Cloning and expression of biologically active Plantago lanceolata pollen allergen Pla l 1 in the yeast Pichia pastoris.
The glycoprotein Pla l 1 is the major allergen from English plantain (Plantago lanceolata) pollen, which is a common cause of pollinosis in temperate areas. Three complete cDNAs for Pla l 1 isoforms were isolated by PCR using specific 3' and 5' primers. All three Pla l 1 cDNAs code for a 25-residue leader peptide and a 131-residue mature protein that contains two polymorphic positions, an N-glycosylation site at position 107 and six cysteine residues involved in three disulphide bridges. The allergen variant Pla l 1.0101 was produced in Pichia pastoris at a yield of 20 mg per litre of culture as a mixture of non-glycosylated (17 kDa), glycosylated (23 kDa) and dimeric (32-39 kDa) forms. Recombinant Pla l 1 (rPla l 1) was purified by affinity chromatography with an anti-natural Pla l 1 (anti-nPla l 1) monoclonal antibody, and its molecular and immunological properties were compared with the natural allergen by CD spectroscopic analysis, enzymic deglycosylation, lectin-binding assay, immunodetection and ELISA-inhibition assays using sera from plantain-allergic patients. The recombinant allergen is properly folded, as deduced from CD spectra, and the immunodominant allergenic epitopes of the natural allergen are preserved in rPla l 1. These results allow us to conclude that P. pastoris is a convenient system for the efficient production of biologically active rPla l 1, which could have a potential use for clinical purposes. Furthermore, a sequence similarity of Pla l 1 to the major allergen from the olive tree pollen, Ole e 1, is revealed in this work, and the allergenic cross-reactivity between both allergens has been studied.
Calabozo
Belén
B
Departamento I + D, ALK-Abelló, S.A., c/Miguel Fleta 19, 28037 Madrid, Spain.
Díaz-Perales
Araceli
A
Salcedo
Gabriel
G
Barber
Domingo
D
Polo
Florentino
F
D000485:Allergens / Q000096:biosynthesis* / Q000235:genetics* / Q000276:immunology; D000595:Amino Acid Sequence; D001483:Base Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D000939:Epitopes / Q000235:genetics; D014644:Genetic Variation / Q000235:genetics; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics* / Q000276:immunology; D006031:Glycosylation; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology / Q000378:metabolism; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins; D010943:Plantago / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D011058:Pollen / Q000737:chemistry / Q000276:immunology; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D000485:Allergens; D018076:DNA, Complementary; D000939:Epitopes; D006023:Glycoproteins; C473663:Pla l 1 allergen, Plantago lanceolata; D010940:Plant Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D007073:Immunoglobulin E