae (CMCC(B) 46117), Aeromonas hydrophila (ATCC 7966), Pseudomonas aeruginosa (ATCC 27853), Salmonella (ATCC 12022), and Trichoderma reesei (RUT-C30). Staphylococcus aureus ATCC 25923, Bacillus subtilis CMCC (B) 63501, Bacillus cereus CMCC (B) 63303, Escherichia coli ATCC 25923, Klebsiella pneumoniae CMCC (B) 46117, Pseudomonas aeruginosa ATCC 27853, Pseudomonas fluorescens ATCC 7966, Salmonella ATCC 12022, and Trichoderma reesei Rut-C30 (maintained in our laboratory). For the agar diffusion assay, 2% agar medium was poured into plates and allowed to solidify. Oxford cups were placed on the solidified medium, and bacterial suspensions were prepared at 1×10 − 6 CFU/mL and inoculated into the agar at 1% (v/v). After thorough mixing and solidification, Oxford cups were removed, and 100 µL of purified peptide solution was added to each well. Plates were incubated at 37°C overnight, and inhibition zones were measured using a caliper. Experiments were performed in triplicate, and mean values were reported. For MIC determination, logarithmic-phase bacterial cultures were diluted to 1×10 − 6 CFU/mL. Purified rt Defb3 was serially diluted two-fold in 96-well plates, and 50 µL of bacterial suspension was added to achieve final peptide concentrations of 1.95–500 µg/mL. The mixture of bacterial suspension with culture medium and the mixture of purified elution buffer containing 250 mM imidazole with sterile medium served as positive and negative controls, respectively. Plates were incubated at 37°C for 6 h, followed by the addition of MTT and a further 3 h incubation at 37°C (Kumar et al.2018). Data Analysis All data were obtained from three independent experiments performed in triplicate and evaluated by one-way ANOVA analysis. The results were displayed as means ± the standard errors (SE). A p value of ≤ 0.05 was considered to be statistically significant. Results PCR Amplification and Sequence Analysis of the Target Gene The first-strand cDNA from the rainbow trout liver was used as the template for PCR amplification with primers Def-1 and Def-2, yielding a 161 bp cDNA fragment encoding the Defensin gene (Fig. 2 a). This fragment served as the template for a second PCR amplification using primers Def-F and Def-R, resulting in a 173 bp target gene containing the D4K cleavage site and a 6×His tag (Fig. 2 b). DNA sequencing confirmed the correct addition of the relevant restriction sites and the 6×His tag. The encoded peptide consists of 50 amino acid residues, including six highly conserved cysteine residues, which form three disulfide bonds to stabilize the protein structure and impart biological activity. The theoretical molecular weight of the Defensin protein, predicted by ExPASy software, is 5.48 kDa with a pI of 7.84. Identification of Recombinant Expression Vector and High-Copy Yeast Transformants Five single colonies with good growth were selected from ampicillin-resistant plates and subjected to colony PCR using primers Def-F and Def-R. The resulting PCR products were analyzed by 1.0% agarose gel electrophoresis, showing a single band at 173 bp (Fig. 3 b), corresponding to the expected size (Fig. 3 a), confirming the presence of the target gene in all selected clones. Positive clones were sent for sequencing, which confirmed successful insertion of the Defensin gene into the pPIC9K expression vector, with no mutations in the nucleotide sequence compared to the designed template (Fig. 3 c). The linearized pPIC9K-Defensin construct was introduced into competent Pichia pastoris GS115 cells, and transformants were selected on MD plates (His⁻). After 4 days, transformants were grew normally. The transformants were then plated on YPD plates containing G418 at concentrations of 0.5, 0.75, 1.0, 2.0, and 3.0 mg/mL to select out the high-copy yeast transformants. PCR using primers 3′AOX and 5′AOX confirmed successful integration of the target gene into the yeast genome, with a clear 640 bp band for positive clones and a 494 bp band for empty vector controls (Fig. 4 a). RT-PCR analysis of total RNA from positive clones confirmed expression of the target gene (Fig. 4 b). Expression, Optimization, and Identification of Recombinant Defensin One positive yeast transformant was selected for protein expression. After collecting and concentrating the culture supernatant by TCA precipitation, Tricine-SDS-PAGE analysis revealed a prominent protein band near 10 kDa in the supernatant of Pichia pastoris GS115 expressing pPIC9K-Defensin, whereas no such band was detected in the empty vector control (Fig. 5 a). The recombinant protein was subsequently purified by IMAC, yielding a single band near 10 kDa that was further analyzed by MALDI-TOF/TOF mass spectrometry. To optimize expression conditions, the effects of methanol concentration, incubation temperature, and induction time were evaluated. As shown in Fig. 5 , the use of 1.0% methanol resulted in the clearest band with minimal contamination by other proteins, indicating this as the optimal concentration. Similarly, expression occurred at all tested temperatures (26°C, 28°C, and 30°C), with the highest level observed at 30°C (Fig. 5 b). Time-course analysis from 24h to 168 h showed detectable expression at all time points, with the maximum yield obtained at 96 h and no significant increase beyond this time (Fig. 5 c). Considering the potential effects of excess Defensin on yeast growth and proteolytic degradation, the optimal induction period was determined to be between 96 and 120 h. HPLC analysis of the purified product revealed a retention time of 2.666 mins and a purity of 97.166% (Fig. 7 ). Tricine-SDS-PAGE of both the culture supernatant and purified protein consistently showed a distinct band near 10 kDa (Fig. 6 a, 6 b), and MALDI-TOF/TOF mass spectrometry analysis of the purified protein confirmed a peak at m/z 1019.05 corresponding to an N-terminal peptide fragment. This sequence matched the theoretical sequence, thereby verifying the identity of the purified product as recombinant Defensin (Fig. 8 ). Antimicrobial Activity of Recombinant Defensin Protein The antimicrobial activity of recombinant rt Defb3 was evaluated using the Oxford cup assay. Compared with the negative control (bacterial culture medium) and the positive control (Ampicillin), recombinant rt Defb3 exhibited clear inhibitory effects against a range of Gram-positive and Gram-negative bacteria. The inhibition zone diameters are shown in Figs. 9 . Specifically, rt Defb3 demonstrated inhibition zones of 1.71 ± 0.22 cm for Klebsiella pneumoniae , 1.98 ± 0.05 cm for Staphylococcus aureus , 1.48 ± 0.11 cm for Bacillus subtilis , 2.24 ± 0.13 cm for Bacillus cereus , 1.24 ± 0.13 cm for Escherichia coli , 3.11 ± 0.02 cm for Aeromonas hydrophila , 3.12 ± 0.02 cm for Salmonella spp ., 2.84 ± 0.07 cm for Pseudomonas aeruginosa , and 3.59 ± 0.15 cm for Trichoderma reesei , indicating strong antimicrobial activity. The minimum inhibitory concentrations (MIC) of rt Defb3 against eight bacterial strains were determined using a two-fold serial dilution method and are summarized in Table 2 . The peptide exhibited varying degrees of inhibitory activity against both Gram-positive and Gram-negative bacteria. Notably, Salmonella (MIC = 31.25 µg/mL) and Staphylococcus aureus (MIC = 15.63 µg/mL) showed particularly strong susceptibility, indicating a broad antimicrobial spectrum. Table 2 The minimum inhibitory concentrations of polypeptides against different micropathogens Microorganism MIC value(µg/ml) Defensin Eschericha coli ATCC 25923 > 500 Salmonella ATCC 12022 31.25 Aeromonas hydrophila ATCC 7966 31.25 Pseudomonas aeruginosa ATCC 27853 15.63 Klebsiella pneumoniae CMCC(B) 46117 125 Staphylococcus aureus ATCC 25923 15.63 Bacillus subtilis CMCC(B) 63501 500 Bacillus cereus CMCC(B) 63303 62.5 Discussion Widespread antibiotic use in aquaculture has led to the emergence of drug-resistant fish pathogens, raising concerns about environmental and food safety (Miller and Harbottle 2018 ; Madhulika et al. 2025 ). Consequently, there is growing interest in natural antimicrobial agents that do not leave residues or promote resistance, with the aim of reducing or replacing antibiotics in aquaculture (Okeke et al. 2022 ; Mohammed et al. 2025 ). β-Defensins, as endogenous defense molecules in teleost, represent logical candidates for such applications (Zou et al. 2007 ). This study provides the first experimental evidence for the antimicrobial activity of rainbow trout β-defensin 3 ( rt Defb3) produced in a recombinant expression system. Fish β-defensins are small (~ 5 kDa), highly cationic polypeptides that are traditionally difficult to obtain in heterologous hosts due to host cell toxicity, proteolytic degradation, or misfolding (Deng et al. 2017 ; Assoni et al. 2020 ; Luong et al. 2020 ). By employing P. pastoris with an optimized induction regime, we achieved substantial yields of correctly folded rt Defb3, confirming that this yeast is an effective host for disulfide-rich fish AMPs when coupled with a suitable secretion pathway. Systematic optimization of methanol concentration, induction temperature, and duration was critical for maximizing peptide production. The best yields were obtained at 1.0% methanol and 30°C after 4–5 days, whereas higher methanol concentrations or prolonged induction reduced productivity, likely due to yeast stress or protease-mediated degradation. These findings are consistent with previous reports describing optimal methanol feeding and time-dependent limitations in Pichia expression systems (Mayson et al. 2003 ; Hu et al. 2022 ). Our strategy, combining the α-factor signal peptide with controlled induction, enabled efficient secretion and oxidative folding of rt Defb3, thereby simplifying purification. This approach aligns with recent advances in K. phaffii expression engineering that emphasize optimized secretory pathways for improved yields (Zou et al. 2022 ; Karbalaei et al. 2020 ). Notably, the anomalous migration of rt Defb3 at ~ 10 kDa in SDS-PAGE, nearly double its theoretical mass, reflects its highly basic nature and reduced SDS binding, a phenomenon previously reported for other small cationic proteins (Schägger and von Jagow 1987 ; Peterkofsky 2013 ). MALDI-TOF/TOF analysis confirmed the identity of the peptide, validating that the observed band corresponded to authentic rt Defb3. Functionally, the recombinant rt Defb3 displayed potent broad-spectrum antimicrobial activity, inhibiting Gram-positive and Gram-negative bacteria as well as a filamentous fungus. Particularly noteworthy was its strong activity against Aeromonas hydrophila , a major fish pathogen, and Pseudomonas aeruginosa , a clinically relevant human pathogen. These results are consistent with the general mechanism of defensins, which interact with negatively charged microbial membranes and disrupt membrane integrity (Le et al. 2022 ; Gallo and Hooper 2012 ). This study therefore fills an important gap in the functional characterization of rainbow trout β-defensins and highlights their potential utility as natural antimicrobial agents in aquaculture (Das et al. 2022 ). Conclusion This study successfully cloned and expressed the rainbow trout Defensin gene in Pichia pastoris GS115 using the pPIC9K vector. The recombinant peptide was purified and identified by HPLC and MALDI-TOF/TOF mass spectrometry. Optimization of expression conditions revealed that the highest yield and stable protein quality were achieved at 30°C with 1.0% methanol induction for 96–120 hours. Functional assays demonstrated that the recombinant Defensin exhibited significant and broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria, as well as fungi. These findings suggest that recombinant fish-derived Defensin holds promise as an alternative to traditional antibiotics in aquaculture, particularly in addressing the issue of multi-drug resistance. Future studies should focus on large-scale fermentation, in vivo protective efficacy, and stability assessments under aquaculture conditions to further advance its practical application. Declarations Author contributions JC and BY conceived and designed research. JC, HW and SH conducted experiments. ML, DP and HW contributed new reagents or analytical tools. JC and SH analyzed data. JC wrote the manuscript. All authors read and approved the manuscript. Funding This work was funded by Bo Yao, Chongqing Municipal Technology Innovation and Application Development Special Top Level Project, CSTB2022TIAD-LDX0006 . Data availability The data sets generated or analyzed during this current study are included in this article. Ethical approval This study received ethical approval from the Animal Research Ethics Committee (AREC) of Chongqing University of Science and Technology, Chongqing China, under reference number CQUST/AREC/2024/137, granted on May 7, 2024. Furthermore, this article does not contain any studies with human participants or other animals performed by the authors. Conflicts of interest The authors declare no competing interest. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. References Zheng, S., Tu, Y., Li, B., Qu, G., Li, A., Peng, X., Li, S., & Shao, C. (2025). 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17:42:05","extension":"png","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":14639,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-7618669/v1/0e1ef32ba1a5750b080d3fc2.png"},{"id":93620030,"identity":"a9ab4d41-907c-403e-b815-a49e4ca2c593","added_by":"auto","created_at":"2025-10-15 17:42:05","extension":"xml","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":115098,"visible":true,"origin":"","legend":"","description":"","filename":"de8f5665c3384b5dbb445799e721e3771structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7618669/v1/7d42219ca019cd11375acbf6.xml"},{"id":93620028,"identity":"82142eaf-4451-4461-a094-cd93ff2f3db0","added_by":"auto","created_at":"2025-10-15 17:42:05","extension":"html","order_by":29,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":130267,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7618669/v1/313acdef59f67ae48672bb5d.html"},{"id":93619991,"identity":"42450b68-1e2a-4f78-af98-631b4d347a25","added_by":"auto","created_at":"2025-10-15 17:42:04","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":85952,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe multi-alignment result of Defensins sequences. The lines show the predicted disulfide-linked peptides.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7618669/v1/6f47d48e7e49ea87eee97ccb.jpg"},{"id":93620158,"identity":"a2f2117a-ce9b-478f-8b53-ad1bf685e830","added_by":"auto","created_at":"2025-10-15 17:50:04","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":28499,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAgarose gel electrophoresis of PCR-amplified target gene.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLane M, DL500 DNA ladder; lanes 1-2, first PCR amplification fragment (a); second PCR amplification fragment (b).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7618669/v1/cce1d2238dead0d08e0b70e9.jpg"},{"id":93619992,"identity":"feff0768-795a-45e4-b49b-9db3736ed49c","added_by":"auto","created_at":"2025-10-15 17:42:04","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":83059,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConstruction and identification of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ert\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eDefb3 vector.\u003c/strong\u003e\u003cbr\u003e\nConstruction of the \u003cem\u003ert\u003c/em\u003eDefb3 vector. (a)\u003cbr\u003e\nColony PCR analysis of \u003cem\u003ePichia pastoris\u003c/em\u003e transformants harboring pPIC9K-\u003cem\u003ert\u003c/em\u003eDefb3: lane M, DL500 DNA ladder; lanes 1–2, colony PCR products; RT-PCR result of \u003cem\u003eP. pastoris\u003c/em\u003e. (b) \u003cbr\u003e\nPartial chromatogram of DNA sequencing. (c)\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7618669/v1/2de0fde87f455f3afbbc6877.jpg"},{"id":93619994,"identity":"ce1e8110-eaf6-4e7a-a770-675f3dfc0201","added_by":"auto","created_at":"2025-10-15 17:42:04","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":28009,"visible":true,"origin":"","legend":"\u003cp\u003ePCR and RT-PCR identification of recombinant \u003cem\u003ePichia pastoris\u003c/em\u003e (\u003cem\u003eP. pastoris\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003ePCR analysis: Lane M, DL2000 DNA ladder; lanes 1-4, different yeast transformants; lane 5, yeast transformed with empty vector. (a)\u003c/p\u003e\n\u003cp\u003eRT-PCR analysis: Lane M, DL2000 DNA marker; lane 1, cDNA amplification product (275bp). (b)\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7618669/v1/9796f4f9cc23a2f57252b515.jpg"},{"id":93620912,"identity":"1746c762-8838-44bc-a2c0-e85d1e7cab13","added_by":"auto","created_at":"2025-10-15 17:58:04","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":62831,"visible":true,"origin":"","legend":"\u003cp\u003eTricine-SDS-PAGE analysis of recombinant strain products at different methanol concentrations (a), temperatures (b), and expression times (c). M: protein marker.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7618669/v1/a724b2d238b7cb447ada4f64.jpg"},{"id":93620001,"identity":"b8487fc8-dac5-430c-bfb1-65f368986c19","added_by":"auto","created_at":"2025-10-15 17:42:04","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":30895,"visible":true,"origin":"","legend":"\u003cp\u003eTricine-SDS-PAGE analysis of the purified products.\u003c/p\u003e\n\u003cp\u003eTricine-SDS-PAGE analysis of the purified products. M：protein marker. 1-2：Negative control pPIC9K-GS115 and GS115，3：Lowest expression 4：Highest expression 5-6：purified product. (b)\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7618669/v1/b784459e7d456cc9eeea9353.jpg"},{"id":93620000,"identity":"e10da364-8039-4fdc-9378-85fa7aafdb06","added_by":"auto","created_at":"2025-10-15 17:42:04","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":25968,"visible":true,"origin":"","legend":"\u003cp\u003eMALDI-TOF-TOF analysis for the purified Defensin\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7618669/v1/83bb31ba69119f223395ddee.jpg"},{"id":93620007,"identity":"58167b81-20dd-48f1-8452-ab32422430ad","added_by":"auto","created_at":"2025-10-15 17:42:04","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":17599,"visible":true,"origin":"","legend":"\u003cp\u003eAnalytical results of purified Defensin by HPLC\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7618669/v1/dab2bcbec0a95b97c376c4c5.jpg"},{"id":93622577,"identity":"35ddb26c-8d55-4944-9774-5426497bb303","added_by":"auto","created_at":"2025-10-15 18:14:04","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":80081,"visible":true,"origin":"","legend":"\u003cp\u003eAntibacterial activity of the recombinant Defensin\u003c/p\u003e\n\u003cp\u003eImage of the zone of inhibition (a); Bar chart showing the diameter of the zone of inhibition. (b)\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7618669/v1/7d44070cc57011796fc6ba74.jpg"},{"id":107351012,"identity":"cde56f45-7933-4785-8db8-c29c035771d7","added_by":"auto","created_at":"2026-04-20 16:07:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1038181,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7618669/v1/89035e8c-3e1f-4648-bb94-47e1e8fa6cba.pdf"},{"id":93620032,"identity":"509a7bcb-0c69-4917-bb6d-058ef53daaef","added_by":"auto","created_at":"2025-10-15 17:42:05","extension":"zip","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":21983707,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfile.zip","url":"https://assets-eu.researchsquare.com/files/rs-7618669/v1/9f00ae7af351cdefed7ff65c.zip"},{"id":93621672,"identity":"34a23b91-c91d-4189-9394-853f388d82e2","added_by":"auto","created_at":"2025-10-15 18:06:04","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":10454418,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.docx","url":"https://assets-eu.researchsquare.com/files/rs-7618669/v1/a54b77f6016afe0c1b57fdb7.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eOptimized Secretory Expression, Purification, and Antibacterial Activity Evaluation of Disulfide-Rich Rainbow Trout β-Defensin 3 in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePichia pastoris\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","fulltext":[{"header":"Key points","content":"\u003cul start=\"50\"\u003e\n \u003cli\u003e\u003cem\u003eFirst successful recombinant production of rainbow trout \u0026beta;-defensin 3 in \u003cem\u003ePichia pastoris\u003c/em\u003e via optimized induction strategy.\u003c/em\u003e\u003c/li\u003e\n \u003cli\u003e\u003cem\u003eHigh-yield secretory expression (~7 mg/mL) with \u0026gt;90% purity confirmed by HPLC and biochemical analysis.\u003c/em\u003e\u003c/li\u003e\n \u003cli\u003e\u003cem\u003eRecombinant rtDefb3 displayed potent broad-spectrum antibacterial activity, offering a repeatable and scalable system and functional validation.\u003c/em\u003e\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eThe rapid rise of antimicrobial-resistant (AMR) pathogens has become a critical global threat to human health, creating an urgent need for alternative strategies to traditional antibiotics (Zheng et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In 2021 alone, more than one million deaths were attributed to antibiotic-resistant bacterial infections, and this burden continues to increase (Charani et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Antimicrobial peptides (AMPs), as key effectors of the innate immune system, have attracted considerable attention due to their broad-spectrum antimicrobial activity, immunomodulatory properties, and lower propensity to induce resistance. These features make AMPs promising candidates for novel antimicrobial therapeutics (Mahlapuu et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Le et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Mwangi et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Numerous recent reviews have summarized AMP biological functions, mechanisms of action, and the challenges and opportunities in clinical translation (Magana et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Mookherjee et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eβ-Defensins are a conserved class of small cationic AMPs characterized by six cysteine residues forming three intramolecular disulfide bonds (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). They are widely distributed in vertebrates and function in both direct antimicrobial defense and immunomodulation (Ganz \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Das et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In teleost, β-defensins contribute to mucosal immunity; for example, certain homologues from zebrafish and tilapia exhibit measurable antimicrobial activity (Dong et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In rainbow trout (\u003cem\u003eOncorhynchus mykiss\u003c/em\u003e), four β-defensin genes (\u003cem\u003eom\u003c/em\u003eBD-1 to \u003cem\u003eom\u003c/em\u003eBD-4) have been identified (Casadei et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Previous studies characterized their cDNA sequences and tissue expression profiles, with \u003cem\u003eom\u003c/em\u003eBD-1 showing antiviral activity against viral hemorrhagic septicemia virus (VHSV) (Falco et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). However, \u003cem\u003eom\u003c/em\u003eBD-2, \u003cem\u003eom\u003c/em\u003eBD-3, and \u003cem\u003eom\u003c/em\u003eBD-4 have mainly been investigated at the sequence and expression level (Casadei et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Falco et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). To date, there has been no direct experimental evidence confirming the antimicrobial activity of rainbow trout β-defensin 3 (\u003cem\u003ert\u003c/em\u003eDefb3, also known as \u003cem\u003eom\u003c/em\u003eBD-3).\u003c/p\u003e\u003cp\u003eProducing bioactive disulfide-rich polypeptides in heterologous systems remains challenging. Bacterial expression often results in misfolded products or inclusion bodies due to the reductive cytoplasm, whereas chemical synthesis of cysteine-rich polypeptides is costly and impractical at scale. Eukaryotic secretory expression systems such as \u003cem\u003ePichia pastoris\u003c/em\u003e (recently reclassified as \u003cem\u003eKomagataella phaffii\u003c/em\u003e) provide advantages for these polypeptides, as they allow proper oxidative folding, post-translational modifications, and efficient secretion into the culture medium, thereby simplifying purification. However, high-yield production of correctly folded polypeptides in \u003cem\u003eP. pastoris\u003c/em\u003e requires careful optimization of the expression construct and culture conditions (Lv and Cai \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Critical factors include the choice of signal peptide, methanol induction level, temperature, and induction duration, which together determine the yield of soluble and bioactive product.\u003c/p\u003e\u003cp\u003eIn this study, we cloned the cDNA of mature \u003cem\u003ert\u003c/em\u003eDefb3 and expressed it in \u003cem\u003eP. pastoris\u003c/em\u003e GS115 using the pPIC9K vector containing the yeast α-mating factor signal peptide to direct secretion. Methanol concentration, induction temperature, and induction time were systematically optimized to obtain maximum production of correctly folded \u003cem\u003ert\u003c/em\u003eDefb3. The secreted recombinant polypeptide was purified and subjected to antimicrobial assays against a panel of Gram-positive and Gram-negative bacteria. We report, for the first time, the successful recombinant production of \u003cem\u003ert\u003c/em\u003eDefb3 and demonstrate its potent broad-spectrum antimicrobial activity. This provides critical functional evidence for a fish β-defensin. Furthermore, our strategy-combining a secretion signal with systematic induction optimization-proved effective for producing an active, disulfide-rich polypeptide in a eukaryotic system. This approach offers a reproducible framework for scalable production of cysteine-rich AMPs (Karbalaei et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and is consistent with recent advances in \u003cem\u003eP. pastoris\u003c/em\u003e secretion engineering that enhance yield and economic feasibility (Lv and Cai \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eExperimental Animals, Strains, and Vectors\u003c/h2\u003e\u003cp\u003eTotal RNA was isolated from the liver of rainbow trout (maintained in our laboratory) and subsequently reverse-transcribed into cDNA. \u003cem\u003ePichia pastoris\u003c/em\u003e GS115 (Shanghai RuiChu Biotechnology Co., Ltd.) was employed as the expression host, and the vector pPIC9K (Chongqing Tsingke Biotechnology Co., Ltd., China) was used for construction of the recombinant plasmid. The antimicrobial activity of the purified peptide was evaluated against the following test strains: \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 25923, \u003cem\u003eBacillus subtilis\u003c/em\u003e CMCC (B) 63501, \u003cem\u003eBacillus cereus\u003c/em\u003e CMCC (B) 63303, \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC 25923, \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e CMCC (B) 46117, \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e ATCC 27853, \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e ATCC 7966, \u003cem\u003eSalmonella\u003c/em\u003e ATCC 12022, and \u003cem\u003eTrichoderma reesei\u003c/em\u003e RUT-C30 (maintained in our laboratory).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eReagents and Equipment\u003c/h3\u003e\n\u003cp\u003eThe mRNA RT SuperMix reverse transcription kit (SynScript\u0026reg; III) was purchased from Tsingke Biotechnology Co., Ltd. (Beijing, China). The DNA gel extraction kit, yeast RNA extraction kit, and DNA marker A (25\u0026ndash;500 bp) were obtained from Sangon Biotech Co., Ltd. (Shanghai, China). Restriction enzymes \u003cem\u003eEcoR\u003c/em\u003e I, \u003cem\u003eNot\u003c/em\u003e I, and \u003cem\u003eSac\u003c/em\u003e I-HF were purchased from New England Biolabs (NEB, USA). The DL2000 DNA marker and DNA ligation kit were purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China). Low molecular weight pre-stained protein marker was obtained from Thermo Fisher Scientific (USA). All other reagents were of analytical grade and sourced domestically in China. The A200 thermal cycler was provided by Hangzhou Long Gene Scientific Instrument Co., Ltd. (Hangzhou, China). The UV-Vis spectrophotometer (Model 6132) was supplied by Eppendorf (Germany). The protein electrophoresis system was provided by Bio-Rad (USA). The gel imaging system (Model 1100/26M) was purchased from Quantum ST4 (USA). The incubator shaker (Model ZQTY-70N) was supplied by Zhichu Instrument Co., Ltd. (Shanghai, China). The high-performance liquid chromatography (HPLC) system (Model LC5090) was provided by Fuli Analytical Instruments Co., Ltd. (Zhejiang, China).\u003c/p\u003e\n\u003ch3\u003eConstruction of the Expression Vector\u003c/h3\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003eSynthesis of the First-Strand cDNA\u003c/h2\u003e\u003cp\u003eTotal mRNA was extracted from the liver of rainbow trout and reverse-transcribed into cDNA using the SynScript\u0026reg; III miRNA RT SuperMix reverse transcription kit (Tsingke Biotechnology Co., Ltd., Beijing, China). The reverse transcription reaction was performed with 10 ng\u003csup\u003e\u0026ndash;1\u003c/sup\u003e \u0026micro;g of mRNA as the template, 2 \u0026micro;L of RTase Mix, 10 \u0026micro;L of 2\u0026times; Reaction Buffer, and RNase-free water to a final volume of 20 \u0026micro;L. The mixture was incubated at 37\u0026deg;C for 60 minutes and then treated at 85\u0026deg;C for 5 minutes. The resulting first-strand cDNA was stored at -20\u0026deg;C.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCloning of the cDNA Encoding the D4K and 6×His Tagged Target Gene\u003c/h3\u003e\n\u003cp\u003eTo express the antimicrobial peptide with a native N-terminal, a D4K protease cleavage site was added at the N-terminus, and a 6\u0026times;His tag was introduced at the C-terminus for purification purposes. The first-strand cDNA was used as the template, and specific primers Def-1 (forward) and Def-2 (reverse) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) were designed for PCR amplification of the \u003cem\u003ert\u003c/em\u003eDefb3 cDNA fragment. The PCR reaction mixture and conditions were as follows: 5 \u0026micro;L of 10\u0026times; PCR Buffer, 4 \u0026micro;L of dNTPs (2.5 mmol/L), 1 \u0026micro;L each of forward and reverse primers, 0.2 \u0026micro;L of first-strand cDNA, 0.5 \u0026micro;L of \u003cem\u003ePfu\u003c/em\u003e DNA Polymerase, and RNase-free water to a total volume of 50 \u0026micro;L. The PCR conditions were: 94\u0026deg;C for 4 minutes, followed by 38 cycles of 94\u0026deg;C for 30 seconds, 55\u0026deg;C for 40 seconds, and 72\u0026deg;C for 20 seconds, with a final extension at 72\u0026deg;C for 10 minutes. The amplified product was cloned into the pMD19-T simple vector and transformed into \u003cem\u003eEscherichia coli\u003c/em\u003e DH5α. Positive clones were selected and sequenced by Shanghai Sangon Biotechnology Co., Ltd. The cDNA fragment was further amplified using primers Def-F (forward) and Def-R (reverse) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), introducing an \u003cem\u003eEcoR\u003c/em\u003e I site at the 5\u0026prime; end and a 6\u0026times;His tag, stop codon, and \u003cem\u003eNot\u003c/em\u003e I site at the 3\u0026prime; end. The PCR reaction was carried out under the same conditions, and the amplified target gene was confirmed by sequencing at Tsingke Biotechnology Co., Ltd. The molecular weight and isoelectric point of the encoded protein were predicted using ExPASy software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://web.expasy.org/compute.pi/\u003c/span\u003e\u003cspan address=\"http://web.expasy.org/compute.pi/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePrimer sequences\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePrimer name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePrimer sequence (5\u0026rsquo;-3\u0026rsquo;)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSize (bp)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDef-1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGCGAATTCATGTCTCTACACTTATGTTTCATTAGTGG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDef-2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eATTCGCGGCCGCCATCATCACCATCACCACTCATTTACAGCATACATTCGGCCATG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e56\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDef-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCGGAATTCGACGACGATGACAAGTCTCTACACTTATGTTTCATTAGTGGGGG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e52\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDef-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eATTCGCGGCCGCTTAGTGATGGTGATGGTGGTGTTTACAGCATACATTCGGCCATGTAC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e59\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3'AOX1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGCAAATGGCATTCTGACATCC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5'AOX1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGACTGGTTCCAATTGACAAGC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eConstruction of the Recombinant Expression Vector and Transformation into\u003c/b\u003e \u003cb\u003ePichia pastoris\u003c/b\u003e \u003cb\u003eGS115\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe pMD19-T-Defensin plasmid was double-digested with \u003cem\u003eEcoR\u003c/em\u003e I and \u003cem\u003eNot\u003c/em\u003e I to obtain the target gene fragment. The fragment was then ligated into the pPIC9K vector (pre-digested with the same restriction enzymes) using 5 \u0026micro;L of ligation mix and incubated at 16\u0026deg;C for 30 minutes. The ligation reaction product was transformed into \u003cem\u003eEscherichia coli\u003c/em\u003e DH5α competent cells, and positive clones were confirmed by colony PCR and sequencing. The verified pPIC9K-Defensin plasmid was digested with \u003cem\u003eSac\u003c/em\u003e I-HF and transformed into \u003cem\u003ePichia pastoris\u003c/em\u003e GS115 competent cells using the Yeast Transformation Kit (Biyuntian, China), with a 1:6 (v/v) transformation ratio. An empty pPIC9K vector was also transformed into \u003cem\u003ePichia pastoris\u003c/em\u003e GS115 as a negative control.\u003c/p\u003e\u003cp\u003e\u003cb\u003eScreening of High-Copy\u003c/b\u003e \u003cb\u003ePichia pastoris\u003c/b\u003e \u003cb\u003eTransformants\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe transformed yeast solution was plated on MD plates (15 g/L agar, 13.4 g/L YNB, 22 g/L glucose, 0.07 g/L biotin) and incubated at 30\u0026deg;C for 3\u0026ndash;4 days. Single colonies were selected, resuspended in sterile water, and plated on YPD plates containing geneticin at final concentrations of 0.5, 0.75, 1, 2, and 3 mg/mL. The plates were incubated for 2\u0026ndash;5 days, and high-copy transformants utilizing methanol were screened. Genomic DNA was extracted from the selected yeast transformants, and PCR was performed using the universal primers 5\u0026prime;AOX1 and 3\u0026prime;AOX1 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) for confirmation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eExpression of the Target Protein in\u003c/b\u003e \u003cb\u003ePichia pastoris\u003c/b\u003e \u003cb\u003eGS115 and Optimization of Expression Conditions\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe PCR-confirmed yeast transformants were inoculated into 5 mL YPD medium and cultured overnight at 30\u0026deg;C, 250 rpm, until the OD\u003csub\u003e600\u003c/sub\u003e reached 0.6\u0026ndash;0.8. One milliliter of the culture was then transferred into 100 mL BMGY medium (10 g/L yeast extract, 20 g/L peptone, 13.4 g/L YNB, 2 mL/L 500\u0026times; biotin, 2 mL/L 50% glycerol, 100 mmol/L potassium phosphate buffer, pH 6.0) and cultured for 24 hours under the same conditions. The culture was then shifted to BMMY medium (same formulation as BMGY, except for 5 mL/L methanol replacing glycerol) and cultured at 30\u0026deg;C, 250 rpm for 72 hours. Methanol was added every 24 hours to a final concentration of 0.5%. The culture supernatant was collected, concentrated by TCA precipitation, and subsequently analyzed by Tricine-SDS-PAGE. Electrophoresis was carried out using polyacrylamide gels with concentrations of 4%, 10%, and 16% for the stacking, intermediate, and separating layers, respectively. In parallel, the methanol concentration in BMMY medium was adjusted to 0.5%, 0.75%, 1.0%, and 1.25%, while the cultivation temperature was set to 26\u0026deg;C, 28\u0026deg;C, and 30\u0026deg;C. Yeast transformants carrying pPIC9K-Defensin were cultured under these conditions to determine the optimal methanol concentration and expression temperature. Based on the obtained results, the same transformants were further incubated for different induction periods to identify the optimal expression time.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003ePurification and Characterization of the Recombinant Protein\u003c/h2\u003e\u003cp\u003eThe selected pPIC9K-Defensin transformants were cultured in 500 mL of the optimized medium. After centrifugation, the supernatant was mixed with an equal volume of Binding Buffer (5 mmol/L imidazole) and applied to a column. The column was washed with 15 column volumes of Binding Buffer to remove unbound proteins, followed by elution with Elution Buffer (25 mmol/L imidazole) until the absorbance returned to the baseline of 280 nm. The eluted protein was analyzed by HPLC. The HPLC system used a C\u003csub\u003e18\u003c/sub\u003e column (4.6\u0026times;250 mm, 5 \u0026micro;m) with pure acetonitrile (Pump B) and ultrapure water (Pump C) as mobile phases, at a flow rate of 1 mL/min and a column temperature of 32\u0026deg;C. The injection volume was 10 \u0026micro;L, and detection was performed at 214 nm. The purified product was analyzed by Tricine\u0026ndash;SDS\u0026ndash;PAGE, and the target band was excised for subsequent MALDI-TOF/TOF mass spectrometric analysis.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eAntimicrobial Activity Evaluation of the Recombinant Protein\u003c/h3\u003e\n\u003cp\u003eThe antimicrobial activity and spectrum of recombinant \u003cem\u003ert\u003c/em\u003eDefb3 were evaluated using agar diffusion method and microdilution assays. Laboratory-maintained Gram-positive, Gram-negative bacteria, and fungi were selected as test strains: \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (ATCC 25923), \u003cem\u003eBacillus subtilis\u003c/em\u003e (CMCC(B) 63501), \u003cem\u003eBacillus cereus\u003c/em\u003e (CMCC(B) 63303), \u003cem\u003eEscherichia coli\u003c/em\u003e (ATCC 25923), \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e (CMCC(B) 46117), \u003cem\u003eAeromonas hydrophila\u003c/em\u003e (ATCC 7966), \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (ATCC 27853), \u003cem\u003eSalmonella\u003c/em\u003e (ATCC 12022), and \u003cem\u003eTrichoderma reesei\u003c/em\u003e (RUT-C30).\u003c/p\u003e\u003cp\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 25923, \u003cem\u003eBacillus subtilis\u003c/em\u003e CMCC (B) 63501, \u003cem\u003eBacillus cereus\u003c/em\u003e CMCC (B) 63303, \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC 25923, \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e CMCC (B) 46117, \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e ATCC 27853, \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e ATCC 7966, \u003cem\u003eSalmonella\u003c/em\u003e ATCC 12022, and \u003cem\u003eTrichoderma reesei\u003c/em\u003e Rut-C30 (maintained in our laboratory).\u003c/p\u003e\u003cp\u003eFor the agar diffusion assay, 2% agar medium was poured into plates and allowed to solidify. Oxford cups were placed on the solidified medium, and bacterial suspensions were prepared at 1\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e CFU/mL and inoculated into the agar at 1% (v/v). After thorough mixing and solidification, Oxford cups were removed, and 100 \u0026micro;L of purified peptide solution was added to each well. Plates were incubated at 37\u0026deg;C overnight, and inhibition zones were measured using a caliper. Experiments were performed in triplicate, and mean values were reported.\u003c/p\u003e\u003cp\u003eFor MIC determination, logarithmic-phase bacterial cultures were diluted to 1\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e CFU/mL. Purified \u003cem\u003ert\u003c/em\u003eDefb3 was serially diluted two-fold in 96-well plates, and 50 \u0026micro;L of bacterial suspension was added to achieve final peptide concentrations of 1.95\u0026ndash;500 \u0026micro;g/mL. The mixture of bacterial suspension with culture medium and the mixture of purified elution buffer containing 250 mM imidazole with sterile medium served as positive and negative controls, respectively. Plates were incubated at 37\u0026deg;C for 6 h, followed by the addition of MTT and a further 3 h incubation at 37\u0026deg;C (Kumar et al.2018).\u003c/p\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eData Analysis\u003c/h2\u003e\u003cp\u003eAll data were obtained from three independent experiments performed in triplicate and evaluated by one-way ANOVA analysis. The results were displayed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;the standard errors (SE). A p value of \u0026le;\u0026thinsp;0.05 was considered to be statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003ePCR Amplification and Sequence Analysis of the Target Gene\u003c/h2\u003e\u003cp\u003eThe first-strand cDNA from the rainbow trout liver was used as the template for PCR amplification with primers Def-1 and Def-2, yielding a 161 bp cDNA fragment encoding the Defensin gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). This fragment served as the template for a second PCR amplification using primers Def-F and Def-R, resulting in a 173 bp target gene containing the D4K cleavage site and a 6\u0026times;His tag (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). DNA sequencing confirmed the correct addition of the relevant restriction sites and the 6\u0026times;His tag. The encoded peptide consists of 50 amino acid residues, including six highly conserved cysteine residues, which form three disulfide bonds to stabilize the protein structure and impart biological activity. The theoretical molecular weight of the Defensin protein, predicted by ExPASy software, is 5.48 kDa with a pI of 7.84.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eIdentification of Recombinant Expression Vector and High-Copy Yeast Transformants\u003c/h2\u003e\u003cp\u003eFive single colonies with good growth were selected from ampicillin-resistant plates and subjected to colony PCR using primers Def-F and Def-R. The resulting PCR products were analyzed by 1.0% agarose gel electrophoresis, showing a single band at 173 bp (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), corresponding to the expected size (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), confirming the presence of the target gene in all selected clones. Positive clones were sent for sequencing, which confirmed successful insertion of the Defensin gene into the pPIC9K expression vector, with no mutations in the nucleotide sequence compared to the designed template (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). The linearized pPIC9K-Defensin construct was introduced into competent \u003cem\u003ePichia pastoris\u003c/em\u003e GS115 cells, and transformants were selected on MD plates (His⁻). After 4 days, transformants were grew normally. The transformants were then plated on YPD plates containing G418 at concentrations of 0.5, 0.75, 1.0, 2.0, and 3.0 mg/mL to select out the high-copy yeast transformants. PCR using primers 3\u0026prime;AOX and 5\u0026prime;AOX confirmed successful integration of the target gene into the yeast genome, with a clear 640 bp band for positive clones and a 494 bp band for empty vector controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). RT-PCR analysis of total RNA from positive clones confirmed expression of the target gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eExpression, Optimization, and Identification of Recombinant Defensin\u003c/h2\u003e\u003cp\u003eOne positive yeast transformant was selected for protein expression. After collecting and concentrating the culture supernatant by TCA precipitation, Tricine-SDS-PAGE analysis revealed a prominent protein band near 10 kDa in the supernatant of \u003cem\u003ePichia pastoris\u003c/em\u003e GS115 expressing pPIC9K-Defensin, whereas no such band was detected in the empty vector control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). The recombinant protein was subsequently purified by IMAC, yielding a single band near 10 kDa that was further analyzed by MALDI-TOF/TOF mass spectrometry. To optimize expression conditions, the effects of methanol concentration, incubation temperature, and induction time were evaluated. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e, the use of 1.0% methanol resulted in the clearest band with minimal contamination by other proteins, indicating this as the optimal concentration. Similarly, expression occurred at all tested temperatures (26\u0026deg;C, 28\u0026deg;C, and 30\u0026deg;C), with the highest level observed at 30\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Time-course analysis from 24h to 168 h showed detectable expression at all time points, with the maximum yield obtained at 96 h and no significant increase beyond this time (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Considering the potential effects of excess Defensin on yeast growth and proteolytic degradation, the optimal induction period was determined to be between 96 and 120 h. HPLC analysis of the purified product revealed a retention time of 2.666 mins and a purity of 97.166% (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Tricine-SDS-PAGE of both the culture supernatant and purified protein consistently showed a distinct band near 10 kDa (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eb), and MALDI-TOF/TOF mass spectrometry analysis of the purified protein confirmed a peak at m/z 1019.05 corresponding to an N-terminal peptide fragment. This sequence matched the theoretical sequence, thereby verifying the identity of the purified product as recombinant Defensin (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eAntimicrobial Activity of Recombinant Defensin Protein\u003c/h2\u003e\u003cp\u003eThe antimicrobial activity of recombinant \u003cem\u003ert\u003c/em\u003eDefb3 was evaluated using the Oxford cup assay. Compared with the negative control (bacterial culture medium) and the positive control (Ampicillin), recombinant \u003cem\u003ert\u003c/em\u003eDefb3 exhibited clear inhibitory effects against a range of Gram-positive and Gram-negative bacteria. The inhibition zone diameters are shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e9\u003c/span\u003e. Specifically, \u003cem\u003ert\u003c/em\u003eDefb3 demonstrated inhibition zones of 1.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22 cm for \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e, 1.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 cm for \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, 1.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 cm for \u003cem\u003eBacillus subtilis\u003c/em\u003e, 2.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 cm for \u003cem\u003eBacillus cereus\u003c/em\u003e, 1.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 cm for \u003cem\u003eEscherichia coli\u003c/em\u003e, 3.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 cm for \u003cem\u003eAeromonas hydrophila\u003c/em\u003e, 3.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 cm for \u003cem\u003eSalmonella spp\u003c/em\u003e., 2.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 cm for \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, and 3.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 cm for \u003cem\u003eTrichoderma reesei\u003c/em\u003e, indicating strong antimicrobial activity.\u003c/p\u003e\u003cp\u003eThe minimum inhibitory concentrations (MIC) of \u003cem\u003ert\u003c/em\u003eDefb3 against eight bacterial strains were determined using a two-fold serial dilution method and are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The peptide exhibited varying degrees of inhibitory activity against both Gram-positive and Gram-negative bacteria. Notably, \u003cem\u003eSalmonella\u003c/em\u003e (MIC\u0026thinsp;=\u0026thinsp;31.25 \u0026micro;g/mL) and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MIC\u0026thinsp;=\u0026thinsp;15.63 \u0026micro;g/mL) showed particularly strong susceptibility, indicating a broad antimicrobial spectrum.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe minimum inhibitory concentrations of polypeptides against different micropathogens\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eMicroorganism\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e\u003cp\u003eMIC value(\u0026micro;g/ml)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDefensin\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eEschericha coli\u003c/em\u003e ATCC 25923\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eSalmonella\u003c/em\u003e ATCC 12022\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e31.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eAeromonas hydrophila\u003c/em\u003e ATCC 7966\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e31.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e ATCC 27853\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e15.63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e CMCC(B) 46117\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e125\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 25923\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e15.63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eBacillus subtilis\u003c/em\u003e CMCC(B) 63501\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eBacillus cereus\u003c/em\u003e CMCC(B) 63303\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e62.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eWidespread antibiotic use in aquaculture has led to the emergence of drug-resistant fish pathogens, raising concerns about environmental and food safety (Miller and Harbottle \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Madhulika et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Consequently, there is growing interest in natural antimicrobial agents that do not leave residues or promote resistance, with the aim of reducing or replacing antibiotics in aquaculture (Okeke et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Mohammed et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). β-Defensins, as endogenous defense molecules in teleost, represent logical candidates for such applications (Zou et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThis study provides the first experimental evidence for the antimicrobial activity of rainbow trout β-defensin 3 (\u003cem\u003ert\u003c/em\u003eDefb3) produced in a recombinant expression system. Fish β-defensins are small (~\u0026thinsp;5 kDa), highly cationic polypeptides that are traditionally difficult to obtain in heterologous hosts due to host cell toxicity, proteolytic degradation, or misfolding (Deng et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Assoni et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Luong et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). By employing \u003cem\u003eP. pastoris\u003c/em\u003e with an optimized induction regime, we achieved substantial yields of correctly folded \u003cem\u003ert\u003c/em\u003eDefb3, confirming that this yeast is an effective host for disulfide-rich fish AMPs when coupled with a suitable secretion pathway.\u003c/p\u003e\u003cp\u003eSystematic optimization of methanol concentration, induction temperature, and duration was critical for maximizing peptide production. The best yields were obtained at 1.0% methanol and 30\u0026deg;C after 4\u0026ndash;5 days, whereas higher methanol concentrations or prolonged induction reduced productivity, likely due to yeast stress or protease-mediated degradation. These findings are consistent with previous reports describing optimal methanol feeding and time-dependent limitations in Pichia expression systems (Mayson et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Hu et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOur strategy, combining the α-factor signal peptide with controlled induction, enabled efficient secretion and oxidative folding of \u003cem\u003ert\u003c/em\u003eDefb3, thereby simplifying purification. This approach aligns with recent advances in \u003cem\u003eK. phaffii\u003c/em\u003e expression engineering that emphasize optimized secretory pathways for improved yields (Zou et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Karbalaei et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Notably, the anomalous migration of \u003cem\u003ert\u003c/em\u003eDefb3 at ~\u0026thinsp;10 kDa in SDS-PAGE, nearly double its theoretical mass, reflects its highly basic nature and reduced SDS binding, a phenomenon previously reported for other small cationic proteins (Sch\u0026auml;gger and von Jagow \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Peterkofsky \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). MALDI-TOF/TOF analysis confirmed the identity of the peptide, validating that the observed band corresponded to authentic \u003cem\u003ert\u003c/em\u003eDefb3.\u003c/p\u003e\u003cp\u003eFunctionally, the recombinant \u003cem\u003ert\u003c/em\u003eDefb3 displayed potent broad-spectrum antimicrobial activity, inhibiting Gram-positive and Gram-negative bacteria as well as a filamentous fungus. Particularly noteworthy was its strong activity against \u003cem\u003eAeromonas hydrophila\u003c/em\u003e, a major fish pathogen, and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, a clinically relevant human pathogen. These results are consistent with the general mechanism of defensins, which interact with negatively charged microbial membranes and disrupt membrane integrity (Le et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Gallo and Hooper \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). This study therefore fills an important gap in the functional characterization of rainbow trout β-defensins and highlights their potential utility as natural antimicrobial agents in aquaculture (Das et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study successfully cloned and expressed the rainbow trout Defensin gene in \u003cem\u003ePichia pastoris\u003c/em\u003e GS115 using the pPIC9K vector. The recombinant peptide was purified and identified by HPLC and MALDI-TOF/TOF mass spectrometry. Optimization of expression conditions revealed that the highest yield and stable protein quality were achieved at 30\u0026deg;C with 1.0% methanol induction for 96\u0026ndash;120 hours. Functional assays demonstrated that the recombinant Defensin exhibited significant and broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria, as well as fungi. These findings suggest that recombinant fish-derived Defensin holds promise as an alternative to traditional antibiotics in aquaculture, particularly in addressing the issue of multi-drug resistance. Future studies should focus on large-scale fermentation, in vivo protective efficacy, and stability assessments under aquaculture conditions to further advance its practical application.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJC and BY conceived and designed research. JC, HW and SH conducted experiments. ML, DP and HW contributed new reagents or analytical tools. JC and SH analyzed data. JC wrote the manuscript. All authors read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis work was funded by \u003cstrong\u003eBo Yao, Chongqing Municipal Technology Innovation and Application Development Special Top Level Project, CSTB2022TIAD-LDX0006\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data sets generated or analyzed during this current study are included in this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study received ethical approval from the Animal Research Ethics Committee (AREC) of Chongqing University of Science and Technology, Chongqing China, under reference number CQUST/AREC/2024/137, granted on May 7, 2024. Furthermore, this article does not contain any studies with human participants or other animals performed by the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOpen Access\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003c/strong\u003eThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article\u0026rsquo;s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article\u0026rsquo;s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZheng, S., Tu, Y., Li, B., Qu, G., Li, A., Peng, X., Li, S., \u0026amp; Shao, C. (2025). Antimicrobial peptide biological activity, delivery systems and clinical translation status and challenges. \u003cem\u003eJournal of translational medicine\u003c/em\u003e, \u003cem\u003e23\u003c/em\u003e(1), 292. https://doi.org/10.1186/s12967-025-06321-9\u003c/li\u003e\n\u003cli\u003eCharani, E., McKee, M., Balasegaram, M., Mendelson, M., Singh, S., \u0026amp; Holmes, A. H. (2022). Global burden of antimicrobial resistance: essential pieces of a global puzzle. 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Epithelial antimicrobial defence of the skin and intestine. \u003cem\u003eNature reviews. Immunology\u003c/em\u003e, \u003cem\u003e12\u003c/em\u003e(7), 503\u0026ndash;516.https://doi.org/10.1038/nri3228\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"β-Defensin, Rainbow trout (Oncorhynchus mykiss), Pichia pastoris, pPIC9K, Expression optimization, Antimicrobial activity","lastPublishedDoi":"10.21203/rs.3.rs-7618669/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7618669/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRainbow trout β-defensin 3 (\u003cem\u003ert\u003c/em\u003eDefb3) 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 \u003cem\u003ert\u003c/em\u003eDefb3coding sequence was cloned from trout liver cDNA and heterologously expressed in \u003cem\u003ePichia pastoris\u003c/em\u003e 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 \u0026gt;90% purity using one-step Ni-IDA affinity chromatography. The identity and purity of the recombinant \u003cem\u003ert\u003c/em\u003eDefb3 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 \u003cem\u003ert\u003c/em\u003eDefb3in \u003cem\u003eP. pastoris\u003c/em\u003e, establishing a scalable expression platform for fish β-defensins. The recombinant \u003cem\u003ert\u003c/em\u003eDefb3peptide holds promise as a natural antimicrobial agent for aquaculture and potential therapeutic applications, addressing the urgent demand for alternatives to conventional antibiotics.\u003c/p\u003e","manuscriptTitle":"Optimized Secretory Expression, Purification, and Antibacterial Activity Evaluation of Disulfide-Rich Rainbow Trout β-Defensin 3 in Pichia pastoris","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-15 17:41:59","doi":"10.21203/rs.3.rs-7618669/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0b74b230-f116-4c2d-975b-65739fd7114d","owner":[],"postedDate":"October 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-20T16:05:30+00:00","versionOfRecord":{"articleIdentity":"rs-7618669","link":"https://doi.org/10.1007/s00253-025-13667-z","journal":{"identity":"applied-microbiology-and-biotechnology","isVorOnly":false,"title":"Applied Microbiology and Biotechnology"},"publishedOn":"2026-04-16 15:59:30","publishedOnDateReadable":"April 16th, 2026"},"versionCreatedAt":"2025-10-15 17:41:59","video":"","vorDoi":"10.1007/s00253-025-13667-z","vorDoiUrl":"https://doi.org/10.1007/s00253-025-13667-z","workflowStages":[]},"version":"v1","identity":"rs-7618669","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7618669","identity":"rs-7618669","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}