title: Stable production of peptide antigens in transgenic tobacco chloroplasts by fusion to the p53 tetramerisation domain.
abstract: The production of short peptides as single molecules in recombinant systems is often limited by the low stability of the foreign peptide. In the plant expression system this problem has been solved by translational fusions to recombinant proteins that are highly stable or are able to form complex structures. Previously, we demonstrated that the highly immunogenic 21 amino acid peptide 2L21, which is derived from the canine parvovirus (CPV) VP2 protein, did not accumulate in transgenic tobacco chloroplasts. In this report, we translationally fused the 2L21 peptide to the 42 amino acid tetramerisation domain (TD) from the human transcription factor p53. The chimaeric 2L21-TD protein was expressed in tobacco chloroplasts. Leaves accumulated high levels of the recombinant protein (up to 0.4 mg/g fresh weight of leaf material, equivalent to ~6% of total soluble protein; 2% considering only the 2L21 peptide). The 2L21-TD protein was able to form tetramers in the stroma of the chloroplast. Mice immunised intraperitoneally with partially purified leaf extracts containing the 2L21-TD protein developed specific antibodies with titres similar to those elicited by a previously reported fusion between 2L21 and the B subunit of the cholera toxin. Mouse sera were able to detect both the 2L21 synthetic peptide and the CPV VP2 protein, showing that the antigenicity of the 2L21 epitope was preserved in the chimaeric protein. These results demonstrate that the p53 TD can be used as a carrier molecule for the accumulation of short peptides (such as 2L21) in the chloroplast without altering the immunogenic properties of the peptide.
authors: Instituto de Agrobiotecnología, Universidad Pública de Navarra-CSIC-Gobierno de Navarra, Campus de Arrosadía, 31006, Pamplona, Spain.
authors: Susana M
authors: SM
authors: Ortigosa
authors: Alicia
authors: A
authors: Fernández-San Millán
authors: Jon
authors: J
authors: Veramendi
mesh_terms: D000818:Animals; D000941:Antigens / Q000235:genetics*; D002736:Chloroplasts / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D010455:Peptides / Q000235:genetics / Q000276:immunology; D030821:Plants, Genetically Modified; D054730:Protein Interaction Domains and Motifs / Q000235:genetics* / Q000276:immunology; D055503:Protein Multimerization / Q000235:genetics; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D013997:Time Factors; D014026:Nicotiana / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000235:genetics* / Q000276:immunology
chemical_list: D000941:Antigens; D010455:Peptides; D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53