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Development of a hypoallergenic and immunogenic Pru p 3 proline variant for treatment of peach allergy


Allergic reactions to peach are highly prevalent in the Mediterranean area with persistent and potentially life threatening symptoms. We aimed to develop a save and efficient immunotherapeutics for treatment of peach allergy by targeting the major allergen Pru p 3.


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.


Recombinant Pru p 3 C1 was produced with a yield of 15 mg/l expression volume. The purity and identity of the protein was confirmed in gel electrophoresis and MS. In size exclusion chromatography and dynamic light scattering the protein showed to be >90% monomeric. Circular dichroism spectroscopy showed that the protein is predominantly in an unfolded state. Thermal denaturation led to a shift of the spectrum which reverted to the initial curve after renaturation. After six month of storage Pru p 3 C1 demonstrated high stability and no aggregation or degradation behavior at temperatures ranging from -70°C to +4°C. In ELISA, the IgE binding capacity of Pru p 3 C1 was reduced by 89% compared to WT Pru p 3. In vivo models showed that the use of aluminum phosphate as adjuvant is superior over aluminum hydroxide. Immunization with Pru p 3 C1 induced a humoral immune response in all animals and triggered cross-reactive IgG1 antibodies in 50% of mouse sera.


Pru p 3 C1 shows high stability, strongly reduced IgE binding capacity and immunogenicity. Therefore, the molecule is a very promising candidate for treatment of peach allergy.

The study was supported by the FAST project EU grant 201871.

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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated.

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Eichhorn, S., Steiner, M., Laimer, J. et al. Development of a hypoallergenic and immunogenic Pru p 3 proline variant for treatment of peach allergy. Clin Transl Allergy 4 (Suppl 2), O22 (2014).

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