Person: Veramendi Charola, Jon
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Veramendi Charola
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Jon
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AgronomĆa, BiotecnologĆa y AlimentaciĆ³n
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IMAB. Research Institute for Multidisciplinary Applied Biology
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0000-0002-3214-213X
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539
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Publication Open Access Post-harvest light treatment increases expression levels of recombinant proteins in transformed plastids of potato tubers(Wiley, 2015) Larraya Reta, Luis MarĆa; FernĆ”ndez San MillĆ”n, Alicia; AncĆn RĆpodas, MarĆa; FarrĆ”n Blanch, Inmaculada; Veramendi Charola, Jon; IdAB. Instituto de AgrobiotecnologĆa / Agrobioteknologiako InstitutuaPlastid genetic engineering represents an attractive system for the production of foreign proteins in plants. Although high expression levels can be achieved in leaf chloroplasts, the results for non-photosynthetic plastids are generally discouraging, mainly due to low transcriptional and translational rates in comparison with chloroplasts. Here, we report the expression of two thioredoxin genes (trx f and m) from the potato plastid genome to study transgene expression in amyloplasts. As expected, the highest transgene expression was detected in the leaf (up to 4.2% of TSP). The Trx protein content in the tuber was approximately 2-3 orders of magnitude lower than in the leaf. However, we demonstrate that a simple post-harvest light treatment of microtubers developed in vitro or soil-grown tubers induces up to 55 times higher accumulation of the recombinant protein in just 7-10 days. The promoter and 5ā-UTR of the psbA gene displayed higher light induction than the rrn promoter. After the applied treatment, the Trx f levels in microtubers and soil-grown tubers increased to 0.14% and 0.11% of TSP, respectively. Moreover, tubers stored for 8 months maintained the capacity of increasing the foreign protein levels after the light treatment. Post-harvest cold induction (up to 5 times) at 4 ĀŗC was also detected in microtubers. We conclude that plastid transformation and post-harvest light treatment could be an interesting approach for the production of foreign proteins in potato.Publication Open Access Functional improvement of human cardiotrophin 1 produced in tobacco chloroplasts by co-expression with plastid thioredoxin m(MDPI, 2020) AncĆn RĆpodas, MarĆa; Sanz Barrio, Ruth; SantamarĆa, Eva; FernĆ”ndez San MillĆ”n, Alicia; Larraya Reta, Luis MarĆa; Veramendi Charola, Jon; FarrĆ”n Blanch, Inmaculada; Institute for Multidisciplinary Research in Applied Biology - IMABHuman cardiotrophin 1 (CT1), a cytokine with excellent therapeutic potential, was previously expressed in tobacco chloroplasts. However, the growth conditions required to reach the highest expression levels resulted in an impairment of its bioactivity. In the present study, we have examined new strategies to modulate the expression of this recombinant protein in chloroplasts so as to enhance its production and bioactivity. In particular, we assessed the effect of both the fusion and co-expression of Trx m with CT1 on the production of a functional CT1 by using plastid transformation. Our data revealed that the Trx m fusion strategy was useful to increase the expression levels of CT1 inside the chloroplasts, although CT1 bioactivity was significantly impaired, and this was likely due to steric hindrance between both proteins. By contrast, the expression of functional CT1 was increased when co-expressed with Trx m, because we demonstrated that recombinant CT1 was functionally active during an in vitro signaling assay. While Trx m/CT1 co-expression did not increase the amount of CT1 in young leaves, our results revealed an increase in CT1 protein stability as the leaves aged in this genotype, which also improved the recombinant proteinās overall production. This strategy might be useful to produce other functional biopharmaceuticals in chloroplasts.Publication Open Access Human papillomavirus L1 protein expressed in tobacco chloroplasts self-assembles into virus-like particles that are highly immunogenic(Wiley, 2008) FernĆ”ndez San MillĆ”n, Alicia; MartĆn Ortigosa, Susana; HervĆ”s Stubbs, Sandra; Corral-MartĆnez, Patricia; SeguĆ-Simarro, JosĆ© M.; GaĆ©tan, Julien; Coursaget, Pierre; Veramendi Charola, Jon; IdAB. Instituto de AgrobiotecnologĆa / Agrobioteknologiako InstitutuaCervical cancer is the second most prevalent cancer in women worldwide. It is linked to infection with human papillomavirus (HPV). As the virus cannot be propagated in culture, vaccines based on virusālike particles have been developed and recently marketed. However, their high costs constitute an important drawback for widespread use in developing countries, where the incidence of cervical cancer is highest. In a search for alternative production systems, the major structural protein of the HPVā16 capsid, L1, was expressed in tobacco chloroplasts. A very high yield of production was achieved in mature plants (~3 mg L1/g fresh weight; equivalent to 24% of total soluble protein). This is the highest expression level of HPV L1 protein reported in plants. A single mature plant synthesized ~240 mg of L1. The chloroplastāderived L1 protein displayed conformationāspecific epitopes and assembled into virusālike particles, visible by transmission electron microscopy. Furthermore, leaf protein extracts from L1 transgenic plants were highly immunogenic in mice after intraperitoneal injection, and neutralizing antibodies were detected. Taken together, these results predict a promising future for the development of a plantābased vaccine against HPV.Publication Open Access Physiological performance of transplastomic tobacco plants overexpressing aquaporin AQP1 in chloroplast membranes(Oxford University Press, 2018) FernĆ”ndez San MillĆ”n, Alicia; Aranjuelo Michelena, Iker; AncĆn RĆpodas, MarĆa; Larraya Reta, Luis MarĆa; FarrĆ”n Blanch, Inmaculada; Veramendi Charola, Jon; Agronomia, Bioteknologia eta Elikadura; AgronomĆa, BiotecnologĆa y AlimentaciĆ³n; IdAB. Instituto de AgrobiotecnologĆa / Agrobioteknologiako InstitutuaThe leaf mesophyll CO2 conductance and the concentration of CO2 within the chloroplast are major factors affecting photosynthetic performance. Previous studies have shown that the aquaporin NtAQP1 (which localizes to the plasma membrane and chloroplast inner envelope membrane) is involved in CO2 permeability in the chloroplast. Levels of NtAQP1 in plants genetically engineered to overexpress the protein correlated positively with leaf mesophyll CO2 conductance and photosynthetic rate. In these studies, the nuclear transformation method used led to changes in NtAQP1 levels in the plasma membrane and the chloroplast inner envelope membrane. In the present work, NtAQP1 levels were increased up to 16-fold in the chloroplast membranes alone by the overexpression of NtAQP1 from the plastid genome. Despite the high NtAQP1 levels achieved, transplastomic plants showed lower photosynthetic rates than wild-type plants. This result was associated with lower Rubisco maximum carboxylation rate and ribulose 1,5-bisphosphate regeneration. Transplastomic plants showed reduced mesophyll CO2 conductance but no changes in chloroplast CO2 concentration. The absence of differences in chloroplast CO2 concentration was associated with the lower CO2 fixation activity of the transplastomic plants. These findings suggest that non-functional pores of recombinant NtAQP1 may be produced in the chloroplast inner envelope membrane.