TY - JOUR
T1 - Isolation, cloning, and characterization of the 2S albumin: A new allergen from hazelnut
AU - Garino, Cristiano
AU - Zuidmeer, Laurian
AU - Marsh, Justin
AU - Lovegrove, Alison
AU - Morati, Maria
AU - Versteeg, Serge
AU - Schilte, Piet
AU - Shewry, Peter
AU - Arlorio, Marco
AU - van Ree, Ronald
PY - 2010
Y1 - 2010
N2 - 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 similar to 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
AB - 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 similar to 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
U2 - https://doi.org/10.1002/mnfr.200900456
DO - https://doi.org/10.1002/mnfr.200900456
M3 - Article
C2 - 20373288
SN - 1613-4125
VL - 54
SP - 1257
EP - 1265
JO - Molecular nutrition & food research
JF - Molecular nutrition & food research
IS - 9
ER -