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ABSTRACT: Birch pollen allergy is predominantly caused by the major allergen Bet v 1 and can lead to crossreactions with homologous proteins in food. Two major cross-reactive food allergens are Dau c 1 from carrot and Api g 1 from celery, which have never been purified from their natural source. Here, we describe a non-denaturing purification method for obtaining natural Bet v 1, Dau c 1 and Api g 1, comprising of ammonium sulfate precipitation, hydrophobic interaction chromatography and size exclusion chromatography. This method resulted in 98-99% pure isoform mixtures for each allergen. Characterization of these isoform mixtures with Q-TOF MS/MS clearly showed earlier reported isoforms of Bet v 1, Dau c 1 and Api g 1, but also new isoforms. The presence of secondary structure in the three purified allergens was demonstrated via circular dichroism and showed high similarity. The immune reactivity of the natural allergens was compared with recombinant proteins by Western blot and ELISA and showed similar reactivity.
Molecular Nutrition & Food Research 51 (2007) 12.
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ABSTRACT: Mal d 1, the major apple allergen, is heat labile and easily oxidized. Oxidative reactions catalyzed by polyphenol oxidase (PPO) and/or peroxidase (POD), present in apple, may be involved in decreasing its allergenicity. PPO and POD convert phenolic compounds into o-quinones. In this study the effect of PPO and POD, the polyphenol catechin and the antioxidant DIECA on IgE-binding by Mal d 1 was analyzed. Golden Delicious peel was selected for its high PPO and POD contents. IgE-binding was analyzed by competitive ELISA. IgE-binding by Mal d 1 decreased by adding oxidative enzymes, this decrease was most pronounced when PPO was used. Catechin induced a reduction in IgE binding when POD was used. The combination of catechin and PPO causes the strongest decrease of the allergenicity of Mal d 1. DIECA protected the IgE-binding by the allergen, protection being less strong in the presence of exogenous PPO and POD. The decrease of immunoreactivity is likely to be due to o-quinones, as active species or other intermediates modifying the tertiary structure of the allergens and cross-linking of the proteins, thus reducing their allergenicity.
Food Chemistry 103 (2007) 1.
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ABSTRACT: Natural and recombinant Bet v 1, the major birch pollen allergen, and homologous allergens, Api g 1 and Dau c 1, from celery and carrot, respectively, were studied by CD spectroscopy under conditions of varying denaturant concentration, pH and temperature to determine fundamental thermodynamic parameters for conformational stability. Thermodynamic studies increase basic knowledge regarding differences between birch pollen-related allergens and are of importance in choosing processing conditions. The conformational stability determined from guanidine hydrochloride denaturation curves was similar for rBet v 1.0101 and rApi g 1.0101. Conformational responses to chaotropic salt were different for recombinant allergens from different species, but were similar for the natural isoform mixtures. The conformational stabilities of nApi g 1 and nDau c 1, were shown to be similar to rBet v 1.2801 at pH > 4.4 [Mogensen, J. E., Ipsen, H., Holm, J., & Otzen, D. E. (2004). Elimination of a misfolded folding intermediate by a single point mutation
Food Chemistry 119 (2010) 1.
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[show abstract]
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ABSTRACT: Natural and recombinant Bet v 1, the major birch pollen allergen, and homologous allergens, Api g 1 and Dau c 1, from celery and carrot, respectively, were studied by CD spectroscopy under conditions of varying denaturant concentration, pH and temperature to determine fundamental thermodynamic parameters for conformational stability. Thermodynamic studies increase basic knowledge regarding differences between birch pollen-related allergens and are of importance in choosing processing conditions. The conformational stability determined from guanidine hydrochloride denaturation curves was similar for rBet v 1.0101 and rApi g 1.0101. Conformational responses to chaotropic salt were different for recombinant allergens from different species, but were similar for the natural isoform mixtures. The conformational stabilities of nApi g 1 and nDau c 1, were shown to be similar to rBet v 1.2801 at pH > 4.4 [Mogensen, J. E., Ipsen, H., Holm, J., & Otzen, D. E. (2004). Elimination of a misfolded folding intermediate by a single point mutation. Biochemistry, 43(12), 3357-3367], but nApi g and nDau c 1 were stable to heating at lower pH-values.
Food Chemistry.
