Cloning, expression, and clinical significance of the major allergen from ash pollen, Fra e 1.
BACKGROUND
Ash tree, an Oleaceae member, is considered an important source of pollen allergy in Central Europe. Fra e 1 is a protein of the Ole e 1-like family, which regulates pollen tube growth. It has been suggested to be a relevant allergen from ash pollen.


OBJECTIVE
Cloning Fra e 1-cDNA and overproducing a properly folded recombinant allergen to analyze its clinical significance.


METHODS
Fra e 1-encoding cDNA was amplified by PCR, cloned in Escherichia coli , and sequenced. The recombinant allergen was produced in Pichia pastoris and used in immunoblotting, ELISA, histamine release, and skin prick tests. Sera and blood cells from patients sensitized to ash pollen as well as anti-Ole e 1 monoclonal and polyclonal antisera were used.


RESULTS
Recombinant Fra e 1 (rFra e 1) is a glycoprotein of 145 amino acids exhibiting 82%, 88%, and 91% identity with Syr v 1, Ole e 1, and Lig v 1, allergens of the Oleaceae family. It was secreted to the extracellular medium of the yeast cultures and purified by means of 3 chromatographic steps. IgG from Ole e 1-specific antibodies recognized rFra e 1. IgE antibodies from ash-sensitized patients bound to rFra e 1 with a prevalence of 75%. The recombinant allergen induced histamine release. Twenty-nine of 30 ash-sensitized patients were positive to rFra e 1 by skin prick tests.


CONCLUSIONS
Fra e 1 is a relevant allergen in ash pollen sensitization. It has been efficiently produced in P pastoris and could be used in diagnosis.
Barderas
Rodrigo
R
Departamento Bioquímica y Biologia Molecular, Universidad Complutense de Madrid, Spain.
Purohit
Ashok
A
Papanikolaou
Ioanna
I
Rodríguez
Rosalía
R
Pauli
Gabrielle
G
Villalba
Mayte
M
D000485:Allergens / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D052179:Antigens, Plant; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D006636:Histamine Release; D006801:Humans; D010843:Pichia / Q000378:metabolism; D011058:Pollen / Q000276:immunology*; D011994:Recombinant Proteins / Q000378:metabolism; D012882:Skin Tests; D014197:Trees / Q000276:immunology*
D000485:Allergens; D000911:Antibodies, Monoclonal; D052179:Antigens, Plant; D018076:DNA, Complementary; C497840:Fra e 1 allergen, Fraxinum excelsior; D011994:Recombinant Proteins