Overexpression And Characterization Of10kDa Allergen In Tartary Buckwheat

Nowadays, there is growing interest in buckwheat because of its nutritive values andmedical values. But many people may have a hypersensitive reaction after ingesting fooddishes containing buckwheat. In this study, the tartary buckwheat10kDa allergen gene(TBAP10) was obtained from the full-length cDNA library of seed-filling period by PCRmethod, and a prokaryotic recombinant expression vector pET47b inserted with TBAP10genewas constructed. Overexpression of the target protein TBAP10was achieved in BL21Star(DE3) cells by the induction with IPTG. After renaturation of isolated inclusion body andcobalt-ion chelating affinity chromatography, the properties of TBAP10were analyed.Online bioinformatics softwares were used to predicte the amino acid sequence ofTBAP10, the results showed that the signal peptide was1-19amino acid residues, themolecular weight was14.87kDa, pI was6.43and the protein was a hydrophobic protein.BLAST comparison results indicated the similarity of gene sequence between TBAP10andTatary buckwheat8kDa allergy was96%and the similarity of primary structure betweenTBAP10and Fag t2(Tatary buckwheat16kDa allergen) was53%. ClustalX2comparisonresult indicated parts of amino acid sequence were the same between TBAP10and Fag t2.The gene of TBAP10deleting N-terminal signal peptide sequence was successfullyamplified from recombinant pTriplEx2-TBAP10, and TBAP10gene was cloned into theprokaryotic expression vector pET47b. We constructed a prokaryotic expression vectorpET47b-TBAP10and then transferred it into BL21Star (DE3).The recombinant protein TBAP10was expressed in E.coli BL21Star (DE3) as aninclusion body form. Ultrasonic crushing and washing was used to purify the inclusion body,and then the inclusion bodies solubilized in the8M/L urea were diluted with renaturationsolutions. After renaturation of isolated inclusion body and cobalt-ion chelating affinitychromatography, TBAP10was purified to homogeneity. His-tag of TBAP10was identified by6xHis Protein Tag Staining Kit.Western blotting assays using rabbit serum against tartary buckwheat16kDa allergenshowed there was cross-reactivity between the two proteins, many amino acids between Fagt2and TBAP10were the same. Competitive ELISA demonstrated that recombinant proteincould bind the IgE of the buckwheat-allergic patient’s; the competitive inhibition rate of TBAP10was49.1%.The thermal stability of TBAP10was detected by SDS-PAGE, and the result showed thatTBAP10could tolerate15min boiling water. According to the United States pharmacopeia,we prepared the in vitro simulated gastric fluid and simulated intestinal fluid. Simulatedgastrointestinal digestion experiments indicated that the recombinant protein had a strongtolerance to pepsin while it was totally degraded after trypsin digestion.SMART software was used to predicte the functional domain of the amino acid sequenceof TBAP10; the result showed there was an AAI domain in the primary structure. AAI domainexists in the α-amylase inhibitor and can inhibit the function of α-amylase. In this study, weadded TBAP10into the reaction system to explore the relationship between TBAP10andα-amylase. The result indicted that TBAP10could activate the activity of α-amylase.