Production of native and modified recombinant der p 1 molecules in tobacco plants

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.