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Mingo Castel, Ángel

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https://academica-e.unavarra.es/handle/2454/49584

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Mingo Castel

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Ángel

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Producción Agraria

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27

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    PublicationOpen Access
    High-density seedling expression system for the production of bioactive human cardiotrophin-1, a potential therapeutic cytokine, in transgenic tobacco chloroplasts
    (Wiley, 2008) Farrán Blanch, Inmaculada; Río-Manterola, Francisco; Íñiguez, María; Gárate, Sonia; Prieto, Jesús; Mingo Castel, Ángel; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua, “Formación de Tecnólogos” 055/01/11. M.N.C
    Histidine‐tagged human cardiotrophin‐1 (hCT‐1), a recently discovered cytokine with excellent therapeutic potential, was expressed in tobacco chloroplasts under the transcriptional and translational control of two different promoters (rrn and psbA) and 5′‐untranslated regions (5′‐UTRs) (psbA and phage T7 gene 10). The psbA 5′‐UTR promotes recombinant hCT‐1 (rhCT‐1) accumulation in chloroplasts at higher levels (eight‐fold) than those obtained for the phage T7 gene 10 5′‐UTR, regardless of the promoter used, indicating that the correct choice of translational control element is most important for protein production in chloroplasts. The maximum level of rhCT‐1 achieved was 1.14 mg/g fresh weight (equivalent to 5% of total soluble protein) with the psbA promoter and 5′‐UTR in young leaves harvested after 32 h of continuous light, although the bioactivity was significantly lower (~35%) than that of commercial hCT‐1. However, harvesting in the dark or after 12 h of light did not result in a significant decrease in the bioactivity of rhCT‐1, suggesting that 32 h of over‐lighting affects the biological activity of rhCT‐1. Because high levels of rhCT‐1 accumulation took place mainly in young leaves, it is proposed that seedlings should be used in a ‘closed system’ unit, yielding up to 3.2 kg per year of rhCT‐1. This amount would be sufficient to meet the estimated annual worldwide needs of hCT‐1 for liver transplantation surgery in a cost‐effective manner. Furthermore, our strategy is an environmentally friendly method for the production of plant‐based biopharmaceuticals.
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    PublicationOpen Access
    Potato genetic resources in Spain
    (International Plant Genetic Resources Institute, 2001) Ritter, E.; Ruiz de Galarreta, José Ignacio; Carrasco, A.; Ruiz De Arcaute Rivero, Roberto; Veramendi Charola, Jon; Mingo Castel, Ángel; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
    Plant genetic resources activities in Spain are globally organized by the Instituto Nacional de Investigación Agraria (INIA) and in particular by one of its institutes, Centro de Recursos Fitogenéticos (CRF). Collections of beans, maize, cereals and many other crops are maintained, evaluated and characterized in the station at Alcala de Henares near Madrid. However, the situation is different for potato. Germplasm collections of potato are maintained in collaborating institutes or private companies. The largest collection with 604 accessions is held at NEIKER (former CIMA, Centro de Investigación y Mejora Agraria), which has been traditionally, as the Station for potato improvement (Estación de la Mejora de la Patata), the cradle of seed potatoes in Spain. Other remarkable collections are maintained at the Public University of Navarra (UPNA), the Instituto de Agrobiotecnología y Recursos Naturales (116 accessions) and the public enterprise APPACALE (213 accessions), which produces seed potatoes and also performs potato breeding in Spain.
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    PublicationOpen Access
    A chloroplast transgenic approach to hyper-express and purify human serum albumin, a protein highly susceptible to proteolytic degradation
    (Wiley / Blackwell, 2003) Fernández San Millán, Alicia; Mingo Castel, Ángel; Miller, Michael; Daniell, Henry; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua
    Human Serum Albumin (HSA) accounts for 60% of the total protein in blood serum and it is the most widely used intravenous protein in a number of human therapies. HSA, however, is currently extracted only from blood because of a lack of commercially feasible recombinant expression systems. HSA is highly susceptible to proteolytic degradation in recombinant systems and is expensive to purify. Expression of HSA in transgenic chloroplasts using Shine-Dalgarno sequence (SD), which usually facilitates hyper-expression of transgenes, resulted only in 0.02% HSA in total protein (tp). Modification of HSA regulatory sequences using chloroplast untranslated regions (UTRs) resulted in hyper-expression of HSA (up to 11.1% tp), compensating for excessive proteolytic degradation. This is the highest expression of a pharmaceutical protein in transgenic plants and 500-fold greater than previous reports on HSA expression in transgenic leaves. Electron micrographs of immunogold labelled transgenic chloroplasts revealed HSA inclusion bodies, which provided a simple method for purification from other cellular proteins. HSA inclusion bodies could be readily solubilized to obtain a monomeric form using appropriate reagents. The regulatory elements used in this study should serve as a model system for enhancing expression of foreign proteins that are highly susceptible to proteolytic degradation and provide advantages in purification, when inclusion bodies are formed.