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.[1]References
- Cloning and expression of biologically active Plantago lanceolata pollen allergen Pla l 1 in the yeast Pichia pastoris. Calabozo, B., Díaz-Perales, A., Salcedo, G., Barber, D., Polo, F. Biochem. J. (2003) [Pubmed]
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