Combination of two epitope identification techniques enables the rational design of soy allergen Gly m 4 mutants

Abstract

Detailed IgE-binding epitope analysis is a key requirement for the understanding and development of diagnostic and therapeutic agents to address food allergies. An IgE-specific linear peptide microarray with random phage peptide display for the high-resolution mapping of IgE-binding epitopes of the major soybean allergen Gly m 4, which is a homologue to the birch pollen allergen Bet v 1 is combined. Three epitopes are identified and mapped to a resolution of four key amino acids, allowing the rational design and the production of three Gly m 4 mutants with the aim to abolish or reduce the binding of epitope-specific IgE. In ELISA, the binding of the mutant allergens to polyclonal rabbit-anti Gly m 4 serum as well as IgE purified from Gly m 4-reactive soybean allergy patient sera is reduced by up to 63% compared to the wild-type allergen. Basophil stimulation experiments using RBL-SX38 cells loaded with patient IgE are showed a decreased stimulation from 25% for the wild-type Gly m 4 to 13% for one mutant. The presented approach demonstrates the feasibility of precise mapping of allergy-related IgE-binding epitopes, allowing the rational design of less allergenic mutants as potential therapeutic agents.

Anti-Drug Antibodies: Emerging Approaches to Predict, Reduce or Reverse Biotherapeutic Immunogenicity

“…Some promising recent developments include combination of peptide array data with results of phage display, protein structural information, B-cell epitope prediction algorithms, etc. [121]. Use of microarrays consisting of immobilized proteins, e.g., auto- or allo-antigens, is an emerging proteomics approach to characterize pathologic antibodies, potentially yielding information characterizing conformational epitopes that is complementary to the finer mapping of epitopes achievable with peptide microarrays [122,123,124].…”

The immunome of soy bean allergy: Comprehensive identification and characterization of epitopes

“…As recently published by our group, we were able to generate variants of Gly m 4 with reduced binding by patient sera on the basis of data achieved using this experimental approach. 26 2 | MATERIALS AND METHODS 28 in dYT for 7 hours for phagemid production. On average 10 5 -10 6 phagemid clones were recovered.…”

“…26 In brief, peptides coated to the microarray covered 26 In brief, peptides coated to the microarray covered …”

“…The PEPperCHIP ® peptide microarray has previously been described. 26 In brief, peptides coated to the microarray covered 10…”

Critical Analysis of the Commercial Potential of Plants for the Production of Recombinant Proteins

“…Recently, we described a new cell-free lysate based on tobacco BY-2 cells. The BY-2 lysate (BYL) achieves yields of up to 270 μg/ml when producing the fluorescent protein eYFP and involves a coupled transcription-translation reaction in a simple 18-h batch process (Buntru et al, 2014, 2015; Havenith et al, 2017). The productivity of the BYL system has been increased to 3,000 μg eYFP per ml by optimizing lysate preparation and the reaction components, and by extending the transcription-translation process to 24–48 h by including active mitochondria that deliver energy for protein biosynthesis (unpublished data).…”