A folded and immunogenic IgE-hyporeactive variant of the major allergen Phl p 1 produced in Escherichia coli

BMC Biotechnology

Research Article

Harm Otten¹  Mattias Levin²  Mats Ohlin²  Claes von Wachenfeldt³ 

[1]Crystallization facility at the MAX IV laboratory and Lund University, Lund, Sweden

[2]Lund Protein Production Platform (LP3), Lund University, Lund, Sweden

[3]Dept. of Immunotechnology, Lund University, Medicon Village building 406, S-223 81, Lund, Sweden

[4]Lund Protein Production Platform (LP3), Lund University, Lund, Sweden

关键词: Group 1 grass pollen allergen;    Hypoallergen;    IgE;    Immunogenicity;    Production;    Protein fold;   

DOI  :  10.1186/s12896-015-0150-z

 received in 2014-09-02, accepted in 2015-04-22,  发布年份 2015

来源: Springer

【 摘 要 】

BackgroundGroup 1 grass pollen allergens are a major cause of allergic disease. Specific immunotherapy involving controlled administration of allergens can be used as a disease-modifying treatment for such disease. Recombinant allergen variants with reduced IgE binding capacity may be used as component in such vaccines, as they may induce fewer treatment side effects than materials currently in use. A mutated variant of the immunodominant C-terminal domain of the group 1 grass pollen allergen Phl p 1 was recently established through an approach that used a set of human monoclonal IgE as a guide to identify mutations that disturbed IgE-allergen interactions. Further analysis of this domain is required to establish its potential for use in treatment.MethodsGST-tagged wild-type and mutated C-terminal domains of Phl p 1 were produced in Escherichia coli TUNER(DE3). The products were purified by affinity chromatography on immobilized glutathione. GST was removed by enzymatic cleavage and tag-free products were purified by size exclusion chromatography. Products were assessed by SDS-PAGE, circular dichroism spectroscopy, differential scanning fluorimetry and dynamic light scattering. Rats were immunized with GST-tagged and tag-free mutated C-terminal domain of Phl p 1. Antigen-binding properties of induced antibodies were assessed by immunochemical analysis.ResultsThe mutated domain has a structure very similar to that of the wild-type domain as determined by circular dichroism, but a reduced thermal stability. Immunization of rats demonstrates that this IgE-hyporeactive domain, despite its three sequence modifications (K8A, N11A, D55A), is able to induce antibodies that substantially block the binding of allergic subjects’ IgE to the wild-type allergen.ConclusionsIt is concluded that this IgE-hyporeactive molecule can be produced in folded form and that it is able to induce an antibody response that efficiently competes with IgE recognition of Phl p 1. These findings suggest that it, or a further evolved variant thereof, is a candidate for use as a component in specific immunotherapy against grass pollen allergy.

【 授权许可】

Unknown   
© Levin et al. 2015. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.