Production of plant-made molecules for disease diagnosis, therapy and prevention

Plants have emerged as a convenient, safe and economical way to produce high amounts of recombinant proteins (‘Molecular Farming’). Proofs of principle and efficacy exist for many proteins. Recombinant proteins can be expressed in plants either following stable plant transformation or using transient expression systems, and can be delivered as plant material or following purification. The aim of this project is to evaluate the efficiency of the ‘Molecular Farming’ in the production of recombinant Humanin, a small animal mitochondrial polypeptide. Humanin exhibits protective effects against stress conditions and apoptosis in numerous cell types through regulation of signalling processes and is currently investigated as a potential therapeutic target in the treatment of different diseases, including neurodegenerative ones. In this study, two constructs were prepared for transient and stable expression of recombinant Humanin in plants. For transient expression, Humanin gene was introduced in a plant viral vector in a way to express the recombinant protein in fusion with two tags (an N-terminal and a C-terminal) which may facilitate purification and confer protection from proteolysis. This vector will be used to agroinfiltrate tobacco plants, and the recombinant protein, following purification, will be analyzed for its neuroprotective effect on cell lines modelling Parkinson’s disease. For stable expression, Humanin gene in fusion with a sequence encoding a signal peptide for targeting to the apoplast was introduced in a binary vector for Agrobacterium tumefaciens-mediated transformation of tomato plants. Tomato fruits expressing recombinant humanin will be used in strategies for disease prevention and therapy through oral delivery.