The Project
What would you take to space? Imagine you where about to participate in an expedition to a planet far away from Earth and think in the things that you would take with you. Oxygen, water and food would be interesting things to begin with. Maybe some of you are also thinking of your favourite books, music, some electronic devices, something to write, to draw... You should also take medicines. Maybe you or another member of the crew needs them on a daily basis, or just in case anyone becomes ill during the expedition. Now, think: what if your trip was of 5, 10 or 15 years long? It may be possible to carry supplies for some months in the spaceship, but in those years everything will spoil. Its conservation would be hard and costly, and it would take too much space.
To avoid this, scientists from different parts of the world are working in making plants grow and survive in other habitats. Astronauts in the International Space Station have already eaten plants they had grown themselves in space. So, it looks like people participating in future long-term space missions would be able to grow and eat their own plants. However, how about nutrients that aren’t found naturally in plants? Or how about those medicines that we’ve recently talked about? How could astronauts produce specific protein compounds? Well, that is what our project is about.
Broadly speaking, large-scale production of recombinant proteins involves two steps: (i) synthesis of proteins using genetically modified organisms, and (ii) purification of the proteins. The first step has been mainly limited to Escherichia coli and Saccharomyces cerevisiae, although increasingly popular plant-based systems offer the potential for safe, economical and high-capacity production for many proteins of pharmaceutical and nutritional interest. Protein purification processes involve multiple steps. Various systems based on the production of genetically engineered fusion proteins (i.e. an affinity tag covalently linked to a target protein) have been developed to simplify these costly processes. Among them, the plant-based technology involving targeting of oleosin-fused proteins to organelles known as oilbodies has been shown to be cost- effective and enable high levels of production and purification of recombinant proteins.
Starch is the main storage carbohydrate in vascular plants, its abundance as a naturally occurring compound of living terrestrial biomass being surpassed only by cellulose. Synthesized by different isoforms of starch synthases (SS) this polyglucan accumulates as dense and insoluble granules in the plastids. One SS isoform (the GBSS) is bound to the starch granule. In this project we propose to develop a simple and cost- effective plant-based method for production and purification of recombinant proteins. The system is based on the production of plants transiently expressing a target protein (the green fluorescence protein, GFP) fused to GBSS. Transformed plant tissues will be milled in a suitable aqueous buffer and the starch granules will be purified from plant tissue-derived impurities through a series of simple centrifugation and wash/elution steps as in this aqueous environment the starch granule can be made to precipitate. The GBSS::GFP will be engineered to contain a unique cleavage site recognized by a specific protease, enabling the GFP to be separated from the GBSS into the aqueous buffer, while the GBSS remains embedded the starch granule. Once treated with the protease, the starch granules will be removed by centrifugation while the highly purified cleaved GFP can be further purified using conventional downstream processing.
