Molecular cloning and expression of active Ole e 3, a major allergen from olive-tree pollen and member of a novel family of Ca2+-binding proteins (polcalcins) involved in allergy

A cDNA encoding Ole e 3, a major allergen from olive-tree pollen, has been cloned and sequenced. A strategy based on two-step PCR amplification toward s the 5' end and 3' end, with an internal specific primer, has been used. T he isolated cDNA contains an open reading frame coding for a. polypeptide o f 84 amino acids, which is in agreement with the composition and molecular mass of the natural allergen, exhibiting two 12-residue segments homologous to Ca2+-binding sites of EF-hand type. The cDNA was inserted into the pET- 11b expression vector and over-expressed in Escherichia coli. The purified recombinant protein shows identical secondary structure to that of the natu ral allergen and is able to bind both IEE from sera of patients allergic to olive pollen and polyclonal antibodies raised against olive-pollen Ole e 3 . The capacity of binding Ca2+ has been demonstrated for both natural and r ecombinant allergens. RNA transcripts of Ole e 3 were only detected in poll en tissue. Northern-blot and Western-blot analyses of poly(A)(+)RNA and pro tein extracts, respectively, obtained from a variety of olive-tree-related and nonrelated mature pollens demonstrated the presence of Ole e 3 homologo us proteins. This indicates a sequence conservation and widespread distribu tion for this family of Ca2+-binding proteins that can be responsible for a llergenic cross-reactivity. We suggest the tentative generic name of polcal cins for the members of this family of Ca2+-binding proteins from pollen.