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- Title
Production of native and modified recombinant Der p 1 molecules in tobacco plants.
- Authors
Burtin, D.; Chabre, H.; Olagnier, B.; Didierlaurent, A.; Couret, M.-N.; Comeau, D.; Wambre, E.; Laparra, H.; Van Overtvelt, L.; Montandon, F.; Batard, T.; Jonval, V.; Lorphelin, A.; Merle, C.; Berrouet, C.; Parry, L.; Gomord, V.; Van Ree, R.; Moingeon, P.
- Abstract
Background As a complex molecule requiring post-translational processing, it has been difficult to produce the Der p 1 major allergen from the Dermatophagoides pteronyssinus house dust mite in a recombinant form. Objective Here, we tested whether transgenic tobacco plants are suitable to express Der p 1, either as a wild-type molecule or as variants lacking N-glycosylation sites (Gly−) and/or cysteine protease activity (Enz−). Methods Using Agrobacterium tumefaciens-based transformation, pro Der p 1 molecules bearing mutations within either the N-glycosylation sites (N34Q, N150Q) and/or the cysteine protease-active site (C132V) were expressed in tobacco plants. After purification by ion exchange chromatography, allergens were characterized using immunoblotting, circular dichroism (CD), as well as basophil and T lymphocyte stimulation assays. Results Four forms of recombinant Der p 1 (i.e. wild-type Gly+/Enz+, as well as Gly−/Enz+, Gly+/Enz− or Gly−/Enz− variants) were successfully expressed in tobacco leaves as pro Der p 1 molecules. Spontaneous cleavage of the pro-peptide was observed in tobacco leaf extracts for all forms of recombinant Der p 1 (r Der p 1). CD confirmed that all r Der p 1 molecules, with the exception of the Gly−/Enz− variant, exhibited secondary structures comparable to the natural protein. A cysteine protease activity was associated only with the Gly+/Enz+ form. All these molecules exhibit a profile similar to natural Der p 1 with respect to IgE immunoreactivity, basophil activation and T cell recognition. Conclusion A tobacco plant expression system allows the production of various forms of mature Der p 1, which could be used for diagnostic or immunotherapeutic purposes.
- Subjects
HOUSE dust mites; RISK assessment of transgenic plants; ESTERIFICATION; CYSTEINE proteinases; CHROMATOGRAPHIC analysis; ANTIGEN analysis; IMMUNITY; RHIZOBIACEAE; CIRCULAR dichroism; CLINICAL immunology
- Publication
Clinical & Experimental Allergy, 2009, Vol 39, Issue 5, p760
- ISSN
0954-7894
- Publication type
Article
- DOI
10.1111/j.1365-2222.2009.03201.x