A non-allergenic Ole e 1-like protein from birch pollen as a tool to design hypoallergenic vaccine candidates
ABSTRACT Recombinant DNA technology offers several approaches to convert allergens into hypoallergenic derivatives that can represent the basis of novel, safer and more effective forms of allergy vaccines. In this context, we used a new strategy for the design of a hypoallergenic derivative of Ole e 1, the main allergen of olive pollen. By screening a cDNA library from birch pollen, the clone BB18, encoding the birch counterpart of Ole e 1, was identified. In this study, BB18 has been produce in Pichia pastoris as a recombinant protein and immunologically characterized. The well-established non-allergenic properties of BB18 were used to generate a genetic variant of Ole e 1, named OB(55-58), by site-direct mutagenesis of four residues (E(55)V(56)G(57)Y(58)) in an IgE/IgG epitope of Ole e 1 by the corresponding ones in BB18 (SDSE). OB(55-58) was expressed in P. pastoris, purified to homogeneity and analyzed for IgE-reactivity by means of ELISA using sera from olive pollen allergic patients and rat basophil activation assay. T cell reactivity was assayed in a mouse model of Ole e 1 sensitization. The mutant OB(55-58) exhibited an impaired IgE reactivity, but not affected T cell reactivity, compared to wild type rOle e 1. This study emphasizes the usefulness of BB18 as a tool for epitope mapping and for engineering hypoallergenic derivatives of Ole e 1 as vaccine candidates for allergy prevention and treatment.
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- "The well-established non-allergenic properties of BB18 were used to generate a genetic variant of Ole e1, named OB 55-58 , by site-direct mutagenesis of four residues (E55V56G57Y58) in an IgE/IgG epitope of Ole e1 by the corresponding ones in BB18 (SDSE). (Marazuela et al., 2012). Their results support the usefulness of BB18 for both epitope mapping of Ole e1 and engineering hypoallergenic derivatives of this allergen as for example the mutant OB55-58, which fulfills the requisites for a suitable hypoallergenic molecule. "
ABSTRACT: Since the 1970s, the establishment and development of the biotech industry has improved exponentially, allowing the commercial production of biopharmaceutical proteins. Nowadays, new recombinant protein production is considered a multibillion-dollar market, in which about 25% of commercial pharmaceuticals are biopharmaceuticals. But to achieve a competitive production process is not an easy task. Any production process has to be highly productive, efficient and economic. Despite that the perfect host is still not discovered, several research groups have chosen Pichia pastoris as expression system for the production of their protein because of its many features. The attempt of this review is to embrace several research lines that have adopted Pichia pastoris as their expression system to produce a protein on an industrial scale in the health care industry.12/2013; 44(4):1043-1048. DOI:10.1590/S1517-83822013000400004
- Clinical & Experimental Allergy 08/2012; 42(8):1146-9. DOI:10.1111/j.1365-2222.2012.04050.x · 4.32 Impact Factor
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ABSTRACT: Allergenic proteins must cross-link specific IgE molecules, bound to the surface of mast cells and basophils, to stimulate an immune response. A structural understanding of the allergen-IgE interface is needed to predict cross-reactivities between allergens and to design hypoallergenic proteins. However, there are less than 90 experimentally determined structures available for the approximately 1500 sequences of allergens and isoallergens catalogued in the Structural Database of Allergenic Proteins (SDAP). To provide reliable structural data for the remaining proteins, we previously produced over 500 3D-models using an automated procedure, with strict controls at template choice and model quality evaluation. Here we assessed how well the fold and residue surface exposure of 10 of these models correlated with recently published experimental 3D structures determined by X-ray crystallography or NMR. We also discuss the impact of intrinsically disordered regions on the structural comparison and epitope prediction. Overall, for seven allergens with sequence identities to the original templates higher than 27%, the backbone root-mean square deviations were less than 2Å between the models and the subsequently determined experimental structures for ordered regions. Further, the surface exposure of known IgE epitopes on the models of three major allergens, from peanut (Ara h 1), latex (Hev b 2) and soy (Gly m 4) was very similar to the experimentally determined structures. For three remaining allergens with lower sequence identities to the modeling templates, the 3D folds were correctly identified. However the accuracy of those models is not sufficient for a reliable epitope mapping. Proteins 2012. © 2012 Wiley Periodicals, Inc.Proteins Structure Function and Bioinformatics 04/2013; 81(4). DOI:10.1002/prot.24239 · 2.92 Impact Factor