Purification of Recombinant Peanut Allergen Ara h 1 and Comparison of IgE Binding to the Natural Protein
AbstractAllergic reactions to food are on the rise worldwide and there is a corresponding increase in interest to understand the molecular mechanisms responsible. Peanut allergies are the most problematic because the reaction often persists into adulthood and can be as severe as anaphylaxis and death. The purpose of the work presented here was to develop a reproducible method to produce large quantities of pure recombinant Ara h 1(rAra h 1) that will enable standardization of immunological tests for patients and allow structural and immunological studies on the wild type and mutagenized forms of the protein. Ara h 1 is initially a pre-pro-protein which, following two endoproteolytic cleavages, becomes the mature form found in peanut. The mature form however has flexible regions that make it refractory to some structural studies including crystallography. Therefore, independent purification of the mature and core regions was desirable. Expression constructs were synthesized cDNA clones for each in a pET plasmid vector without tags. Codons were optimized for expression in E. coli. High-level expression was achieved in BL21 strains. Purification to near homogeneity was achieved by a combination of ammonium sulfate precipitation and ion exchange chromatography. The purified rAra h 1 was then compared with natural Ara h 1 for IgE binding. All patients recognized both the folded natural and rAra h 1, but the IgE binding to the rArah1 was significantly reduced in comparison to the natural allergen, which could potentially make it useful for immunotherapeutic purposes. View Full-Text
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Hurlburt, B.K.; McBride, J.K.; Nesbit, J.B.; Ruan, S.; Maleki, S.J. Purification of Recombinant Peanut Allergen Ara h 1 and Comparison of IgE Binding to the Natural Protein. Foods 2014, 3, 642-657.
Hurlburt BK, McBride JK, Nesbit JB, Ruan S, Maleki SJ. Purification of Recombinant Peanut Allergen Ara h 1 and Comparison of IgE Binding to the Natural Protein. Foods. 2014; 3(4):642-657.Chicago/Turabian Style
Hurlburt, Barry K.; McBride, Jane K.; Nesbit, Jacqueline B.; Ruan, Sanbao; Maleki, Soheila J. 2014. "Purification of Recombinant Peanut Allergen Ara h 1 and Comparison of IgE Binding to the Natural Protein." Foods 3, no. 4: 642-657.