纤连蛋白FNIII8-10在毕赤酵母中的重组表达及发酵优化

Expression of recombinant fibronectin FNIII8-10 in Pichia pastoris and fermentation process optimization

摘要:

纤连蛋白(fibronectin, FN) III型结构域的8-10重复序列(FNIII8-10)具备多个整合素结合位点，是介导细胞黏附的核心模块，目前仅在原核微生物中表达。本研究旨在构建高效表达重组FNIII8-10的毕赤酵母表达体系，采用毕赤酵母系统成功表达重组FNIII8-10，但存在分泌效率低的问题，通过信号肽理性设计与发酵工艺调控相结合的策略显著提高了其分泌表达效率。首先，筛选了多个信号肽，发现α-factor信号肽表达效率最高；随后，对其疏水核心区进行定点突变，获得高效突变体α-V50A，使蛋白表达量提升44.8%。通过响应面法优化发酵条件，发现最佳发酵条件为发酵117.46 h、pH 6.91、甲醇添加量1.91%；同时，以山梨醇作为辅助碳源，以缓解甲醇胁迫，重组FNIII8-10产量达到65.49 mg/L。最终通过高密度细胞发酵，使其产量达到532.82 mg/L，比摇瓶最高产量提升了7倍。本研究为FNIII8-10重组蛋白的工业规模化生产提供了技术基础。

Abstract:

The 8-10 repeat of fibronectin (FN) type III domain (FNIII8-10) contains multiple integrin-binding sites and serves as the core module mediating cell adhesion. Currently, recombinant FNIII8-10 is expressed only in prokaryotic microorganisms. This study aims to establish a Pichia pastoris expression system for the high-level production of recombinant FNIII8-10. Although the recombinant FNIII8-10 was successfully expressed in Pichia pastoris, the secretion efficiency was low. Through rational signal peptides design and fermentation optimization, secretion efficiency was significantly improved. Screening of multiple signal peptides identified the α-factor signal peptide as the most efficient. Site-directed mutagenesis of its hydrophobic core generated the high-efficiency mutant α-V50A, which increased secreted protein yield by 44.8%. Using the response surface methodology, fermentation conditions were optimized to 117.46 h, pH 6.91, and 1.91% (V/V) methanol supplementation. Meanwhile, with sorbitol as an auxiliary carbon source to alleviate methanol stress, the yield of the recombinant FNIII8-10 reached 65.49 mg/L. Finally, high-cell-density fermentation achieved a yield of 532.82 mg/L, representing a 7-fold increase compared with the highest yield in shake flasks. This study lays a technical foundation for the industrial-scale production of recombinant FNIII8-10.