Natural and recombinant molecules of the cherry allergen Pru av 2 show diverse structural and B cell characteristics but similar T cell reactivity

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Abstract
Background Cherry allergy is often reported in the context of allergy to other fruits of the Rosaceae family and pollinosis to trees because of cross‐reactive allergens. Allergic reactions to cherry are reported by 19–29% of birch pollen‐allergic patients. Pru av 2, identified as a thaumatin‐like protein (TLP) from sweet cherry, was recognized by the majority of cherry‐allergic patients in immunoblotting. Objectives In order to investigate the structural characteristics and the immunoglobulin (Ig)E‐ and T cell reactivity of cherry‐derived TLP, recombinant Pru av 2 was expressed in Escherichia coli and natural Pru av 2 was purified. Methods Parallel‐His and FLAG expression vectors were used for recombinant production of Pru av 2 in the cytoplasm and the periplasm of E. coli. Natural Pru av 2 was purified from fresh cherries and verified by N‐terminal sequencing. Structural characterization was performed using circular dichroism (CD) measurements, and the biologic activity was measured in a glucanase assay. Using cherry‐specific sera, the IgE‐binding ability of recombinant and natural Pru av 2 was investigated in IgE‐ELISA and the T cell reactivity was studied in proliferation assays. Results Natural Pru av 2 revealed thaumatin‐like structural features and bound IgE of 50% of cherry‐allergic patients. It was demonstrated to be enzymatically active. Recombinant Pru av 2 expressed in the cytoplasm of E. coli exhibited a slightly different folding compared with the natural protein. It was not recognized by IgE from cherry‐allergic subjects, but retained the ability to stimulate T lymphocytes. Periplasmic recombinant Pru av 2 was able to bind an anti‐grape TLP antibody and cherry‐specific IgE. Conclusions We prepared two recombinant model TLPs from cherry, and compared their molecular characteristics as well as their IgE‐binding activity and T cell interactions in relation to the natural counterpart. The cytoplasmic recombinant Pru av 2 can be used as a hypoallergenic variant in allergen‐specific immunotherapy, whereas the periplasmic protein can be included in a component‐resolved diagnosis.