title: Profiling recombinant major birch pollen allergen Bet v 1a and carbamylated variants with CZE and CIEF.
abstract: 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 pI of the target allergen Bet v 1a was 4.9 as determined by means of two pI 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%.
authors.affiliation: Division of Chemistry, Department of Molecular Biology, University of Salzburg, Salzburg, Austria.
authors.forename: Barbara
authors.initials: B
authors.lastname: Kronsteiner
authors.forename: Hans
authors.initials: H
authors.lastname: Malissa
authors.forename: Hanno
authors.initials: H
authors.lastname: Stutz
mesh_terms: D000485:Allergens / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D052179:Antigens, Plant; D002219:Carbamates / Q000737:chemistry*; D019075:Electrophoresis, Capillary / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000295:instrumentation / Q000379:methods*; D020033:Protein Isoforms / Q000737:chemistry / Q000302:isolation & purification / Q000592:standards; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000592:standards
chemical_list: D000485:Allergens; D052179:Antigens, Plant; D002219:Carbamates; D020033:Protein Isoforms; D011994:Recombinant Proteins