| Over the last ten years, plants have emerged as a promising platform for the production of clinically-useful proteins. Plant molecular farming offers several advantages over other production systems, including a potential for large-scale production and competitive costs at the farm scale. On the other hand, plant biofactones still present major limitations, including notably the problem of protein stability during the recovery process. Production operations in the field or in greenhouses involve several steps, including harvesting, storage and protein extraction, which all may lead to unwanted protein degradation, negatively affecting protein quality and yield. In this context, the main goal of this project was to develop molecular approaches to increase the stability of recombinant proteins during the extraction and purification processes, without the need for adding expensive and often harmful synthetic proteinase inhibitors in the extraction buffer. More specifically, our strategy was to direct induced or ectopic expression of proteinaceous protease inhibitors in the plant platform considered for expression, in such a way that proteases released in the medium after cell disruption would be inhibited, without being able to affect integrity of the proteins extracted. This project included three main steps: (i) studying the potential of a new endogenous inhibitor of alfalfa in protecting proteins in leaf crude extracts; (ii) characterizing alfalfa and potato proteolytic systems in order to identify target proteases and candidate inhibitors for the design of transgenic plants with reduced 'proteolytic power'; and (iii) developing proteinase inhibitor-expressing transgenic lines of potato showing a protein protective effect during the extraction process.; The results presented confirmed the potential of the proposed concept for protecting either endogenous and recombinant proteins recovered in plant leaf extracts. Two patent applications have been submitted based on these conclusions, the first patent related to the protection of recombinant proteins for a direct application in plant molecular farming; the second related to the protection of total endogenous proteins in crude extracts, giving leaf extracts of the host plant a 'plus-value' from an agronomic perspective. |
