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Original Article

 

Characterization of Profilin and Polcalcin Panallergens From Ash Pollen

 

S Mas,1* M Garrido-Arandia,1,2* E Batanero,1 A Purohit,3 G Pauli,3 R Rodríguez,1 R Barderas,1 M Villalba1

1Departamento Bioquímica y Biología Molecular I, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, Madrid, Spain
2Centro de Biotecnología y Genómica de Plantas (UPM-INIA), Campus de Montegancedo, Pozuelo de Alarcón, Madrid, Spain
3Service de Pneumologie, Hôpital Lyautey, Hopitaux Universitaires de Strasbourg, Strasbourg, France
*Both authors contributed equally to this work

J Investig Allergol Clin Immunol 2014; Vol. 24(4): 257-266

 

 Abstract


Background: Ash (Fraxinus excelsior) is an important source of allergenic pollen in temperate areas of Europe. Profilin and polcalcin are 2 important panallergens involved in cross-reactivity between different sources.

Objective: To clone and produce Fra e 2 (profilin) and Fra e 3 (polcalcin) as recombinant proteins and evaluate their immunological properties using the natural forms obtained from ash pollen.

Methods: Total RNA from ash pollen was used as a template to obtain the specific complementary DNA (cDNA) sequences of the 2 panallergens. The cDNA-encoding sequences were cloned into the pET11b expression vector and used to transform BL21 (DE3) Escherichia coli cells. Proteins were expressed, purified by chromatography, and characterized structurally by circular dichroism, mass spectrometry, and immunologically by western blot and ELISA using profilin and polcalcin polyclonal antibodies and human sera from ash pollen–sensitized patients.

Results: Profilin and polcalcin amino acid sequences from ash pollen showed a high degree of identity with homologous allergens from different sources. The cDNA-encoding allergen sequences were expressed as nonfusion recombinant proteins and purified to homogeneity. Secondary structure values were similar to those obtained from other members of these families. Allergenic properties of the recombinant allergens were observed to be equivalent to those of the natural counterparts of F excelsior pollen.

Conclusions: Fra e 2 and Fra e 3 recombinant allergens might be used in clinical diagnosis to determine profilin- and polcalcin-specific IgE levels present in the sera of ash pollen–sensitized patients, thus facilitating the finding of the sensitizing source in areas with complex sensitization profiles.

Key words: Ash pollen. Panallergens. Polcalcin. Profilin. Recombinant allergen.