RT Journal Article T1 Production and detailed characterization of biologically active olive pollen allergen Ole e 1 secreted by the yeast Pichia pastoris A1 Huecas, Sonia A1 Villalba Díaz, María Teresa A1 González Arana, Eva María A1 Martínez Ruiz, Antonio A1 Rodríguez García, Rosalía AB 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 α‐mating factor prepropeptide using the pPIC9 vector, was inserted into the yeast genome under the control of the AOX 1 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. SN 0014-2956 SN 1432-1033 YR 1999 FD 1999-04 LK https://hdl.handle.net/20.500.14352/102617 UL https://hdl.handle.net/20.500.14352/102617 LA eng NO Eur. J. Biochem. 261, 539-545 (1999) NO Dirección General de Investigación Científica y Técnica NO Ministerio de Educación y Ciencia DS Docta Complutense RD 8 abr 2025