游离型与整合型共表达增强毕赤酵母表达抗菌肽的水平研究

Study on the enhancement of antimicrobial peptide expression levels in Pichia pastoris by co-expression of episomal and integrative vectors

中文关键词:  毕赤酵母  抗菌肽  游离型表达  整合型表达  共表达

英文关键词:Pichia pastoris  antimicrobial peptide  episomal expression  integrative expression  co-expression

中文摘要:

      目的 建立游离型与整合型载体共表达系统, 提高抗菌肽在毕赤酵母中的表达效率, 解决其因拷贝数限制和表达不稳定性导致的产量不足问题。方法 利用乳酸克鲁维酵母(Kluyveromyces lactis)来源的panARS自主复制序列, 构建卡那霉素抗性的游离型表达载体pPICKαA-panARS-JS21T和pGAPKαA-panARS-JS21T, 与整合型载体pPICZαA-JS21T共同电转化毕赤酵母X-33菌株。通过博来霉素Zeocin和遗传霉素G418双抗生素平板筛选阳性转化子, 摇瓶发酵120 h以上, 每24 h补加甲醇诱导表达。采用Bradford法测定分泌总蛋白含量, 三羟甲基甘氨酸-十二烷基硫酸钠聚丙烯酰胺凝胶电泳(trimethylglycine-sodium dodecyl sulfate polyacrylamide gel electrophoresis, Tricine-SDS-PAGE)检测目标条带, 琼脂扩散法测定对金黄色葡萄球菌的抑菌活性。结果 发酵120 h后, 整合型与游离型pGAPKαA-panARS-JS21T共表达组的上清总蛋白含量达到107.22 μg/mL, 比单一整合型表达组70.56 μg/mL提高了52%; 抑菌圈分析显示其抑菌活性提升129% (P<0.05)。整合型与pPICKαA-panARS-JS21T共表达组也比单一整合型组提升了15.8%。结论 游离型与整合型载体共表达策略可协同提高抗菌肽JS21T在毕赤酵母中的表达水平, 其效果优于单一载体系统, 为小分子肽类高效生产提供了新思路。

英文摘要:

      Objective To establish a co-expression system combining episomal and integrative vectors, enhance the expression efficiency of antimicrobial peptides in Pichia pastoris, and address the issue of insufficient production caused by copy number limitations and expression instability. Methods The 2 kanamycin-resistant episomal vectors, pPICKαA-panARS-JS21T and pGAPKαA-panARS-JS21T, were constructed using the Kluyveromyces lactis panARS autonomously replicating sequence. These were co-electroporated with the integrative vector pPICZαA-JS21T into Pichia pastoris X-33. Positive transformants were selected on Zeocin plus G418 double-antibiotic plates. Shake-flask cultures were run for more than 120 h with methanol supplementation every 24 h to maintain induction. Secreted total protein was quantified by the Bradford assay, target bands were visualized by trimethylglycine-sodium dodecyl sulfate polyacrylamide gel electrophoresis (Tricine-SDS-PAGE), and antimicrobial activity against Staphylococcus aureus was measured by agar diffusion. Results After 120 h of fermentation, the integrative plus episomal pGAPKαA-panARS-JS21T co-expression group yielded 107.22 μg/mL of extracellular total protein, 52% higher than 70.56 μg/mL of the integrative-only control. The corresponding inhibition-zone assay showed a 129% increase in antimicrobial activity (P<0.05). The integrative plus pPICKαA-panARS-JS21T group also achieved a 15.8% improvement over the integrative-only group. Conclusion A combined episomal/integrative co-expression strategy synergistically elevates JS21T antimicrobial peptide levels in Pichia pastoris, outperforming single-vector systems and offering a new route for high-level production of small peptide molecules.