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Folded or Not? Tracking Bet v 1 Conformation in Recombinant Allergen Preparations

Title data

Husslik, Felix ; Hanschmann, Kay-Martin ; Krämer, Ariane ; Seutter von Loetzen, Christian ; Schweimer, Kristian ; Bellinghausen, Iris ; Treudler, Regina ; Simon, Jan C. ; Vogel, Lothar ; Völker, Elke ; Randow, Stefanie ; Reuter, Andreas ; Rösch, Paul ; Vieths, Stefan ; Holzhauser, Thomas ; Schiller, Dirk:
Folded or Not? Tracking Bet v 1 Conformation in Recombinant Allergen Preparations.
In: PLoS One. Vol. 10 (July 2015) Issue 7 .
ISSN 1932-6203
DOI: https://doi.org/10.1371/journal.pone.0132956

Abstract in another language

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%

CONCLUSION:
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.

Further data

Item Type: Article in a journal
Refereed: Yes
Institutions of the University: Faculties
Faculties > Faculty of Biology, Chemistry and Earth Sciences
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Former Professors
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Chair Biopolymers
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Former Professors > Chair Biopolymers - Univ.-Prof. Dr. Paul Rösch
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Chair Biopolymers > Lehrstuhl Biopolymere - Apl. Prof. Dr. Birgitta Wöhrl
Result of work at the UBT: Yes
DDC Subjects: 500 Science > 540 Chemistry
500 Science > 570 Life sciences, biology
Date Deposited: 30 Jan 2019 11:39
Last Modified: 11 Jun 2019 07:31
URI: https://eref.uni-bayreuth.de/id/eprint/47135