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Int Arch Allergy Immunol. 2012;158(2):157-67. doi: 10.1159/000331143. Epub 2012 Jan 25.

Expression and characterization of natural-like recombinant Der p 2 for sublingual immunotherapy.

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  • 1Stallergènes SA, Antony, France.

Abstract

BACKGROUND:

Recombinant allergens with a native conformation represent an alternative to natural extracts for immunotherapy and diagnostic purposes.

METHODS:

We produced the Der p 2 mite allergen in Pichia pastoris and Escherichia coli. After purification by cation exchange chromatography, recombinant molecules were compared to their natural counterpart based upon structural (disulfide bonds, secondary structure, thermal stability) and immunological properties (antibody reactivity, basophil and T cell activation, tolerance induction in a murine sublingual immunotherapy model).

RESULTS:

The Der p 2.0101 isoform was confirmed to be prevalent in Dermatophagoides pteronyssinus extracts. It was then produced as a secreted molecule in P. pastoris or refolded from E. coli inclusion bodies. The yeast-expressed rDer p 2 molecule exhibits a natural-like disulfide bridge distribution and secondary structure, whereas the E. coli-derived rDer p 2 presents some heterogeneity in cysteine bonds and a lower stability following thermal stress. The two recombinant as well as natural Der p 2 molecules exhibit comparable IgE recognition and activate basophil and CD4+ T cells. Sublingual immunotherapy of nDer p 2- sensitized mice using either one of the rDer p 2 molecules efficiently decreases airway hyperresponsiveness as well as Th2 responses.

CONCLUSIONS:

Natural and recombinant Der p 2 molecules produced in P. pastoris and E. coli exhibit comparable immunological properties despite distinct structural features. Natural-like cysteine pairing is a critical parameter to identify stable, well-folded and homogenous proteins appropriate for immunotherapy and diagnostic purposes.

Copyright © 2012 S. Karger AG, Basel.

PMID:
22286395
[PubMed - indexed for MEDLINE]
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