title: Purification and characterization of natural Bet v 1 from birch pollen and related allergens from carrot and celery.
abstract: Birch pollen allergy is predominantly caused by the major allergen Bet v 1 and can lead to crossreactions with homologous proteins in food. Two major cross-reactive food allergens are Dau c 1 from carrot and Api g 1 from celery, which have never been purified from their natural source. Here, we describe a non-denaturing purification method for obtaining natural Bet v 1, Dau c 1 and Api g 1, comprising of ammonium sulfate precipitation, hydrophobic interaction chromatography and size exclusion chromatography. This method resulted in 98-99% pure isoform mixtures for each allergen. Characterization of these isoform mixtures with Q-TOF MS/MS clearly showed earlier reported isoforms of Bet v 1, Dau c 1 and Api g 1, but also new isoforms. The presence of secondary structure in the three purified allergens was demonstrated via circular dichroism and showed high similarity. The immune reactivity of the natural allergens was compared with recombinant proteins by Western blot and ELISA and showed similar reactivity.
authors.affiliation: Product Design and Quality Management, Department of Agrotechnology and Food Sciences, Wageningen University and Research Centre, Bornsesteeg 59, Wageningen, The Netherlands.
authors.forename: Mirko A
authors.initials: MA
authors.lastname: Bollen
authors.forename: Aranzazu
authors.initials: A
authors.lastname: Garcia
authors.forename: Jan H G
authors.initials: JH
authors.lastname: Cordewener
authors.forename: Harry J
authors.initials: HJ
authors.lastname: Wichers
authors.forename: Johannes P F G
authors.initials: JP
authors.lastname: Helsper
authors.forename: Huub F J
authors.initials: HF
authors.lastname: Savelkoul
authors.forename: Martinus A J S
authors.initials: MA
authors.lastname: van Boekel
mesh_terms: D000485:Allergens / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D052179:Antigens, Plant / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000077102:Apium / Q000737:chemistry; D029662:Betula / Q000737:chemistry; D015153:Blotting, Western; D002942:Circular Dichroism; D018552:Daucus carota / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011058:Pollen / Q000737:chemistry*; D020033:Protein Isoforms / Q000737:chemistry; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000378:metabolism
chemical_list: D000485:Allergens; D052179:Antigens, Plant; C096695:Api g 1 protein, Apium graveolens; C119925:Dau c 1 protein, Daucus carota; D010940:Plant Proteins; D020033:Protein Isoforms; D014357:Trypsin