Cloning and expression of Ole e I, the major allergen from olive tree pollen. Polymorphism analysis and tissue specificity.

M Villalba,E Batanero,R.I Monsalve,M.A González De La Peña,C Lahoz,R Rodríguez

doi:10.1016/s0021-9258(17)36594-8

M Villalba, E Batanero + Show 4 more

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https://doi.org/10.1016/s0021-9258(17)36594-8

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Abstract

Ole e I, the major allergen from the olive tree (Olea europaea), is one of the main causes of allergy in Mediterranean countries and some areas of North America. The cloning and sequencing of several cDNAs coding for the olive allergen have been achieved. cDNA has been synthesized from total pollen RNA and amplified by using the polymerase chain reaction. The nucleotide sequence data demonstrate the existence of microheterogeneities in at least 37 positions out of the 145 amino acids of Ole e I, thus explaining the high degree of polymorphism exhibited by the natural protein. One of the sequenced cDNAs encoding a full-length isoform was inserted into the plasmid vector pGEX-2T and overexpressed. The recombinant Ole e I has been produced in Escherichia coli as a fusion protein with glutathione S-transferase of Schistosoma japonicum. This chimeric protein was purified by affinity chromatography on a glutathione-Sepharose 4B column and digested with thrombin to release the recombinant allergen. Both the fusion protein and the recombinant Ole e I were recognized in Western blot analysis by rabbit polyclonal and mouse monoclonal antisera raised against native Ole e I as well as by the IgE of olive pollen-sensitive human sera. This indicates that the recombinant production of individual isoforms may be useful for the improvement of reagents to be used in diagnosis and therapy of IgE-mediated disorders. In addition, Ole e I mRNA has been observed to be pollen-specific as shown in a Northern blot analysis.

Highlights

The expression of the inserted cDNA fragment generated a recombinant proteinG, ST-OLE3c,with an estimatemd olecular mass of 42 kDa (Fig. 51, which agrees with the molecular mass of 26 kDa for glutathione S-transferase [29] and 16.3 kDa for the polypeptide chain of Ole e I ( 5 ) . the yield was about 100 mgof fusion proteiditer of cell culture, less than 2% of this protein remained soluble after lytic treatment of the transformed cells (Fig. 5 ) .After treatment of the pellet in 8 M urea, spin column separation, andfolding overnight [30],the soluble material was chromatographed over a glutathione-Sepharose affinity column
With afull-length open reading frame nucleotide sequence, a construction was designed to achieve the expression of the recombinant allergen
The sequence data for the amplified cDNA revealed considerable heterogeneity for the Ole e I allergen that would comprise a family of proteins including a t least ninemembers

Summary

POLYMORPHISM ANALYSIS AND TISSUE SPECIFICITY*

(Received forpublication, December 21, 1993, and inrevised form, February 24, 1994). Mayte Villalba, Eva Batanero, Rafael I. More than 70%of olive-allergic patients are sensitive toOle e I, the major allergen from Olea europaea pollen [3] This protein has been isolated,purified to homogeneity, and characterized by biochemical methods, including the determinatioonf its aminoacid sequence[4,5].Ole e I is an acidic protein, that exhibits two variants of the same 145-residue polypeptide chain, glycosylated (20 kDa) andnonglycosylated (18.5 kDa) [6], and shows microheterogeneity a t several positions of its primary structure[5].The biological role of Ole e I in the olive pollen is unknown.

MATERIALS AND METHODS

RESULTS

CElxoapnridensgsion ofPAOollileverengen

DISCUSSION

Findings

FTN FTN