Molecular cloning, recombinant expression and IgE‐binding epitope of ω‐5 gliadin, a major allergen in wheat‐dependent exercise‐induced anaphylaxis

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Abstract
Wheatω-5 gliadin has been identified as a major allergen in wheat-dependent exercise-induced anaphylaxis. We have detected seven IgE-binding epitopes in primary sequence of the protein. We newly identified four additional IgE-binding epitope sequences, QQFHQQQ, QSPEQQQ, YQQYPQQ and QQPPQQ, in three patients with wheat-dependent exercise-induced anaphylaxis in this study. Diagnosis and therapy of food allergy would benefit from the availability of defined recombinant allergens. However, because ω-5 gliadin gene has not been cloned, recombinant protein is currently unavailable. We sought to clone the ω-5 gliadin gene and produce the homogeneous recombinant protein for use in an in vitro diagnostic tool. Using a PCR-based strategy we isolated two full-length ω-5 gliadin genes, designated ω-5 and ω-5b, from wheat genomic DNA and determined the nucleotide sequences. The protein encoded by ω-5a was predicted to be 439 amino acids long with a calculated mass of 53 kDa; the ω-5b gene would encode a 393 amino acid, but it contains two stop codons indicating that ω-5b is pseudogene. The C-terminal half (178 amino acids) of the ω-5a gliadin protein, including all 11 IgE-binding epitope sequences, was expressed in Escherichia coli by means of the pET system and purified using RP-HPLC. Western blot analysis and dot blot inhibition assay of recombinant and native ω-5 gliadin purified from wheat flour demonstrated that recombinant protein had IgE-binding ability. Our results suggest that the recombinant protein can be a useful tool for identifying patients with wheat-dependent exercise-induced anaphylaxis in vitro.