cDNA cloning and heterologous expression of the major allergens from peach and apple belonging to the lipid-transfer protein family

Background Lipid‐transfer proteins (LTPs) have been identified as major allergens of Rosaceae fruits in populations living in areas virtually free of Fagales trees, such as several Mediterranean communities. Pru p 3 and Mal d 3, the allergens from peach and apple, respectively, have a main clinical relevance in these areas. Objetive To isolate and characterize cDNAs for Pru p 3 and Mal d 3,and to produce recombinant Pru p 3 in the yeast Pichia pastoris. Methods cDNAs for both allergens were isolated by polymerase chain reaction using non‐degenerated primers. Expression of Pru p 3 was performed in P. pastoris using the pPIC 9 vector. The recombinant product was purified by gel‐filtration chromatography followed by RP‐HPLC. Immunodetection and immunoblot inhibition assays were carried out with sera from peach‐allergic patients. Results The cDNAs for both Pru p 3 and Mal d 3 showed a 273 open reading frame coding for the 91 amino acid mature polypeptides. The deduced amino acid sequences exhibited N‐terminal regions fully identical to those previously determined for the natural peach and apple allergens. Pru p 3 was expressed in P. pastoris at 20 mg/L of culture medium. The recombinant allergen showed the same N‐terminal sequence (plus a glutamic acid added for proper extracellular expression) and apparent molecular size as natural Pru p 3. Both the recombinant and natural forms of Pru p 3 displayed similar immunoglobulin (Ig)E‐binding capacity in immunodetection and immunoblot inhibition assays. Conclusions Comparison of the complete primary structures of mature Pru p 3 and Mal d 3 deduced from their corresponding cDNA clones supports the close relationship between both allergens. Recombinant Pru p 3 binds IgE in vitro like its natural counterpart. Therefore, it can be a useful tool for specific diagnosis and structural studies.