|RUAN, SANBAO - Louisiana State University|
Submitted to: International Archives of Allergy and Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/29/2014
Publication Date: 12/18/2014
Citation: Hurlburt, B.K., Mcbride, J.K., Nesbit, J.B., Ruan, S., Maleki, S.J. 2014. Purification of recombinant peanut allergen Ara h 1 and comparison of IgE binding to the native protein. International Archives of Allergy and Immunology. 3:642-657.
Interpretive Summary: Allergic reactions to food are on the rise worldwide, and there is a corresponding increase in interest to understand the molecular mechanisms responsible. Peanut is the most problematic of all foods in that the reaction often persists into adulthood, and can be as severe as anaphylaxis and death. Furthermore, peanut products are found in myriad processed foods. There are three dominant allergen proteins in peanut. One of those, Ara h 1, is the subject of this work. A system to express high levels of full-length Ara h 1, and a smaller core part in E. coli and a reproducible method to purify the protein were developed. These proteins were compared to native Ara h 1 for binding of allergy-specific antibodies (IgE) from allergic patients sera. The purified proteins will also be used to standardize clinical tests for peanut allergy, as well as allow further immunological and biochemical characterization. Furthermore, the proteins will be used in development of hypoallergenic treatments for people with peanut allergy.
Technical Abstract: Allergic 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 native Ara h 1 for IgE binding. All patients recognized both the folded native and rAra h 1, but the IgE binding to the rAra h 1 was significantly reduced in comparison to the native allergen, which could potentially make it useful for immunotherapeutic purposes.