Immunological and molecular characterization of the major allergens from lilac and privet pollens overproduced in Pichia pastoris.
The main allergens from privet and lilac pollens, Lig v 1 and Syr v 1, are proteins homologous to Ole e 1 and have been shown to be involved in cross-reactivity. To overproduce the correctly folded Lig v 1 and Syr v 1 allergens and to study their immunological properties in comparison with those of their natural counterparts. The yeast Pichia pastoris was used as an expression system to produce these recombinant allergens. The proteins were isolated by ion-exchange and size-exclusion chromatographies. Amino acid quantifying, Edman degradation, mass spectrometry and circular dichroism were carried out to obtain molecular properties of the recombinant proteins. Anti-Ole e 1 monoclonal and polyclonal antibodies, as well as sera from patients allergic to olive pollen, were used in immunoblotting and ELISA for immunological characterization. Recombinant Lig v 1 and Syr v 1 were secreted at high yield to the extracellular medium of the yeast. The purified proteins displayed the native conformation, as deduced from their spectroscopic properties and binding ability to an IgG monoclonal antibody. The recombinant allergens behaved similarly to their natural counterparts when they were analysed against Ole e 1-specific antibodies. IgE and IgG binding properties of lilac and privet allergens to olive allergic sera and Ole e 1-specific antibodies indicated that these molecules share common B-cell epitopes with Ole e 1. P. pastoris yeast is an appropriate system for the efficient production of Ole e 1-like allergens, which could be used as analogous allergens and predictors of clinical sensitization.
Departamento de Bioquímica y Biología Molecular, Facultad de Química, Universidad Complutense, 28040 Madrid, Spain.
E
E
González
M
M
Villalba
R
R
Rodríguez
D000485:Allergens / Q000235:genetics / Q000276:immunology; D052179:Antigens, Plant; D002942:Circular Dichroism; D003429:Cross Reactions; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D010843:Pichia / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics* / Q000276:immunology*; D011058:Pollen / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology; D006255:Rhinitis, Allergic, Seasonal / Q000276:immunology; D014162:Transfection
D000485:Allergens; D052179:Antigens, Plant; D007074:Immunoglobulin G; C081946:Ole e I protein, Olea europaea; D010940:Plant Proteins; D011994:Recombinant Proteins; C087029:Syr v I protein, Syringa vulgaris; D007073:Immunoglobulin E