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Background <p>Efficient and scalable production of viral antigens remains a key challenge in the development of recombinant subunit vaccines and diagnostic reagents. Microbial expression systems, particularly <i>Pichia pastoris</i> (<i>P. pastoris</i>), offer a promising platform for producing complex viral glycoproteins with appropriate folding and post-translational modifications.</p> Results <p>In this study, the hemagglutinin head domain (HA1) of influenza A (H1N1) virus was expressed as a secreted recombinant protein in <i>P. pastoris</i> GS115. The HA1 gene was codon-optimized and expressed under the control of the methanol-inducible AOX1 promoter with an α-factor signal peptide. Multicopy integrants were enriched using G418 selection, and expression conditions were systematically optimized. Under shake-flask induction, the selected recombinant strain produced up to 0.375&#xa0;g/L of rHA1 in the culture supernatant. The protein was efficiently purified by Ni-NTA affinity chromatography to a purity exceeding 95%. PNGase F digestion confirmed N-linked glycosylation. Limited functional validation demonstrated that the yeast-expressed rHA1 retained antigenic integrity, as evidenced by the induction of rHA1-specific antibodies and hemagglutination-inhibiting activity in a murine model.</p> Conclusions <p>These results establish <i>P. pastoris</i> as an effective microbial cell factory for the high-level secretion of influenza HA1 protein. The optimized expression and purification strategy provides a scalable and cost-efficient framework for microbial production of viral antigens and may be applicable to other glycoproteins of biomedical relevance.</p>

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Scalable secretory production of influenza A (H1N1) hemagglutinin HA1 in Pichia pastoris through expression and process optimization

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年3月17日

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  • Zeliang Guo,
  • Wenting Ding,
  • Chendi Yang,
  • Xiaoying Zhi,
  • Yu Zhang,
  • Fengjia Xi,
  • Rongzeng Liu

摘要

Background

Efficient and scalable production of viral antigens remains a key challenge in the development of recombinant subunit vaccines and diagnostic reagents. Microbial expression systems, particularly Pichia pastoris (P. pastoris), offer a promising platform for producing complex viral glycoproteins with appropriate folding and post-translational modifications.

Results

In this study, the hemagglutinin head domain (HA1) of influenza A (H1N1) virus was expressed as a secreted recombinant protein in P. pastoris GS115. The HA1 gene was codon-optimized and expressed under the control of the methanol-inducible AOX1 promoter with an α-factor signal peptide. Multicopy integrants were enriched using G418 selection, and expression conditions were systematically optimized. Under shake-flask induction, the selected recombinant strain produced up to 0.375 g/L of rHA1 in the culture supernatant. The protein was efficiently purified by Ni-NTA affinity chromatography to a purity exceeding 95%. PNGase F digestion confirmed N-linked glycosylation. Limited functional validation demonstrated that the yeast-expressed rHA1 retained antigenic integrity, as evidenced by the induction of rHA1-specific antibodies and hemagglutination-inhibiting activity in a murine model.

Conclusions

These results establish P. pastoris as an effective microbial cell factory for the high-level secretion of influenza HA1 protein. The optimized expression and purification strategy provides a scalable and cost-efficient framework for microbial production of viral antigens and may be applicable to other glycoproteins of biomedical relevance.