Production of Recombinant Mal d 3, a Major Apple Allergen, in Pichia Pastoris, to Investigate the Impact of the Food Matrix and Post-Translational Modifications on Mal d 3 Immuno-Reactivity

Background. Non-specific lipid transfer proteins (nsLTPs) are important plant food allergens able to induce severe systemic reactions in sensitized individuals. The aim of this project is to evaluate the impact of food matrix interaction and post-translational modifications on the allergenicity of nsLTPs, studying apple as a model food and its nsLTP, Mal d 3, as model food allergen. Notably, to perform these studies, a high amount of well characterized and biologically active allergen is necessary. Therefore, our first goal is to produce rMal d 3 in the yeast Pichia pastoris,which has been shown to be an efficient system for expressing huge amount of soluble and immunologically active nsLTPs.

Methods. The DNA sequence of mature Mal d 3 was codon optimized for P. pastoris, cloned into the pPICZαA vector and propagated in E. coli cells. The linearized pPICZαA-Mal d 3 plasmid was used to transform P. pastoris cells. Positive transformants were selected and the presence of the insert analyzed by PCR. Selected clones were cultured and screened for the expression of the recombinant protein by SDS-PAGE and immunoblotting with rabbit antiserum against nsLTP and allergic patients’ sera. rMal d 3 was purified from the culture supernatant by chromatographic methods (IEC and RP-HPLC) and analyzed by MALDI-TOF mass spectrometry.

Results. Multy-copy P. pastoris clones (n=12) were selected, cultured and analyzed. Immunoblotting results showed that recombinant proteins retain both IgG- and IgE-binding capabilities. Two clones, highly expressing rMal d 3, were selected to perform a large scale production of the recombinant allergen. Purified rMal d 3 migrates in SDS-PAGE as a double band between 10 and 15 kDa. MALDI-TOF MS analysis confirmed the identity of the purified recombinant proteins, providing 9,553 kDa and 9,752 kDa (corresponding to the calculated theoretical masses of 9,560 kDa and 9,761 kDa, respectively), which resulted from different cleavage site of the signal sequence. Both forms were recognized by both anti-nsLTP antiserum and allergic patients’ sera.

Conclusions. Our data confirm the suitability of P. pastoris for the expression of nsLTPs. Purified rMal d 3 will be used to perform specific experiments to evaluate the impact of selected food matrix components (pectins, lipids..) and PTMs on its allergenicity. Supported by Marie-Curie project CARAMEL 626572.