A recombinant precursor of the mustard allergen Sin a 1 retains the biochemical and immunological features of the heterodimeric native protein.
BACKGROUND
Mustard has been an important cause of food allergy of increasing incidence in the last years. Sin a 1, a storage 2S albumin, is the most relevant allergen from this spice.


METHODS
Pichia pastoris has been used as host for the recombinant production of the precursor form of Sin a 1 (rproSin a 1). rproSin a 1 was purified to homogeneity by three chromatographic steps: gel filtration, anion exchange and reverse-phase HPLC. Molecular characterization was performed using Edman degradation, mass spectrometry, amino acid composition, and circular dichroism. Immunological properties were analyzed by immunoblotting, ELISA, and ELISA inhibition experiments.


RESULTS
We overexpressed rproSin a 1 as a single polypeptide with both large and small chains linked by an internal processed fragment at high yield. The purified rproSin a 1 (>95%) was obtained as a monomeric and soluble protein. rproSin a 1 showed equivalent structural and immunological properties to natural heterodimeric Sin a 1 allergen. rproSin a 1 was recognized by 75% of the patients allergic to mustard. The inhibitory capacity of rproSin a 1 to the total allergenicity of mustard extracts varied from 13 to 83% in different patients, with a mean value of 54%.


CONCLUSIONS
rproSin a 1 is a good candidate to replace natural allergen in diagnosis protocols of mustard allergy. P. pastoris has been demonstrated to be a suitable expression system for the production of allergenic derivates of Sin a 1 that could be used for immunotherapy purposes in future.
Palomares
Oscar
O
Departamento de Bioquímica y Biología Molecular I, Facultad de Ciencias Químicas, Universidad Complutense, Madrid, Spain.
Cuesta-Herranz
Javier
J
Rodríguez
Rosalía
R
Villalba
Mayte
M
D000485:Allergens / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D000595:Amino Acid Sequence; D052179:Antigens, Plant; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000097:blood; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D016133:Polymerase Chain Reaction; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D020539:Sequence Analysis, Protein; D031226:Sinapis / Q000276:immunology*
D000485:Allergens; D052179:Antigens, Plant; D018076:DNA, Complementary; D010940:Plant Proteins; D011498:Protein Precursors; D011994:Recombinant Proteins; C079479:Sin a I protein, Sinapis alba; D007073:Immunoglobulin E