Production and detailed characterization of biologically active olive pollen allergen Ole e 1 secreted by the yeast Pichia pastoris.
The glycoprotein Ole e 1 is a significant aeroallergen from the olive tree (Olea europaea) pollen, with great clinical relevance in the Mediterranean area. To produce a biologically active form of recombinant Ole e 1, heterologous expression in the methylotrophic yeast Pichia pastoris was carried out. A cDNA encoding Ole e 1, fused to a Saccharomyces cerevisiae alpha-mating factor prepropeptide using the pPIC9 vector, was inserted into the yeast genome under the control of the AOX1 promoter. After induction with methanol, the protein secreted into the extracellular medium was purified by ion-exchange and size-exclusion chromatography. The structure of the isolated recombinant Ole e 1 was determined by chemical and spectroscopic techniques, and its immunological properties analysed by blotting and ELISA inhibition with Ole e 1-specific monoclonal antibodies and IgE from sera of allergic patients. The allergen was produced at a yield of 60 mg per litre of culture as a homogeneous glycosylated protein of around 18.5 kDa. Recombinant Ole e 1 appears to be properly folded, as it displays spectroscopic properties (CD and fluorescence) and immunological reactivities (IgG binding to monoclonal antibodies sensitive to denaturation and IgE from sera of allergic patients) indistinguishable from those of the natural protein. This approach gives high-yield production of homogeneous and biologically active allergen, which should be useful for scientific and clinical purposes.
Huecas
S
S
Departamento de Bioquímica y Biología Molecular, Facultad de Química, Universidad Complutense, Madrid, Spain.
Villalba
M
M
González
E
E
Martínez-Ruiz
A
A
Rodríguez
R
R
D000485:Allergens / Q000276:immunology*; D052179:Antigens, Plant; D002942:Circular Dichroism; D005656:Fungal Proteins / Q000276:immunology; D006023:Glycoproteins / Q000737:chemistry / Q000276:immunology; D006801:Humans; D006967:Hypersensitivity / Q000209:etiology; D007073:Immunoglobulin E / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D013058:Mass Spectrometry; D019083:Mediterranean Region; D010446:Peptide Fragments / Q000737:chemistry; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D010940:Plant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology; D011058:Pollen / Q000276:immunology*; D011134:Polysaccharides / Q000032:analysis; D011994:Recombinant Proteins / Q000276:immunology; D013050:Spectrometry, Fluorescence; D014170:Transformation, Genetic
D000485:Allergens; D052179:Antigens, Plant; D005656:Fungal Proteins; D006023:Glycoproteins; D007074:Immunoglobulin G; C081946:Ole e I protein, Olea europaea; D010446:Peptide Fragments; D010940:Plant Proteins; D011134:Polysaccharides; D011994:Recombinant Proteins; D007073:Immunoglobulin E