Open AccessDevelopment of a hypoallergenic and immunogenic Pru p 3 proline variant for treatment of peach allergy
Author(s) -
Eichhorn Stephanie,
Steiner Markus,
Laimer Josef,
Lackner Peter,
ZuidmeerJongejan Laurian,
Briza Peter,
Mari Adriano,
Ree Ronald,
Ferreira Fatima,
Gadermaier Gabriele
Publication year - 2014
Publication title -
clinical and translational allergy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.979
H-Index - 37
ISSN - 2045-7022
DOI - 10.1186/2045-7022-4-s2-o22
Subject(s) - hypoallergenic , chromatography , circular dichroism , proline , recombinant dna , size exclusion chromatography , chemistry , amino acid , microbiology and biotechnology , allergy , biochemistry , medicine , allergen , enzyme , biology , immunology , gene
Methods We deployed an in-silico mutagenesis approach to design a fold variant of recombinant Pru p 3. Four stability hot spot residues were identified and proline was predicted as the most effective replacement amino acid. Pru p 3 C1 was produced in E.coli using a pET-based expression system. After refolding and purification by cation exchange chromatography, the protein was characterized by reducing and non-reducing gel electrophoresis, circular dichroism spectroscopy, size exclusion chromatography, dynamic light scattering and mass spectrometry (MS). An accelerated stability test was performed by storing the protein up to 6 month at temperatures ranging from -70°C to +40°C.The IgE binding capacity of Pru p 3 C1 and WT Pru p 3 was tested in ELISA using sera from peach allergic patients. Different adjuvants and adsorption conditions were evaluated for use in a mouse model. Mice were s.c. immunized with Pru p 3 C1 and WT Pru p 3 and sera were analyzed for IgG reactivity.