| Abstract [eng] |
An allergy is an altered immune response to an allergen involving allergen-specific IgE antibodies and T helper cells. Conventional diagnostics rely on allergen extracts derived from natural sources, but their variability and limited standardization reduce assay accuracy and reproducibility. Recombinant allergens provide defined allergen molecules that enable the precise determination of a patient’s molecular sensitization profile. This component-resolved diagnostics (CRD) approach forms the basis of molecular allergology, but its application requires comprehensive molecular characterization and robust production systems. In this work, the black tiger shrimp allergen Pen m 4, dog allergen Can f 6, and common wasp allergen Ves v 5 were produced and characterized using the yeast Pichia pastoris, while also providing comparative data against the traditional E. coli system. During protein expression optimization, the effects of the maltose-binding protein (MBP) on allergen expression, stability, and IgE reactivity were evaluated. Analysis of MBP-allergen fusion proteins revealed known and previously unreported proteolytic cleavage patterns. The effects of glycosylation, lyophilization, calcium chelation, and purification strategies on the stability, antigenicity, and IgE reactivity of recombinant allergens were also investigated. These findings contribute to CRD and identify preanalytical and stability-related factors that should be controlled to improve diagnostic reliability. |
