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Production of native and modified recombinant Der p 1 molecules in tobacco plants
Author(s) -
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.
Publication year - 2009
Publication title -
clinical and experimental allergy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.462
H-Index - 154
eISSN - 1365-2222
pISSN - 0954-7894
DOI - 10.1111/j.1365-2222.2009.03201.x
Subject(s) - agrobacterium tumefaciens , cysteine protease , recombinant dna , protease , microbiology and biotechnology , cysteine , biochemistry , chemistry , allergen , biology , enzyme , transgene , immunology , allergy , gene
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.