Profiling recombinant major birch pollen allergen Bet v 1a and carbamylated variants with CZE and CIEF

A preparation of recombinant birch pollen allergen of Betula verrucosa isoform 1a (Bet v 1a) containing chemically modified (carbamylated) variants has been analyzed by CZE and CIEF. In CZE, employing a 100 mmol/L MES buffer at pH 6.50, with 0.4 mmol/L tetraethylenepentamine (TEPA) added, allowed for the resolution of 17 protein fractions. The CIEF profiling of the allergen preparation required a combination of a wide‐pH‐range carrier ampholyte (CA) of pH 3–10 with two narrow‐range CAs of pH 5–6 and 5–7. For CIEF, 91 mmol/L of glycine at pH 2.12 and 20 mmol/L of CHES at pH 10.00 were applied as anolyte and catholyte, respectively. The generated pH gradient was nonlinear with a flat slope for pH 4–6, thus providing an improved resolution. In CIEF, up to 18 protein fractions were distinguished as well. The p I of the target allergen Bet v 1a was 4.9 as determined by means of two p I marker compounds flanking the allergen. Relative purity of the target allergen within the preparation containing carbamylated variants was in accordance for both separation systems and varied between 40.7 and 42.8%.