Production of native and modified recombinant Der p 1 molecules in tobacco plants
| Autor: | A. Lorphelin, H. Laparra, D. Comeau, M.-N. Couret, Philippe Moingeon, B. Olagnier, R. van Ree, L. Parry, Erik Wambre, F. Montandon, C. Berrouet, D. Burtin, L. Van Overtvelt, Véronique Gomord, Henri Chabre, Alain Didierlaurent, V. Jonval, Thierry Batard, C. Merle |
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| Přispěvatelé: | Amsterdam institute for Infection and Immunity, Amsterdam Public Health, Experimental Immunology |
| Rok vydání: | 2009 |
| Předmět: |
Circular dichroism
T-Lymphocytes Immunology Basophil Arthropod Proteins Cell Line law.invention chemistry.chemical_compound Biosynthesis law Tobacco Gene expression medicine Humans Immunology and Allergy Antigens Dermatophagoides Cloning Molecular Pyrophosphatases biology Phosphoric Diester Hydrolases Agrobacterium tumefaciens Plants Genetically Modified biology.organism_classification Molecular biology Recombinant Proteins Basophils Plant Leaves Cysteine Endopeptidases medicine.anatomical_structure chemistry Cell culture Recombinant DNA Cysteine |
| Zdroj: | Clinical and experimental allergy, 39(5), 760-770. Wiley-Blackwell |
| ISSN: | 1365-2222 0954-7894 |
| DOI: | 10.1111/j.1365-2222.2009.03201.x |
| Popis: | Summary 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. |
| Databáze: | OpenAIRE |
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