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
Potential contamination of animal-derived collagen with pathogens has led to the demand for safe recombinant sources of this complex molecule. In continuation of our previous work [Ruggiero et al. (2000) FEBS Lett. 469, 132-136], here we show that it is possible to produce recombinant hydroxylated homotrimeric collagen in tobacco plants that are co-transformed with a human type I collagen and a chimeric proline-4-hydroxylase (P4H). This is to our knowledge the first time that transient expression in tobacco was used to improve the quality of a recombinant protein produced in plants through co-expression with an animal cell-derived modifying enzyme. We demonstrated the functionality of the new chimeric P4H and thus improved the thermal stability of recombinant collagen I from plants to 37 degrees C.
Asunto(s)
Agrobacterium tumefaciens/metabolismo , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , Aminoácidos/análisis , Bioensayo , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Colágeno Tipo I/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Expresión Génica , Humanos , Hidroxilación , Pepsina A/química , Plantas Modificadas Genéticamente , Procolágeno-Prolina Dioxigenasa/genética , Procolágeno-Prolina Dioxigenasa/metabolismo , Conformación Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Temperatura , Nicotiana/química , Transformación GenéticaRESUMEN
The replacement of beta 5(A2)Pro by Arg in Hb Warwickshire appears to be without an effect on the functional properties of human Hb A, despite adding two external positive charges close to the central cavity of the hemoglobin tetramer, along the dyad axis. To clarify the role of this portion of the molecule involved in oxygen-linked anion binding, we have engineered the recombinant hemoglobin alpha 2 beta (2)5(A2)Pro-->Ala[rHb beta 5(A2)Pro-->Ala]. The rHb beta 5(A2)Pro-->Ala exhibits an increased oxygen affinity compared to Hb A, with normal heterotropic effects in standard conditions. The increased oxygen affinity may be attributed to the absence of proline, which would render the A helix more flexible, thus destabilizing the T structure. The normal functional properties of Hb Warwickshire may be due to the regulation of oxygen affinity by electrostatic effects involving diffusible anions not bound to any specific site.