N-terminal Sequencing Ara h 2 original files

<b>Introduction</b>: Recombinant allergens are an important diagnostic tool for determining the IgE-sensitization profile of patients, assessing the risk of symptom severity and potential clinical cross-reactivity. In this context, the use of different host cells for recombinant expression must be evaluated in terms of IgE reactivity and diagnostic value. Therefore, recombinant Ara h 2, the major peanut allergen, was produced in yeast,<i> </i>structurally characterized<i> </i>and investigated in respect to IgE-binding and allergenic properties.<b>Methods</b>: Ara h 2 was produced as recombinant protein using yeast and <i>E. coli</i> expression systems. Purified proteins were assessed using SDS-PAGE (reducing and non-reducing conditions), CD-spectroscopy and IgE reactivity.<b>Results</b>: Recombinant Ara h 2^wt expressed in yeast resulted in an additional predominant band of approximately 12 kDa upon DTT treatment. In contrast, the molecular mass of rAra h 2 expressed in <i>E. coli</i> (Ara h 2^{<em>E.coli</em>}) remained unaffected by reduction. Analysis of rAra h 2^wt confirmed the presence of two Ara h 2-derived peptides, one with the expected N-terminus and the other with an N-terminal glycine residue. <i>In silico</i> analysis revealed the presence of a Kex2 cleavage site (R₅₈R₅₉*G₆₀). To test whether Kex2 cleavage affects an IgE-epitope, mutagenesis of this cleavage site from R₅₈ to E₅₈ (Ara h 2^mut) was performed. DTT treatment of rAra h 2^mut purified from yeast showed that no cleavage of the protein had occurred. No effect on IgE binding could be observed as all rAra h 2 preparations showed IgE-reactivity. Cross-linking of human serum IgE and monoclonal human Ara h 2-specific IgE antibodies showed comparable mediator release in response to Ara h 2^wt and rAra h 2^mut. However, utilizing a specific combination of human Ara h 2-specific IgE antibodies revealed slight epitope diversity between wildtype and mutated rAra h 2.<b>Discussion</b>: An endogenous protease, like Kex2 from the expression system, can affect the structural integrity of the target protein, leading to a slightly altered epitope structure. Even this finding has little or no impact for diagnosis, a suitable expression system and a detailed physico- and immunochemical characterization of recombinant allergens prior to their use as a diagnostic tool are of great importance.