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

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 .