Enhancing production and assessing IgE reactivity of dog allergen Can f 6 in Pichia pastoris and Escherichia coli.

Pet allergies are increasingly prevalent in developed nations, significantly affecting humans and strongly linked with asthma and rhinitis. Allergic reactions to cats and dogs affect 15.7% of Americans and 27.2% of Europeans, with sensitization rates to dog allergens reaching 56.0% in Denmark. Despite these concerns, dog ownership remains widespread, with 25% of European and 45.5% of US households owning at least one dog. With sensitization on the rise and current diagnostic and therapeutic approaches predominantly relying on inherently inconsistent allergen extracts derived from natural sources, recombinant allergen production offers a pathway to component-resolved diagnostics, improving specificity and reliability in allergy diagnosis. The present research explored, for the first time, the production of the allergen component glycoprotein Can f 6 in the eukaryotic expression system Pichia pastoris and compared its IgE antigenicity to recombinant Can f 6 (rCan f 6) variants produced in Escherichia coli. Yields were significantly increased by fusing Can f 6 with the maltose binding protein (MBP), resulting in a 1.8-fold increase in production in E. coli and a threefold increase in P. pastoris. Antigenicity analysis showed that N-glycosylation is not critical for folding or IgE recognition of Can f 6, making both systems equally suitable for producing the allergen. Notably, P. pastoris-produced MBP fused protein purified through cation exchange chromatography yielded a lower protein quantity. Still, it exhibited stronger IgE reactivity than the same protein purified using anion exchange chromatography.