RESUMEN
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.
Asunto(s)
Antígenos Dermatofagoides/biosíntesis , Clonación Molecular , Nicotiana/genética , Antígenos Dermatofagoides/genética , Antígenos Dermatofagoides/inmunología , Proteínas de Artrópodos , Basófilos/inmunología , Basófilos/metabolismo , Línea Celular , Cisteína Endopeptidasas , Humanos , Hidrolasas Diéster Fosfóricas/inmunología , Hidrolasas Diéster Fosfóricas/metabolismo , Hojas de la Planta/genética , Plantas Modificadas Genéticamente , Pirofosfatasas/inmunología , Pirofosfatasas/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
The use of tobacco plants as a novel expression system for the production of human homotrimeric collagen I is presented in this report. Constructs were engineered from cDNA encoding the human proalpha1(I) chain to generate transgenic tobacco plants expressing collagen I. The recombinant proalpha1(I) chains were expressed as disulfide-bonded trimers and were shown to fold into a stable homotrimeric triple helix. Moreover, the recombinant procollagen was subsequently processed to collagen as it occurs in animals. Large amounts of recombinant collagen were purified from field grown plant material. The data suggest that plants are a valuable alternative for the recombinant production of collagen for various medical and scientific purposes.
Asunto(s)
Nicotiana/genética , Plantas Tóxicas , Procolágeno/genética , Secuencia de Aminoácidos , Dicroismo Circular , Electroforesis en Gel de Poliacrilamida , Humanos , Microscopía Electrónica , Datos de Secuencia Molecular , Plantas Modificadas Genéticamente , Procolágeno/química , Conformación Proteica , Desnaturalización Proteica , Pliegue de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/ultraestructura , TripsinaRESUMEN
Production and characterization of human lactoferrin (hLf) in transgenic tobacco is reported. We have engineered two constructs containing either the native signal peptide from human lactoferrin or the signal peptide from sweet potato sporamin fused to human lactoferrin encoding cDNA. N-terminal sequences of rhLf purified from tobacco were identical to Lf from human milk for both constructs. The tobacco rhLf presents a molecular mass closely identical to native protein. Overall sugar composition shows the presence of plant specific xylose while sialic acid is absent. Binding parameters of the recombinant molecule to both Jurkat lymphoblastic T-cells or HT29-18-C1 enterocytes are similar to those of human lactoferrin isolated from milk.