Molecular Cloning of cDNA, Recombinant Protein Expression and Characterization of a Buckwheat 16-kDa Major Allergen

Abstract

BACKGROUND: Buckwheat is a common food in Japan, Korea and other countries. A candidate major buckwheat allergen, a 16-kDa protein (BWp16), was previously characterized as a pepsin-resistant protein associated with immediate-type allergies to buckwheat. However, whether recombinant BWp16 can react with a patient's IgE remains uncertain. METHODS: The cDNA encoding BWp16 from Japanese buckwheat seeds was cloned based on the sequences obtained by the 5'-rapid amplification of cDNA ends (RACE) and 3'-RACE PCR. Recombinant BWp16 protein expressed in Escherichia coli was purified using affinity chromatography. Western blotting, ELISA and cross inhibition tests of the purified recombinant BWp16 were performed using sera from patients with positive IgE binding to buckwheat and controls. Pepsin digestion experiments were also performed. RESULTS: The full-length cDNA encodes 149 amino acid residues with a calculated molecular mass of 16.9 kDa. The deduced amino acid sequence included a putative signal peptide sequence. BWp16 showed significant homologies to the buckwheat 8-kDa allergen and Ricinus communis (castor bean) 2S albumin. Sera from patients with positive IgE binding to buckwheat reacted with the purified BWp16. Cross inhibition tests revealed immunological equivalence of the purified recombinant and natural BWp16. The recombinant and natural BWp16 were comparably resistant to pepsin digestion. CONCLUSIONS: BWp16 belongs to the 2S albumin family and is a buckwheat allergen. This purified recombinant BWp16 could be used in the diagnosis of buckwheat allergy.