{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,21]],"date-time":"2026-08-21T11:53:22Z","timestamp":1787313202521,"version":"build-2736575974"},"reference-count":44,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,12,18]],"date-time":"2025-12-18T00:00:00Z","timestamp":1766016000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"\n Introduction<\/jats:title>\n Recombinant allergens are an important diagnostic tool for determining the IgE-sensitization profile of patients, assessing the risk of symptom severity and potential clinical cross-reactivity. In this context, the use of different host cells for recombinant expression must be evaluated in terms of IgE reactivity and diagnostic value. Therefore, recombinant Ara h 2, the major peanut allergen, was produced in yeast, structurally characterized and investigated in respect to IgE-binding and allergenic properties.<\/jats:p>\n <\/jats:sec>\n \n Methods<\/jats:title>\n \n Ara h 2 was produced as recombinant protein using yeast and\n E. coli<\/jats:italic>\n expression systems. Purified proteins were assessed using SDS-PAGE (reducing and non-reducing conditions), CD-spectroscopy and IgE-reactivity.\n <\/jats:p>\n <\/jats:sec>\n \n Results<\/jats:title>\n \n Recombinant Ara h 2\n wt<\/jats:sup>\n expressed in yeast resulted in an additional predominant band of approximately 12 kDa upon DTT treatment. In contrast, the molecular mass of rAra h 2 expressed in\n E. coli<\/jats:italic>\n (Ara h 2\n \n E.coli<\/jats:sup>\n <\/jats:italic>\n ) remained unaffected by reduction. Analysis of rAra h 2\n wt<\/jats:sup>\n confirmed the presence of two Ara h 2-derived peptides, one with the expected N-terminus and the other with an N-terminal glycine residue.\n In silico<\/jats:italic>\n analysis revealed the presence of a Kex2 cleavage site (R\n 58<\/jats:sub>\n R\n 59<\/jats:sub>\n *G\n 60<\/jats:sub>\n ). To test whether Kex2 cleavage affects an IgE-epitope, mutagenesis of this cleavage site from R\n 58<\/jats:sub>\n to E\n 58<\/jats:sub>\n (Ara h 2\n mut<\/jats:sup>\n ) was performed. DTT treatment of rAra h 2\n mut<\/jats:sup>\n purified from yeast showed that no cleavage of the protein had occurred. No effect on IgE binding could be observed as all rAra h 2 preparations showed IgE-reactivity. Cross-linking of human serum IgE and monoclonal human Ara h 2-specific IgE antibodies showed comparable mediator release in response to Ara h 2\n wt<\/jats:sup>\n and rAra h 2\n mut<\/jats:sup>\n . However, utilizing a specific combination of human Ara h 2-specific IgE antibodies revealed slight epitope diversity between wildtype and mutated rAra h 2.\n <\/jats:p>\n <\/jats:sec>\n \n Discussion<\/jats:title>\n An endogenous protease, like Kex2 from the expression system, can affect the structural integrity of the target protein, leading to a slightly altered epitope structure. Even this finding has little or no impact for diagnosis, a suitable expression system and a detailed physico- and immunochemical characterization of recombinant allergens prior to their use as a diagnostic tool are of great importance.<\/jats:p>\n <\/jats:sec>","DOI":"10.3389\/fimmu.2025.1713823","type":"journal-article","created":{"date-parts":[[2025,12,18]],"date-time":"2025-12-18T06:43:02Z","timestamp":1766040182000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":1,"title":["Impact of recombinant expression in Komagataella phaffii on the allergenic properties of the peanut allergen Ara h 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