Isolation,cloning, and characterization of the 2S albumin: A new allergen from hazelnut |
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Authors: | Cristiano Garino Laurian Zuidmeer Justin Marsh Alison Lovegrove Maria Morati Serge Versteeg Piet Schilte Peter Shewry Marco Arlorio Ronald van Ree |
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Affiliation: | 1. Università del Piemonte Orientale “A. Avogadro”, DiSCAFF & Drug and Food Biotechnological Center, Novara, Italy;2. These authors have contributed equally to this study.;3. Academic Medical Center, Department of Experimental Immunology, Amsterdam, The Netherlands;4. Rothamsted Research, Harpenden, Hertfordshire, UK;5. Medical Center Alkmaar, Department of Pediatrics, Alkmaar, The Netherlands;6. Academic Medical Center, Department of Otorhinolaryngology, Amsterdam, The Netherlands |
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Abstract: | Scope: 2S albumins are the major allergens involved in severe food allergy to nuts, seeds, and legumes. We aimed to isolate, clone, and express 2S albumin from hazelnut and determine its allergenicity. Methods: 2S albumin from hazelnut extract was purified using size exclusion chromatography and RP‐HPLC. After N‐terminal sequencing, degenerated and poly‐d(T) primers were used to clone the 2S albumin sequence from hazelnut cDNA. After expression in Escherichia coli and affinity purification, IgE reactivity was evaluated by Immunoblot/ImmunoCAP (inhibition) analyses using sera of nut‐allergic patients. Results: N‐terminal sequencing of a ~10 kDa peak from size exclusion chromatography/RP‐HPLC gave two sequences highly homologous to pecan 2S albumin, an 11 amino acid (aa) N‐terminal and a 10aa internal peptide. The obtained clone (441 bp) encoded a 147aa hazelnut 2S albumin consisting of a putative signal peptide (22 aa), a linker peptide (20 aa), and the mature protein sequence (105 aa). The latter was successfully expressed in E. coli. Both recombinant and natural 2S albumin demonstrated similar IgE reactivity in Immunoblot/ImmunoCAP (inhibition) analyses. Conclusion: We confirmed the postulated role of hazelnut 2S albumin as an allergen. The availability of recombinant molecules will allow establishing the importance of hazelnut 2S albumin for hazelnut allergy. |
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Keywords: | 2S albumin EuroPrevall Food allergy Hazelnut (Corylus avellana L.) Recombinant |
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