Purification, Crystallization and Preliminary X-ray Characterization of Prunin-1, a Major Component of the Almond (Prunus dulcis) Allergen Amandin

Abstract

The 11S globulins from plant seeds account for a number of major food allergens. Because of the interest in the structural basis underlying the allergenicity of food allergens, we sought to crystallize the main 11S seed storage protein from almond ( Prunus dulcis). Prunin-1 (Pru1) was purified from defatted almond flour by water extraction, cryoprecipitation, followed by sequential anion exchange, hydrophobic interaction, and size exclusion chromatography. Single crystals of Pru1 were obtained in a screening with a crystal screen kit, using the hanging-drop vapor diffusion method. Diffraction quality crystals were grown after optimization. The Pru1 crystals diffracted to at least 3.0 A and belong to the tetragonal space group P4(1)22, with unit cell parameters of a = b = 150.912 A, c = 165.248 A. Self-rotation functions and molecular replacement calculations showed that there are three molecules in the asymmetry unit with water content of 51.41%. The three Pru1 protomers are related by a noncrystallographic 3-fold axis and they form a doughnut-shaped trimer. Two prunin trimers form a homohexamer. Elucidation of prunin structure will allow further characterization of the allergenic features of the 11S protein allergens at the molecular level.

Recombinant monoclonal human immunoglobulin E to investigate the allergenic activity of major grass pollen allergen Phl p 5

“…Several explanations have been discussed how allergens can efficiently cross‐link effector cell‐bound antibodies. They include the idea that allergens can form oligomers which can present the same epitopes in a repetitive manner [14–20], the possibility that allergens occur as monomers containing repetitive epitope sequences [21–23] and finally, the model that allergens contain several different IgE epitopes which cluster on certain surface‐exposed areas [10, 15, 45]. In an earlier study we have suggested that the majority of IgE epitopes and of the allergenic activity of Phl p 5 resides in an allergen fragment which is defined by rhuMabEP5 [10, 46].…”

“…Several explanations for this high allergenic activity can be found in the literature. Several studies report that allergens form oligomers in solution [14–19]. Furthermore, it has been claimed that such oligomers contain repetitions of the same epitope which can cross‐link effector cell‐bound IgE and are responsible for degranulation [20].…”

Epigenetic regulation of peanut allergen gene Ara h 3 in developing embryos

“…It is known that the 11S protein has multiple isoforms in many plant species. Each isoform of the 11S globulin is encoded by a single gene (Wang et al 2002) that produces a precursor which is post-translationally cleaved by an asparaginyl endopeptidase after the formation of the interchain disulWde bond between the N-terminal and the C-terminal subunits (Albillos et al 2008). The 11S storage protein from soybean is known to be a hexameric globulin composed of various mixtures of Wve diVerent isoforms (Badley et al 1975).…”

Identification and characterisation of the proteins bound by specific phage‐displayed recombinant antibodies (scFv) obtained against Brazil nut and almond extracts

“…The sample was centrifuged at 10 000 × g for 30 min, and the supernatant was filtered through a 0.20 μm syringe filter. Chromatography was performed in an ÄKTA purifier FPLC system (GE Healthcare, Uppsala, Sweden) following the procedure described by Albillos et al ., with some modifications. Briefly, 1 mL of protein extract was diluted in 10 mL of 0.01 mol L −1 Tris–HCl buffer, pH 7.9, and loaded onto a 1 mL Mono Q HR 5/5 anion exchange column (GE Healthcare) previously equilibrated with Tris–HCl buffer.…”