Expression, purification, characterization, and patient IgE reactivity of new macadamia nut iso-allergen

• Published in: Protein expression and purification 2023-03, Vol.203, p.106211-106211, Article 106211
• Main Authors: Zhang, Yuzhu, Bhardwaj, Shilpa R., Lyu, Shu-Chen, Chinthrajah, Sharon, Nadeau, Kari C., Li, Caiming
• Format: Article
• Language: English
• Subjects: Allergens - chemistry; Antigens, Plant - chemistry; Antigens, Plant - genetics; Escherichia coli - genetics; Humans; Immunoglobulin E - chemistry; Legumain; Legumins; Mac i 2; Macadamia - genetics; Nut Hypersensitivity - diagnosis; Nut Hypersensitivity - metabolism; Plant Proteins - chemistry; Plant Proteins - genetics; Post-translational modification; Protein Isoforms; Recombinant allergen; Tree nut allergen
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2022.106211

Structural and functional information about food allergens is essential for understanding the allergenicity of food proteins. All allergens belong to a small number of protein families. Various allergens from different families have been successfully produced recombinantly in E. coli for their characterization and applications in allergy diagnosis and treatment. However, recombinant hexameric 11S seed storage protein has not been reported, although numerous 11S legumins are known to be food allergens, including the recently identified macadamia nut allergen Mac i 2. Here we report the production of a macadamia nut legumin by expressing it in E. coli with a substrate site of HRV 3C protease and cleaving the purified protein with HRV 3C protease. The protease divided the protein into two chains and left a native terminus for the C-terminal chain, resulting in a recombinant hexameric 11S allergen for the first time after the residues upstream to the cleavage site flipped out of the way of the trimer-trimer interaction. The 11S allergens are known to have multiple isoforms in many species. The present study removed an obstacle in obtaining homogeneous allergens needed for studying allergens and mitigating allergenicity. Immunoreactivity of the protein with serum IgE confirmed it to be a new isoform of Mac i 2. •A new isoform of a recently identified macadamia nut allergen was expressed in E. coli.•An HRV 3C protease substrate site was incorporated into the recombinant protein.•HRV 3C protease processing of the expressed protein mimics the natural post-translational protease modification of the 11S proteins.•A hexameric 11S legumin was recombinantly produced for the first time.•Serum IgE reactions identified the protein as a new isoform of Mac i 2.