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.