IDENTIFICATION AND CLONING OF PRS-A-1, A 32-KDA ENDOCHITINASE AND MAJOR ALLERGEN OF AVOCADO, AND ITS EXPRESSION IN THE YEAST PICHIA-PASTORIS

Avocado, the fruit of the tropical tree Persea americana, is a source of allergens that can elicit diverse IgE-mediated reactions including anaphylaxis in sensitized individuals. We characterized a 32-kDa major avocado allergen, Prs a 1, which is recognized by 15 out of 20 avocad o- and/or latex-allergic patients. Natural Prs a 1 was purified, and i ts N-terminal and two tryptic peptide sequences were determined. We is olated the Prs al encoding cDNA by PCR using degenerate primers and 5' -rapid amplification of cDNA ends. The Prs a 1 cDNA coded for an endoc hitinase of 326 amino acids with a leader peptide of 25 amino acids. W e expressed Prs a 1 in the yeast Pichia pastoris at 50 mg/liter of cul ture medium. The recombinant Prs a I showed endochitinase activity, in hibited growth and branching of Fusarium oxysporum hyphae, and possess ed IgE binding capacity. IgE cross-reactivity with latex proteins incl uding a 20-kDa allergen, most likely prohevein, was demonstrated, prov iding an explanation for the commonly observed cross-sensitization bet ween avocado and latex proteins. Sequence comparison showed that Prs a 1 and prohevein had 70% similarity in their chitin-binding domains. C haracterization of chitinases as allergens has implications for engine ering transgenic crops with increased levels of chitinases.