The Major Peanut Allergen Ara h 2 Produced in Contains Hydroxyprolines and Is a Viable Alternative to the Product in Allergy Diagnosis.

Peanut allergy is a potentially life-threatening disease that is mediated by allergen-specific immunoglobulin E (IgE) antibodies. The major peanut allergen Ara h 2, a 2S albumin seed storage protein, is one of the most dangerous and potent plant allergens. Ara h 2 is posttranslationally modified to harbor four disulfide bridges and three hydroxyprolines. These hydroxyproline residues are required for optimal IgE-binding to the DPYSPS motifs representing an immunodominant IgE epitope. So far, recombinant Ara h 2 has been produced in insect cell, and chloroplast expression systems, which were all incapable of proline hydroxylation. However, molecular diagnosis of peanut allergy is performed using either natural or -produced major peanut allergens. As IgE from the majority of patients is directed to Ara h 2, it is of great importance that the recombinant Ara h 2 harbors all of its eukaryotic posttranslational modifications. We produced hydroxyproline-containing and correctly folded Ara h 2 in the endoplasmic reticulum of leaf cells of plants, using the plant virus-based magnICON transient expression system with a yield of 200 mg/kg fresh biomass. To compare prokaryotic with eukaryotic expression methods, Ara h 2 was expressed in together with the disulfide-bond isomerase DsbC and thus harbored disulfide bridges but no hydroxyprolines. The recombinant allergens from and were characterized and compared to the natural Ara h 2 isolated from roasted peanuts. Natural Ara h 2 outperformed both recombinant proteins in IgE-binding and activation of basophils IgE cross-linking, the latter indicating the potency of the allergen. Interestingly, significantly more efficient IgE cross-linking by the -produced allergen was observed in comparison to the one induced by the product. Ara h 2 from plants displayed a higher similarity to the natural allergen in terms of basophil activation due to the presence of hydroxyproline residues, supporting so far published data on their contribution to the immunodominant IgE epitope. Our study advocates the use of plants instead of prokaryotic expression hosts for the production of the major peanut allergen Ara h 2.