title: Production of native and modified recombinant Der p 1 molecules in tobacco plants.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Meristem Therapeutics, Clermont-Ferrand, France.
authors.forename: D
authors.initials: D
authors.lastname: Burtin
authors.forename: H
authors.initials: H
authors.lastname: Chabre
authors.forename: B
authors.initials: B
authors.lastname: Olagnier
authors.forename: A
authors.initials: A
authors.lastname: Didierlaurent
authors.forename: M-N
authors.initials: MN
authors.lastname: Couret
authors.forename: D
authors.initials: D
authors.lastname: Comeau
authors.forename: E
authors.initials: E
authors.lastname: Wambre
authors.forename: H
authors.initials: H
authors.lastname: Laparra
authors.forename: L
authors.initials: L
authors.lastname: Van Overtvelt
authors.forename: F
authors.initials: F
authors.lastname: Montandon
authors.forename: T
authors.initials: T
authors.lastname: Batard
authors.forename: V
authors.initials: V
authors.lastname: Jonval
authors.forename: A
authors.initials: A
authors.lastname: Lorphelin
authors.forename: C
authors.initials: C
authors.lastname: Merle
authors.forename: C
authors.initials: C
authors.lastname: Berrouet
authors.forename: L
authors.initials: L
authors.lastname: Parry
authors.forename: V
authors.initials: V
authors.lastname: Gomord
authors.forename: R
authors.initials: R
authors.lastname: Van Ree
authors.forename: P
authors.initials: P
authors.lastname: Moingeon
mesh_terms: D039741:Antigens, Dermatophagoides / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D060829:Arthropod Proteins; D001491:Basophils / Q000276:immunology / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases; D006801:Humans; D010727:Phosphoric Diester Hydrolases / Q000276:immunology / Q000378:metabolism; D018515:Plant Leaves / Q000235:genetics; D030821:Plants, Genetically Modified; D011755:Pyrophosphatases / Q000276:immunology / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics*
chemical_list: D039741:Antigens, Dermatophagoides; D060829:Arthropod Proteins; C427944:ENPP3 protein, human; D011994:Recombinant Proteins; D010727:Phosphoric Diester Hydrolases; D003546:Cysteine Endopeptidases; C451162:Dermatophagoides pteronyssinus antigen p 1; D011755:Pyrophosphatases