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

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. 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. 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.