Cloning and expression of biologically active Plantago lanceolata pollen allergen Pla l 1 in the yeast Pichia pastoris

Abstract

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.

Che a 1: Recombinant Expression, Purification and Correspondence to the Natural Form

“…cDNA encoding Che a 1 has been efficiently expressed in P. pastoris cells and secreted in the extracellular culture medium, which has low levels of contaminants and facilitates the isolation of the allergen. The amount of purified rChe a 1 from the P. pastoris culture is in agreement with the yield reported for other proteins of the Ole e 1-like family in the same expression system [18, 22], which provides a well-defined soluble protein that would be suitable for clinical and scientific purposes.…”

“…Several members of this family of proteins have been recognized as relevant allergens: Che a 1 affects >70% of patients sensitized to chenopod pollen, Ole e 1 >70% of patients allergic to olive pollen, Lol p 11 >65% of ryegrass-allergic subjects and Pla l 1 >80% of English plantain-allergic patients [12, 17, 18, 19]. Che a 1 displays very low cross-reactivity with Ole e 1 as well as with Pla l 1 [12, 20].…”

“…Because of the low yield obtained during the purification of the natural allergen (nChe a 1) from the pollen [12], the production of rChe a 1 with immunological and structural features of the natural form can be useful for several purposes. Pichia pastoris yeast was used in the production of different members of the Ole e 1-like family such as Ole e 1, Lig v 1, Syr v 1, Pla l 1 [18, 21, 22]and Fra e 1 [unpubl. data], which were secreted as soluble and active allergens with high yields.…”

The Principle of Homologous Groups in Regulatory Affairs of Allergen Products – A Proposal

“…The only identified pollen allergen of Plantago lanceolata in the IUIS list, Pla l 1, is a member of the Ole e 1 family with an amino acid sequence identity of 40% with Ole e 1, but natural Pla a 1 is to a negligible degree cross-reactive to natural Ole e 1 [78]. …”

Challenges for Allergy Diagnosis in Regions with Complex Pollen Exposures

“…There is a specific marker, Pla l 1 [63], an Ole e 1-like member, that is available for specific diagnosis. Epidemiological studies suggest that plantain allergy is relevant in some areas [2,3].…”