cDNA cloning and heterologous expression of the major allergens from peach and apple belonging to the lipid-transfer protein family
Article first published online: 28 FEB 2002
Clinical & Experimental Allergy
Volume 32, Issue 1, pages 87–92, January 2002
How to Cite
Diaz-Perales, A., Garcia-Casado, G., Sanchez-Monge, R., Garcia-Selles, F. J., Barber, D. and Salcedo, G. (2002), cDNA cloning and heterologous expression of the major allergens from peach and apple belonging to the lipid-transfer protein family. Clinical & Experimental Allergy, 32: 87–92. doi: 10.1046/j.0022-0477.2001.01257.x
- Issue published online: 28 FEB 2002
- Article first published online: 28 FEB 2002
- Submitted 8 February 2001; revised 19 June 2001; accepted 16 July 2001
- lipid-transfer proteins;
- plant allergen;
- recombinant allergen;
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.