Che a 1: recombinant expression, purification and correspondence to the natural form.
BACKGROUND
Pollinosis to chenopods is one of the main causes of allergy in desertic regions and it is increasing in the South of Europe and Western USA. Che a 1 is a major allergen for chenopod-allergic subjects and belongs to the Ole-e-1-like family of proteins.


METHODS
Pichia pastoris yeast has been used as expression system to produce the recombinant form of Che a 1 (rChe a 1). The allergen was isolated using a gel permeation column and reverse-phase/high-performance liquid chromatography. Molecular characterization was performed using Edman degradation, mass spectrometry and concanavalin A staining. Sera from patients allergic to chenopod pollen, as well as polyclonal and monoclonal antibodies raised against Ole e 1, were used in immunoblotting, ELISA and inhibition assays for immunological characterization of rChe a 1.


RESULTS
The allergen was purified to homogeneity with a final yield of 15 mg/l of cell culture and showed a glycosylated character. N-terminal amino acid sequence of rChe a 1 and molecular mass were according to those of the protein isolated from chenopod pollen. The recombinant allergen maintained the IgG and IgE epitopes of the natural allergen deduced from the immunological assays.


CONCLUSIONS
Structural and in vitro immunological properties of rChe a 1 produced in P. pastoris were equivalent to those of the natural form of the allergen and, thus, it could be used in testing patients allergic to chenopods.
Barderas
Rodrigo
R
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, Madrid, Spain.
Villalba
Mayte
M
Rodríguez
Rosalía
R
D000485:Allergens / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D052179:Antigens, Plant; D015153:Blotting, Western; D027463:Chenopodium album / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006967:Hypersensitivity / Q000097:blood / Q000276:immunology*; D007073:Immunoglobulin E / Q000097:blood; D010843:Pichia / Q000235:genetics; D011058:Pollen / Q000737:chemistry / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000485:Allergens; D052179:Antigens, Plant; C451660:Che a 1 allergen; D011994:Recombinant Proteins; D007073:Immunoglobulin E