Isolation of the main allergen Fra e 1 from ash (Fraxinus excelsior) pollen: comparison of the natural and recombinant forms Rodrigo Barderas, PhD*; Ashok Purohit, MD†; Rosalı ´a Rodrı ´guez, PhD*; Gabrielle Pauli, MD†; and Mayte Villalba, PhD* Background: Fra e 1 is a major allergen for ash pollen–sensitized individuals in northern and central Europe. It belongs to the Ole e 1–like family and displays high cross-reactivity with taxonomically related members. Objectives: To isolate and characterize natural Fra e 1 (nFra e 1) from ash pollen and to compare its structural, antigenic, and allergenic properties with those of its recombinant form (rFra e 1). Methods: The allergen was isolated by means of gel permeation chromatography and reverse-phase high-performance liquid chromatography columns. Molecular characterization was performed by means of Edman degradation, mass spectrometry, circular dichroism, concanavalin A lectin reaction, and anti– horseradish peroxidase polyclonal antibody. Immunologic charac- terization was performed using immunoblotting and enzyme-linked immunosorbent assay, inhibition experiments, and histamine release assays with serum samples from allergic patients with well-known reactivity to Fra e 1 or Ole e 1 and with polyclonal antiserum and monoclonal antibodies against Ole e 1. The protein used as a reference was rFra e 1, which was produced in the yeast Pichia pastoris. Results: Purified nFra e 1 appeared as 5 variants with different glycosylation degrees. Both nFra e 1 and rFra e 1 were equivalently folded as deduced from the spectroscopic analysis using circular dichroism. Both molecules share the antigenic and allergenic epitopes after the purification process, and the glycan group of nFra e 1 is a potential epitope. Natural Fra e 1 displayed strong cross-reactivity with Ole e 1. Conclusions: Natural Fra e 1 is a heterogeneously glycosylated protein with high allergenic relevance. It displays structural, antigenic, and allergenic similarity with rFra e 1. Both proteins could be used for clinical purposes. Ann Allergy Asthma Immunol. 2006;96:557–563. INTRODUCTION Ash (Fraxinus excelsior) tree is widely distributed across Europe, and its pollen constitutes an important cause of allergy in temperate zones in Europe, including northern Spain, the British Islands, central Europe, Scandinavia, and Russia. 1–3 The concomitant presence of ash pollen in the atmosphere together with other allergenic pollens, such as birch, has originated the absence of a deep knowledge about its relevance and allergenic profile. The presence of the major allergen Fra e 1 and panaller- gens such as profilin and polcalcin has been determined. 4,5 The recombinant form of Fra e 1 (rFra e 1) has recently been cloned, expressed in the methylotrophic yeast Pichia pastoris and purified. 6 The amino acid sequence of Fra e 1 was found to be highly similar to that of the major allergen Ole e 1 from olive pollen, and, therefore, it has been included in the Ole e 1–like family of proteins, to which Lig v 1 from privet and Syr v 1 from lilac belong. The immunologic response of rFra e 1 using in vitro and in vivo methods has reasonably con- firmed its correct folding and potential usefulness in clinical studies. Glycosylation and polymorphism are 2 common character- istics of natural allergens. Frequently, many variants are represented in the natural allergens in contrast to the recom- binant ones, in which only 1 homogeneous form of the allergen is obtained. Moreover, the purification of natural allergens involves important problems, such as the scarce availability of the biologic source and the low yield during the isolation process. The use of recombinant allergens, after their validation, could solve these difficulties. However, the practical availability of recombinant allergens tolerated a main inconvenience, such as the incorrect protein folding, perhaps owing to the lack of posttranslational modifications or to the instability of the allergen during the isolation pro- cess. These problems would produce a decrease in the rec- ognition of the recombinant allergen by the patients’ sera compared with the natural forms. Independent of the choice of the most representative form of the allergen, the natural form is essential for the validation of the recombinant form. The aims of this work are to isolate natural Fra e 1 (nFra e 1), to detect the glycan moiety, and to study its equivalence * Departmento Bioquı ´mica y Biologı ´a Molecular I, Facultad de Ciencias Quı ´micas, Universidad Complutense de Madrid, Madrid, Spain. † The Service de Pneumologie, Hopital Lyauteil, Hopitaux Universitaires de Strasbourg, Strasbourg, France. This work was supported by grant SAF2002-02711 (DGI) from the Minis- terio de Ciencia y Tecnologı ´a (Spain) and the French Society of Aerobiology (France). Received for publication July 14, 2005. Accepted for publication in revised form September 22, 2005. VOLUME 96, APRIL, 2006 557