Abstract
A 16 kDa buckwheat protein (BWp16) is a major allergen responsible for immediate hypersensitivity reactions including anaphylaxis. A deletion mutant of BWp16 (rBWp16DeltaN) was overproduced and purified and was shown to be immunologically active. A three-wavelength MAD data set was collected from a crystal of selenomethionine-labelled rBWp16DeltaN. The crystal belonged to the triclinic space group P1, with unit-cell parameters a = 28.39, b = 31.54, c = 32.20 A, alpha = 111.92, beta = 108.91, gamma = 98.74 degrees . One monomer was expected to be present in the asymmetric unit based on the calculated Matthews coefficient of 1.76 A(3) Da(-1).
Highlights
Buckwheat (Fagopyrum esculentum) belongs to the Polygonaceae family
The codon AGC for the last amino acid (Ser) of the thrombin recognition site overlapped with that for the 13th amino acid of BWp16 (Ser13).] The resulting fragment was cut and cloned into pGEX-6P-2 (GE Healthcare) vector. rBWp16ÁN consisted of residues 13–127 of BWp16 and the N-terminal vector-derived Gly residue left after removal of the glutathione S-transferase (GST) tag by thrombin cleavage
After coating 96-well plates (Nunc LockWell Module Plate, Nunc) with recombinant BWp16 (rBWp16) or rBWp16ÁN (1.0 mg per 50 ml of 50 mM sodium carbonate buffer pH 9.6 in the well) and incubating overnight at 277 K, they were washed with phosphate-buffered saline (PBS) containing 0.05% Tween 20 (PBS-T) and blocked with 0.1% casein-PBS for 1 h at room temperature
Summary
Buckwheat (Fagopyrum esculentum) belongs to the Polygonaceae family. Buckwheat noodles are a popular food in East Asian countries. Investigators have reported that 24, 19, 16, 14, 10 and 9 kDa buckwheat proteins can react with IgE antibodies from allergic patients (Nagata et al, 2000; Park et al, 2000; Tanaka et al, 2002; Yoshimasu et al, 2000; Matsumoto et al, 2004). Tanaka and coworkers demonstrated that the 16 kDa buckwheat protein BWp16, a member of the 2S albumin family (Shewry et al, 1995), was resistant to pepsin and preserved its reactivity with IgE antibodies from allergic patients with immediate hypersensitivity reactions (Tanaka et al, 2002). The colorimetric intensity at 450 nm was measured according to the manufacturer’s protocol
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