Site-directed Mutagenesis And Analysis Of The Major Allergen In Tartary Buckwheat

As a kind of healthy food, buckwheat seeds contain abundant protein and are rich in essential amino acids, such as lysine, methionine and tryptophan. Holding rutin, flavonol and glycoside, the seeds perform functions of an antagonist to capillary fragility diseases and hypertension. However, buckwheat could trigger allergy reaction towards ingestion and inhalation, and the symptom including asthma, dermatitis, eczema and even anaphylactic shock, which badly restrict its comprehensive application as a new alternative food and popular additive. As a result, the demand of identifying the allergen molecule and clarifying the mechanism of buckwheat allergy is severely urgent, for development of the accurate diagnosis and safer immunotherapy.In the previous work, we have obtained a natural protein namely TBa (tartary buckwheat allergen a) from the Yunnan tartary buckwheat seeds by separation and purification. It was identified as one of the major allergens in tartary buckwheat by immunological identification, and the molecular weight is approximately 24 kDa from SDS-PAGE. Registered in the GenBank (Accession No. AY044918), the gene of TBa was obtained for the first time by cDNA cloning. Predicted by Antigenic program, full-length tartary buckwheat allergen (TBa) was divided into six fragments (E1:40 amino acids from 27 to 67, E2:aa 87-137, E12: 27-137, E3:120-156, E6:155-191, E36:120-191), and the results of immunological determination revealed that E1 fragment reacted stronger with the patients'serum IgE than the other fragments, which indicated that E1 was the major epitope.The homology between the amino acid sequence of TBa and other proteins was determined by comparing their sequences using BlASTp in the NCBI website. The schematic location of putative conserved domain in TBa indicated that the region (amino acid 20-160) including E1 fragment (amino acids 27-67) was the most important, which belongs to cupin super family. Based on our BLASTp search results, alignment of the amino acid sequences of E1 and other plant seed storage proteins was carried adopting DNAStar software. The results showed that Leu39, Leu42, Leu47 and Leu54 were homologous, while the secondary structure prediction revealed that most of these sites located in the flexible region andβ-sheets.Five variants of E1 were generated with a mutation at sites 39,42,47, 52 and 54, in which it was replaced by an Arg residue, and we refer to them as L39R, L42R, L47R, V52R, and L54R, respectively. Gene of the mutants were cloned into the expression vector pET-32m, and expressed in host cells, E. coli BL21 (DE3). After the proteins were purified by affinity chromatography, SDS-PAGE analysis showed that the molecular weight was accordant to WT E1, and the purity were>95%. E1 and the five mutant proteins were studied for their IgE binding by ELISA and dot blot using sera from patients with positive IgE binding activity to buckwheat, and healthy control. The results showed that all the five mutants exhibited decreased IgE binding activity in comparison with E1 WT. However, L42R, L47R and L54R showed remarkable decrease by nearly 50%, considering the data with healthy control. These results indicated that Leu42, Leu47 and Leu54 contribute more significant towards the IgE binding of the allergen TBa, which will provide evidence for clarifying the mechanism of buckwheat allergy.