Expression and characterization of three important panallergens from hazelnut.

Area of Science:

• Food Allergy Research
• Protein Biochemistry
• Immunology

Background:

• Several hazelnut allergens have been identified, exhibiting varying clinical relevance and cross-reactivity.
• Accurate diagnosis of hazelnut allergy relies on well-characterized, pure allergens for component-resolved testing.

Purpose of the Study:

• To develop scalable protocols for producing three key recombinant hazelnut allergens: Cor a 1.04, Cor a 2, and Cor a 8.
• To ensure these recombinant allergens are folded correctly and retain immunological activity for diagnostic applications.

Main Methods:

• Heterologous protein expression strategies (fusion and non-fusion) were optimized for each allergen.
• Proteins were purified using a two-step Fast Protein Liquid Chromatography (FPLC) method.
• Identity, structure, and IgE reactivity were confirmed via N-terminal sequencing, mass spectrometry, CD-spectroscopy, and NMR analysis.

Main Results:

• Scalable protocols yielded approximately 10 mg/L of pure rCor a 1.04 and rCor a 2 (as fusion proteins in E. coli).
• Expression of rCor a 8 as a non-fusion protein in yeast yielded a higher amount of 30 mg/L.
• Purified recombinant allergens demonstrated verified IgE antibody reactivity.

Conclusions:

• Successfully established production protocols for key recombinant hazelnut allergens.
• The availability of these pure, active allergens will significantly advance in vitro diagnostic tests for hazelnut allergy.
• This work supports the development of more sensitive and specific component-resolved diagnostic approaches.