D2.41 - Production of recombinant Cuc ma 5: A Step Towards Molecular Diagnosis of Pumpkin Seed Allergy

The consumption of edible seeds has increased due to their recognized nutritional benefits, paralleled by a rising prevalence of seed allergies. Pumpkin seed allergy remains poorly characterized at the molecular level. Seed storage proteins, particularly 2S albumins, are major allergens due to their structural stability and resistance to heat and digestion. Cuc ma 5, a 2S albumin from pumpkin seed, has been proposed as a clinically relevant allergen associated with severe reactions. However, recombinant Cuc ma 5 has not previously been reported, limiting its application in molecular diagnostics.

The DNA sequence encoding an IL-2 signal peptide followed by the small and large chain of Cuc ma 5 with a linker in between, and a C-terminal 6xhistidine tag was synthesized and cloned into the expression plasmid pcDNA3.1(+). The sequence was codon optimized for mammalian cells. Expi293 HEK cells were transfected with the plasmid and the supernatant was harvested 4 days later. Recombinant Cuc ma 5 was purified using Ni-NTA affinity chromatography. Elution fractions were analyzed by SDS-PAGE. Protein identity was analyzed by Western blot and ELISA using an anti-His antibody and serum from a patient with confirmed pumpkin seed allergy, respectively.

The expression plasmid was amplified at high purity. Recombinant Cuc ma 5 was expressed in transfected Expi293 HEK cells as a soluble protein, which was secreted to the culture supernatant. The recombinant protein was eluted from a Ni-NTA column at high purity. SDS-PAGE demonstrated a distinct band, consistent with the expected molecular weight of 13607 Dalton. Western blot analysis confirmed the presence of the C-terminal histidine tag on Cuc ma 5. Recombinant Cuc ma 5 bound IgE from a pumpkin seed allergic patient as demonstrated by ELISA.

We successfully expressed recombinant Cuc ma 5 in mamalian cells. To our knowledge, this represents the first reported recombinant production of this allergen. The availability of purified rCuc ma 5 provides an essential tool for future IgE-binding studies and component-resolved diagnostic approaches in pumpkin seed allergy.