Person: Gil Dones, Félix
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First Name
Félix
Last Name
Gil Dones
Affiliation
Universidad Complutense de Madrid
Faculty / Institute
Ciencias Biológicas
Department
Genética, Fisiología y Microbiología
Area
Genética
Identifiers
8 results
Search Results
Now showing 1 - 8 of 8
Item Multimerization of peptide antigens for production of stable immunogens in transgenic plants(Journal of Biotechnology, 2007) Gil Dones, Félix; Reytor, Edel; Pérez-Filgueira, Daniel Mariano; Escribano, José M.Previous literature addressing the production of recombinant proteins in heterologous systems has consistently shown that proteins capable of forming complex structures tend to accumulate within host cells at relatively higher levels than monomeric forms. In this report, we translationally fused a 21-aminoacids long highly immunogenic peptide (2L21), derived from canine parvovirus (CPV) VP2 protein to a 41-aminoacid long tetramerization domain (TD) from the transcriptional factor p53. The chimerical DNA construction 2L21-TD was cloned in a binary plant transformation vector and used to transform Arabidopsis thaliana plants. Fifteen of the 25 transgenic lines obtained in the experiment showed detectable 2L21-TD RNA accumulation and from these we chose 4 to study 2L21-TD protein accumulation. Non-denaturing immunoblotting assays revealed that 2L21-TD chimeras effectively formed tetrameric complexes with yields reaching up to 12 μg/mg of soluble protein. Mice immunized by oral or intraperitoneal routes with crude protein extracts containing 2L21-TD protein were able to detect both 2L21-synthetic peptide and CPV VP2 proteins, with titers similar to those elicited by a previously reported fusion between 2L21 and the β-glucuronidase protein. These results demonstrate that multimerization directed by the small TD domain contributed to the stabilization and consequently to the accumulation of the 2L21 peptide in transgenic plants, without altering its native antigenicity and immunogenicity.Item Self-aggregation of a recombinant form of the propeptide NH2-terminal of the precursor of pulmonary surfactant protein SP-B: a conformational study(Journal of Industrial Microbiology & Biotechnology, 2008) Bañares-Hidalgo, A.; Bolaños-Gutiérrez, A.; Pérez Gil, Jesús; Gil Dones, Félix; Estrada, P.; Estrada, P.; Society for Industrial MicrobiologyA recombinant form of the peptide N-terminally positioned from proSP-B (SP-BN) has been produced in Escherichia coli as fusion with the Maltose Binding Protein, separated from it by Factor Xa cleavage and purified thereafter. This protein module is thought to control assembly of mature SP-B, a protein essential for respiration, in pulmonary surfactant as it progress through the progressively acidified secretory pathway of pneumocytes. Self-aggregation studies of the recombinant propeptide have been carried out as the pH of the medium evolved from neutral to moderately acid, again to neutral and finally basic. The profile of aggregation versus subsequent changes in pH showed differences depending on the ionic strength of the medium, low or moderate, and the presence of additives such as L-arginine (a known aggregation suppressor) and Ficoll 70 (a macromolecular crowder). Circular dichroism studies of SP-BN samples along the aggregation process showed a decrease in α-helical content and a concomitant increase in β-sheet. Intrinsic fluorescence emission of SP-BN was dominated by the emission of Trp residues in neutral medium, being its emission maximum shifted to red at low pH, suggesting that the protein undergoes a pH-dependent conformational change that increases the exposure of their Trp to the environment. A marked increase in the fluorescence emission of the extrinsic probe bis-ANS indicated the exposure of hydrophobic regions of SP-BN at pH 5. The fluorescence of bis-ANS decreased slightly at low ionic strength, but to a great extent at moderate ionic strength when the pH was reversed to neutrality, suggesting that self-aggregation properties of the SP-BN module could be tightly modulated by the conditions of pH and the ionic environment encountered by pulmonary surfactant during assembly and secretion.Item Tissue proteomics in atherosclerosis: elucidating the molecular mechanisms of cardiovascular diseases(Expert Review of Proteomics, 2009) Cuesta, Fernando de la; Álvarez Llamas, Gloria; Gil Dones, Félix; Martin-Rojas, Tatiana; Zubiri, Irene; Pastor Vargas, Carlos; Barderas, Maria ; Vivanco Martínez, FernandoAtherosclerosis is a disease with higher levels of mortality in developed countries. Comprehension of the molecular mechanisms can yield very useful information in clinics for prevention, diagnosis and recovery monitoring. Proteomics represents an ideal methodology for this purpose, as proteins constitute the effectors of the different biological processes running during pathogenesis. To date, studies in atherosclerosis have been mainly focused on the search for plasma biomarkers. However, tissue proteomics allows going deeper into tissue secretomes, arterial layers or particular cells of interest, which, in turn, constitutes a more direct approximation to in vivo operating mechanisms. The aim of this review is to report latest advances in tissue proteomics in atherosclerosis and related diseases (e.g., aortic stenosis and ischemic injury).Item Oral immunogenicity of the plant derived spike protein from swine-transmissible gastroenteritis coronavirus(2000) Gómez, N.; Wigdorovitz, A.; Castañón, S.; Gil Dones, Félix; Ordá, R.; Borca, M. V.; Escribano, J. M.Transgenic plants represent an inexpensive alternative to classical fermentation systems for production of recombinant subunit vaccines. Transgenic potato plants were created that express the N-terminal domain of the glycoprotein S (N-gS) from Transmissible gastroenteritis coronavirus (TGEV), containing the major antigenic sites of the protein. Extracts from potato tubers expressing N-gS were inoculated intraperitoneally to mice, and the vaccinated mice developed serum IgG specific for TGEV. Furthermore, when potato tubers expressing N-gS were fed directly to mice, they developed serum antibodies specific for gS protein, demonstrating the oral immunogenicity of the plant derived spike protein from TGEV.Item High‐yield expression of a viral peptide vaccine in transgenic plants(FEBS Letters, 2001) Gil Dones, Félix; Brun, Alejandro; Wigdorovitz, Andrés; Catalá, Rafael; Martı́nez-Torrecuadrada, Jorge L.; Casal, Ignacio; Salinas, Julio; Borca, Manuel V.; Escribano, José M.A high-yield production of a peptide vaccine in transgenic plants is described here. A21-mer peptide, which confersprotectiontodogsagainstchallengewithvirulentcanine parvovirus,hasbeenexpressedintransgenicplantsasanaminoterminal translational fusionwiththeGUSgene.Transformants were selected on the basis of theirGUS activities, showing expressionlevelsoftherecombinantproteinupto3%ofthetotal leaf soluble protein, a production yield comparable to that obtainedwith the same epitope expressed by chimeric plant viruses. The immunogenicity of the plant-derived peptidewas demonstrated in mice immunized either intraperitoneally or orallywithtransgenicplantextracts,providingthesuitabilityof theGUS fusions approach for low-cost productionof peptide vaccines.Item Successful oral prime‐immunization with VP60 from rabbit haemorrhagic disease virus produced in transgenic plants using different fusion strategies(Plant Biotechnology Journal, 2005) Elena Titarenko; Estela Terrada; Elsa Arcalís; José M. Escribano; Gil Dones, FélixExpression levels of vaccine antigens in transgenic plants have important consequences in their use as edible vaccines. The major structural protein VP60 from the rabbit haemorrhagic disease virus (RHDV) has been produced in transgenic plants using different strategies to compare its accumulation in plant tissues. The highest expressing plants were those presenting stable, complex, high-density structures formed by VP60, suggesting the importance of multisubunit structures for the stability of this protein in plant cells. Mice fed with leaves of transgenic plants expressing VP60 were primed to a subimmunogenic baculovirus-derived vaccine single dose. This indicates that plants expressing VP60 antigen may be a new means for oral RHDV immunization.Item A novel methodology to develop a foot and mouth disease virus (FMDV) peptide-based vaccine in transgenic plants(2002) Dus Santos, Mar´ ıaJ.; Wigdorovitz, Andrés; Trono, Karina; Rı́os, Raúl D.; Franzone, Pascual M.; Gil Dones, Félix; Moreno, Javier; Carrillo, Consuelo; Escribano, José M.; Borca, Manuel V.The expression of antigens in transgenic plants has been increasingly used as an alternative to the classical methodologies for antigen expression in the development of experimental vaccines. However, an important limitation in most cases is the low concentration of the recombinant antigens in the plant tissues, which reduces the possibilities of practical applications. Because the site of insertion of the transferred DNA into the cellular chromosomal DNA is at random, different levels of foreign protein expression in independent transformants is expected. Strategies to allow the evaluation of a high number of the transgenic individuals, usually an expensive and very time consuming process, would permit the selection of those plants presenting the highest levels of recombinant protein expression. Here, we present the development of an experimental immunogen based in the expression of a highly immunogenic epitope from foot and mouth disease virus (FMDV) fused to the glucuronidase (gus A) reporter gene, which allows selection of the transgenic plants by the ß-glucuronidase (ßGUS) enzymatic activity. We produced transgenic plants of alfalfa expressing the immunogenic site between amino acid residues 135–160 of structural protein VP1 (VP135–160), fused to the ßGUS protein. Plants expressing the highest levels of the immunogenic epitope VP135–160, analyzed by Western blot, were efficiently selected based on their levels of ßGUS enzymatic activity. The FMDV epitope expressed in plants was highly immunogenic in mice which developed, after immunization, a strong anti-FMDV antibody response against a synthetic peptide representing the region VP135–160, to native virus VP1, and to purified FMDV particles. Additionally, these mice were completely protected against experimental challenge with the virulent virus. To our knowledge, this constitutes the first report of a peptide-based vaccine produced in transgenic plants that induces a protective immune response when used in experimental hosts. Also, these results demonstrated the possibility of using a novel and simple methodology for obtaining transgenic plants expressing high levels of foreign immunogenic epitopes, which could be directly applied in the development of plant-based vaccines.Item An Optimal Protocol to Analyze the Rat Spinal Cord Proteome(Biomarker Insights, 2009) Gil Dones, Félix; Alonso-Orgaz, Sergio; Avila, Gerardo; Martin-Rojas, Tatiana; Moral-Darde, Veronica; Barroso, Gemma; Vivanco Martínez, Fernando; Scott-Taylor, Julian; Barderas, MariaSince the function of the spinal cord depends on the proteins found there, better defing the normal Spinal Cord Proteome is an important and challenging task. Although brain and cerebrospinal fluid samples from patients with different central nervous system (CNS) disorders have been studied, a thorough examination of specific spinal cord proteins and the changes induced by injury or associated to conditions such as neurodegeneration, spasticity and neuropathies has yet to be performed. In the present study, we aimed to describe total protein content in the spinal cord of healthy rats, employing different proteomics tools. Accordingly, we have developed a fast, easy, and reproducible sequential protocol for protein extraction from rat spinal cords. We employed conventional two dimensional electrophoresis (2DE) in different pH ranges (eg. 4–7, 3–11 NL) combined with identification by mass spectrometry (MALDI-TOF/TOF), as well as first dimension protein separation combined with Liquid Chromatography Mass Spectrometry/Mass Spectrometry (LC-MS/MS) to maximise the benefits of this technology. The value of these techniques is demonstrated here by the identification of several proteins known to be associated with neuroglial structures, neurotransmission, cell survival and nerve growth in the central nervous system. Furthermore this study identified many spinal proteins that have not previously been described in the literature and which may play an important role as either sensitive biomarkers of dysfunction or of recovery after Spinal Cord Injury.