title: Purification of Recombinant Peanut Allergen Ara h 1 and Comparison of IgE Binding to the Natural Protein.
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 inE. 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.
authors.affiliation: Southern Regional Research Center, Agricultural Research Service, United States Department of Agriculture, 1100 Robert E. Lee, Blvd., New Orleans, LA 70124, USA. barry.hurlburt@ars.usda.gov.
authors.forename: Barry K
authors.initials: BK
authors.lastname: Hurlburt
authors.affiliation: Southern Regional Research Center, Agricultural Research Service, United States Department of Agriculture, 1100 Robert E. Lee, Blvd., New Orleans, LA 70124, USA. jane.mcbride@ars.usda.gov.
authors.forename: Jane K
authors.initials: JK
authors.lastname: McBride
authors.affiliation: Southern Regional Research Center, Agricultural Research Service, United States Department of Agriculture, 1100 Robert E. Lee, Blvd., New Orleans, LA 70124, USA. jbnesbit@uno.edu.
authors.forename: Jacqueline B
authors.initials: JB
authors.lastname: Nesbit
authors.affiliation: Southern Regional Research Center, Agricultural Research Service, United States Department of Agriculture, 1100 Robert E. Lee, Blvd., New Orleans, LA 70124, USA. cyoumc@yahoo.com.
authors.forename: Sanbao
authors.initials: S
authors.lastname: Ruan
authors.affiliation: Southern Regional Research Center, Agricultural Research Service, United States Department of Agriculture, 1100 Robert E. Lee, Blvd., New Orleans, LA 70124, USA. soheila.maleki@ars.usda.gov.
authors.forename: Soheila J
authors.initials: SJ
authors.lastname: Maleki
keywords: Ara h 1; IgE binding; immunodominant; peanut allergy