Recombinant allergens from tobacco root cell cultures

Molecular farming is the production of pharmaceutical important and commercially valuable proteins in plants or plant cell cultures. Compared with animal or microbial expression systems the plant-based expression of proteins has several advantages: potential for large-scale, low-cost biomass production, correct eukaryote-specific post- translational modifications (phosphorylation, glycosylation), low risk of contaminations by mammalian viruses, oncogenes, prions or bacterial toxins, correct folding and assembly of multimeric proteins, low downstream processing affords and costs, introduction of multiple transgenes and finally, no ethical problems with transgenic animals and higher acceptance in the public. In recent years a variety of foreign proteins were expressed in plants including recombinant antibodies and antibody fragments, human interleukins, ß-glucuronidase etc., and different plant species were used for the heterologous expression of proteins including tobacco, potato, soybean or Arabidopsis thaliana. Depending on the expressed protein and the plant system used for production up to 500 g recombinant protein per gram plant tissue could be produced and costs were very low (<25 US $ g-1 protein) compared to mammalian cell cultures and transgenic goats (1.000 - 10.000 US $ g-1 protein). Thus, development of molecular farming strategies will enable new therapies that are now too expensive for wide use of medical practitioners, e.g. tumor-specific antibodies or recombinant allergens for specific immunotherapies. If recombinant allergens are going to play an important role in diagnosis or even in therapy of allergies as suggested by allergologists, the only way to produce the required large amounts on a cost-effective base is the production in plants. Recent results on the expression of Art v 1 in Agrobacterium-transformed tobacco plants look very promising, so that a general strategy will be designed (plasmids, promoter, reporter genes, localization and purification) for the production of plant-derived allergens by molecular farming using plant cell cultures expressing the recombinant allergens identical to the respective native proteins. However, the most expensive step in the production of recombinant allergens is the purification of the recombinant protein. The development of simple and cheap purification strategies will dramatically reduce the production costs. Transgenic tobacco plant root cell cultures over-expressing the pollen allergen Art v 1 and/or the latex allergen Hev b 2, will be generated in a way that the recombinant allergen enters the secretory pathway of these cells and is secreted into the external culture medium thus reducing the purification costs. Parallel to the allergen a fluorescent protein ("production marker") is secreted by the same cellular mechanisms allowing monitoring of the enrichment of the recombinant allergen in the culture medium using fluorescence detection methods. In addition, the recombinant allergens may be labelled with cleavable tags to further reduce the costs of the following purification steps of the recombinant protein. Finally, by using the respective patients` sera, the immunological properties of the recombinant allergens produced by the plant cell cultures will be characterised in comparison to the native and bacteria-produced allergens to test their suitability for applications in diagnostic assays or specific immunotherapy. The aim of this project is (i) to optimise and improve molecular farming strategies transferable to biotechnological production companies for the production of recombinant allergens immunological identical to their native proteins, (ii) the development of low-cost purification procedures of the recombinant allergen to realise the use of recombinant allergens in clinical diagnosis, (iii) to create a basis for the estimation/calculation of the putative production costs per gram of recombinant allergen.