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<p>In the gastro-intestinal tract, <i>Escherichia coli</i> (<i>E.coli</i>) infections can trigger programmed cell death of intestinal epithelial cells, through mechanisms such as oxidative stress and ferroptosis, which compromise gut barrier integrity. Given the rising prevalence of antibiotic-resistant <i>E. coli</i> strains, there is an urgent need to develop innovative antimicrobial therapies that go beyond conventional antibiotics. Antimicrobial peptides represent a promising alternative for combating resistant bacterial strains due to their dual role in antimicrobial activity and immune modulation. In this study, we constructed multiple expression cassettes to express porcine β-defensin 2 (PBD2)-derived peptide RH in <i>Pichia pastoris</i> (<i>P. pastoris</i>), purified the peptide using nickel column affinity chromatography, and assessed its in vivo and in vitro activity. The results indicated that under the optimal condition (3% methanol), the total secreted protein concentration reached 306.5&#xa0;mg/L after 120&#xa0;h of fermentation. Following purification, the yield of recombinant active peptide RH (rRH) can reached 59.34&#xa0;mg/L. The rRH exhibits strong antimicrobial activity and resistance to oxidation, and by inhibiting oxidative stress-mediated ferroptosis it reduces <i>E. coli</i>-induced cell death and injury in the jejunum. This dual functionality of rRH positions it as a potential therapeutic candidate for treating gastrointestinal infections and improving gut health, providing a crucial alternative to traditional antibiotics.</p>

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Production and Functional Evaluation of Recombinant Active Peptide RH in Pichia Pastoris: Protection Against Escherichia Coli Induced Cell Death

  • 期刊论文

  • 发表日期：

    2025年3月14日

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  • Lu Zhao,
  • Mingyang Hu,
  • Yuwen Li,
  • Jiaoyu Xin,
  • Yuxin Fang,
  • Chenyu Xue,
  • Na Dong

摘要

In the gastro-intestinal tract, Escherichia coli (E.coli) infections can trigger programmed cell death of intestinal epithelial cells, through mechanisms such as oxidative stress and ferroptosis, which compromise gut barrier integrity. Given the rising prevalence of antibiotic-resistant E. coli strains, there is an urgent need to develop innovative antimicrobial therapies that go beyond conventional antibiotics. Antimicrobial peptides represent a promising alternative for combating resistant bacterial strains due to their dual role in antimicrobial activity and immune modulation. In this study, we constructed multiple expression cassettes to express porcine β-defensin 2 (PBD2)-derived peptide RH in Pichia pastoris (P. pastoris), purified the peptide using nickel column affinity chromatography, and assessed its in vivo and in vitro activity. The results indicated that under the optimal condition (3% methanol), the total secreted protein concentration reached 306.5 mg/L after 120 h of fermentation. Following purification, the yield of recombinant active peptide RH (rRH) can reached 59.34 mg/L. The rRH exhibits strong antimicrobial activity and resistance to oxidation, and by inhibiting oxidative stress-mediated ferroptosis it reduces E. coli-induced cell death and injury in the jejunum. This dual functionality of rRH positions it as a potential therapeutic candidate for treating gastrointestinal infections and improving gut health, providing a crucial alternative to traditional antibiotics.