title: Folded or Not? Tracking Bet v 1 Conformation in Recombinant Allergen Preparations.
abstract: BACKGROUND
  Recombinant Bet v 1a (rBet v 1a) has been used in allergy research for more than three decades, including clinical application of so-called hypoallergens. Quantitative IgE binding to rBet v 1a depends on its native protein conformation, which might be compromised upon heterologous expression, purification, or mutational engineering of rBet v 1a.
  
  
  OBJECTIVE
  To correlate experimental/theoretical comparisons of IgE binding of defined molar ratios of folded/misfolded recombinant Bet v 1a variants and to determine accuracy and precision of immuno- and physicochemical assays routinely used to assess the quality of recombinant allergen preparations.
  
  
  METHODS
  rBet v 1a and its misfolded variant rBet v 1aS112P/R145P were heterologously expressed and purified from Escherichia coli. Structural integrities and oligomerisation of the recombinant allergens were evaluated by 1H-nuclear magnetic resonance (1H-NMR), circular dichroism (CD) spectroscopy, and dynamic light scattering (DLS). IgE binding of defined combinations of rBet v 1a and rBet v 1aS112P/R145P was assessed using immunoblotting (IB), enzyme-linked immunosorbent assay (ELISA) and mediator release (MR) of humanized rat basophilic leukemia cells sensitized with serum IgE of subjects allergic to birch pollen. Experimental and theoretically expected results of the analyses were compared.
  
  
  RESULTS
  1H-NMR spectra of rBet v 1a and rBet v 1aS112P/R145P demonstrate a native and highly disordered protein conformations, respectively. The CD spectra suggested typical alpha-helical and beta-sheet secondary structure content of rBet v 1a and random coil for rBet v 1aS112P/R145P. The hydrodynamic radii (RH) of 2.49 ± 0.39 nm (rBet v 1a) and 3.1 ± 0.56 nm (rBet v 1aS112P/R145P) showed monomeric dispersion of both allergens in solution. Serum IgE of birch pollen allergic subjects bound to 0.1% rBet v 1a in the presence of 99.9% of non-IgE binding rBet v 1aS112P/R145P. Immunoblot analysis overestimated, whereas ELISA and mediator release assay underestimated the actual quantity of IgE-reactive rBet v 1a in mixtures of rBet v 1a/rBet v 1aS112P/R145P with a molar ratio of rBet v 1a ≤ 10%.
  
  
  CONCLUSIONS
  Valid conclusions on quantitative IgE binding of recombinant Bet v 1a preparations depend on the accuracy and precision of physico- and immunochemical assays with which natively folded allergen is detected.
authors.affiliation: Division of Allergology, Paul-Ehrlich-Institut, Langen, Hesse, Germany.
authors.forename: Felix
authors.initials: F
authors.lastname: Husslik
authors.affiliation: Division of EU Cooperation/Microbiology, Paul-Ehrlich-Institut, Section Biostatistics, Langen, Hesse, Germany.
authors.forename: Kay-Martin
authors.initials: KM
authors.lastname: Hanschmann
authors.affiliation: Division of Allergology, Paul-Ehrlich-Institut, Langen, Hesse, Germany.
authors.forename: Ariane
authors.initials: A
authors.lastname: Krämer
authors.affiliation: Department of Biopolymers, University of Bayreuth, Bayreuth, Bavaria, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Seutter von Loetzen
authors.affiliation: Department of Biopolymers, University of Bayreuth, Bayreuth, Bavaria, Germany.
authors.forename: Kristian
authors.initials: K
authors.lastname: Schweimer
authors.affiliation: Department of Dermatology, University Medical Center, Mainz, Rhineland-Palatinate, Germany.
authors.forename: Iris
authors.initials: I
authors.lastname: Bellinghausen
authors.affiliation: Department of Dermatology, Venerology and Allergology, University of Leipzig, Leipzig, Saxony, Germany.
authors.forename: Regina
authors.initials: R
authors.lastname: Treudler
authors.affiliation: Department of Dermatology, Venerology and Allergology, University of Leipzig, Leipzig, Saxony, Germany.
authors.forename: Jan C
authors.initials: JC
authors.lastname: Simon
authors.affiliation: Division of Allergology, Paul-Ehrlich-Institut, Langen, Hesse, Germany.
authors.forename: Lothar
authors.initials: L
authors.lastname: Vogel
authors.affiliation: Division of Allergology, Paul-Ehrlich-Institut, Langen, Hesse, Germany.
authors.forename: Elke
authors.initials: E
authors.lastname: Völker
authors.affiliation: Division of Allergology, Paul-Ehrlich-Institut, Langen, Hesse, Germany.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Randow
authors.affiliation: Division of Allergology, Paul-Ehrlich-Institut, Langen, Hesse, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Reuter
authors.affiliation: Department of Biopolymers, University of Bayreuth, Bayreuth, Bavaria, Germany.
authors.forename: Paul
authors.initials: P
authors.lastname: Rösch
authors.affiliation: Division of Allergology, Paul-Ehrlich-Institut, Langen, Hesse, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Vieths
authors.affiliation: Division of Allergology, Paul-Ehrlich-Institut, Langen, Hesse, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Holzhauser
authors.affiliation: Division of Allergology, Paul-Ehrlich-Institut, Langen, Hesse, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Schiller
mesh_terms: D000485:Allergens / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D052179:Antigens, Plant / Q000737:chemistry* / Q000378:metabolism*; D001491:Basophils / Q000378:metabolism; D002942:Circular Dichroism; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D055767:Immobilized Proteins / Q000378:metabolism; D015151:Immunoblotting; D007073:Immunoglobulin E; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D000485:Allergens; D052179:Antigens, Plant; D055767:Immobilized Proteins; D011994:Recombinant Proteins; C061312:Bet v 1 allergen, Betula; D007073:Immunoglobulin E