Alicia Fernández-San Millán

Human papillomavirus L1 protein expressed in tobacco chloroplasts self-assembles into virus-like particles that are highly immunogenic.

Cervical 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 virus-like 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 HPV-16 capsid, L1, was expressed in tobacco chloroplasts. A very high yield of production was achieved in mature plants (approximately 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 approximately 240 mg of L1. The chloroplast-derived L1 protein displayed conformation-specific epitopes and assembled into virus-like 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 plant-based vaccine against HPV.

Chaperone-like properties of tobacco plastid thioredoxins f and m

Thioredoxins (Trxs) are ubiquitous disulphide reductases that play important roles in the redox regulation of many cellular processes. However, some redox-independent functions, such as chaperone activity, have also been attributed to Trxs in recent years. The focus of our study is on the putative chaperone function of the well-described plastid Trxs f and m. To that end, the cDNA of both Trxs, designated as NtTrxf and NtTrxm, was isolated from Nicotiana tabacum plants. It was found that bacterially expressed tobacco Trx f and Trx m, in addition to their disulphide reductase activity, possessed chaperone-like properties. In vitro, Trx f and Trx m could both facilitate the reactivation of the cysteine-free form of chemically denatured glucose-6 phosphate dehydrogenase (foldase chaperone activity) and prevent heat-induced malate dehydrogenase aggregation (holdase chaperone activity). Our results led us to infer that the disulphide reductase and foldase chaperone functions prevail when the proteins occur as monomers and the well-conserved non-active cysteine present in Trx f is critical for both functions. By contrast, the holdase chaperone activity of both Trxs depended on their oligomeric status: the proteins were functional only when they were associated with high molecular mass protein complexes. Because the oligomeric status of both Trxs was induced by salt and temperature, our data suggest that plastid Trxs could operate as molecular holdase chaperones upon oxidative stress, acting as a type of small stress protein.

Tobacco plastidial thioredoxins as modulators of recombinant protein production in transgenic chloroplasts

Thioredoxins (Trxs) are small ubiquitous disulphide proteins widely known to enhance expression and solubility of recombinant proteins in microbial expression systems. Given the common evolutionary heritage of chloroplasts and bacteria, we attempted to analyse whether plastid Trxs could also act as modulators of recombinant protein expression in transgenic chloroplasts. For that purpose, two tobacco Trxs (m and f) with different phylogenetic origins were assessed. Using plastid transformation, we assayed two strategies: the fusion and the co-expression of Trxs with human serum albumin (HSA), which was previously observed to form large protein bodies in tobacco chloroplasts. Our results indicate that both Trxs behave similarly as regards HSA accumulation, although they act differently when fused or co-expressed with HSA. Trxs-HSA fusions markedly increased the final yield of HSA (up to 26% of total protein) when compared with control lines that only expressed HSA; this increase was mainly caused by higher HSA stability of the fused proteins. However, the fusion strategy failed to prevent the formation of protein bodies within chloroplasts. On the other hand, the co-expression constructs gave rise to an absence of large protein bodies although no more soluble HSA was accumulated. In these plants, electron micrographs showed HSA and Trxs co-localization in small protein bodies with fibrillar texture, suggesting a possible influence of Trxs on HSA solubilization. Moreover, the in vitro chaperone activity of Trx m and f was demonstrated, which supports the hypothesis of a direct relationship between Trx presence and HSA aggregates solubilization in plants co-expressing both proteins.

Human papillomavirus-like particles vaccine efficiently produced in a non-fermentative system based on insect larva

a Instituto de Agrobiotecnologia (Universidad Publica de Navarra–CSIC–Gobierno de Navarra), Campus Arrosadia, 31006 Pamplona, Spain Alternative Gene Expression S.L. (ALGENEX), Centro empresarial Parque Cientifico y Tecnologico de la Universidad Politecnica de Madrid Campus de Montegancedo, Pozuelo deAlarcon, 28223 Madrid, Spain Departamento de Biotecnologia, INIA, Autovia A6 Km 7, 28040 Madrid, Spain

Over-expression of peptide deformylase in chloroplasts confers actinonin resistance, but is not a suitable selective marker system for plastid transformation

Arabidopsis thaliana peptide deformylase PDF1B was expressed in tobacco chloroplasts using spectinomycin as the selective agent. The foreign protein accumulated in chloroplasts (6% of the total soluble protein) and was enzymatically active. Transplastomic plants were evaluated for resistance to the peptide deformylase inhibitor actinonin. In vitro seed germination in the presence of actinonin and in planta application of the inhibitor demonstrated the resistance of the transformed plants. In addition, transgenic leaf explants were able to develop shoots via organogenesis in the presence of actinonin. However, when the combination of the PDF1B gene and actinonin was used as the primary selective marker system for chloroplast transformation of tobacco, all developed shoots were escapes. Therefore, under the experimental conditions tested, the use of this system for plastid transformation would be limited to function as a secondary selective marker.

Post-harvest light treatment increases expression levels of recombinant proteins in transformed plastids of potato tubers

Plastid 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. Here, we report the expression of two thioredoxin genes (trx f and trx 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 two to three 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 seven to ten days. 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 eight months maintained the capacity of increasing the foreign protein levels after the light treatment. Post‐harvest cold induction (up to five times) at 4°C 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.

Physiological performance of transplastomic tobacco plants overexpressing aquaporin AQP1 in chloroplast membranes

Overexpression of NtAQP1 from the tobacco plastid genome resulted in impaired photosynthetic performance of transplastomic tobacco plants.