Isolation, cloning, and characterization of the 2S albumin: a new allergen from hazelnut.
METHODS
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 approximately 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 10 aa internal peptide. The obtained clone (441 bp) encoded a 147 aa 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.


CONCLUSIONS
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.
Garino
Cristiano
C
Università del Piemonte Orientale "A. Avogadro", DiSCAFF & Drug and Food Biotechnological Center, Novara, Italy.
Zuidmeer
Laurian
L
Marsh
Justin
J
Lovegrove
Alison
A
Morati
Maria
M
Versteeg
Serge
S
Schilte
Piet
P
Shewry
Peter
P
Arlorio
Marco
M
van Ree
Ronald
R
D055516:2S Albumins, Plant / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D000293:Adolescent; D000328:Adult; D000368:Aged; D000485:Allergens / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D000595:Amino Acid Sequence; D031211:Corylus / Q000276:immunology* / Q000378:metabolism; D005260:Female; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D021184:Nut Hypersensitivity / Q000097:blood / Q000276:immunology*; D009754:Nuts / Q000276:immunology* / Q000378:metabolism; D010936:Plant Extracts / Q000378:metabolism; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D016415:Sequence Alignment; D017385:Sequence Homology; D055815:Young Adult
D055516:2S Albumins, Plant; D000485:Allergens; D010936:Plant Extracts; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D007073:Immunoglobulin E