Development of a general system for the production of controlled levels of proteins in eukaryotic cells

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The objective of the project is to develop the delRNA-2 system into a practical system for the controlled expression of proteins in eukaryotes, with particular emphasis on making it suitable for high-throughput analysis. To achieve this, a number of critical improvements to the system will be made: (1) At present, insertion of foreign sequences is carried out using conventional restriction enzyme-based technology which is not suitable for the expression screening of libraries. We will therefore make the system GatewayTM - and In-FusionTM- compatible. (2) For many purposes, such as the expression of protein complexes and the sequential steps in a metabolic pathway, it is highly desirable that a system is developed for the co-expression of multiple proteins in different amounts in the same cell. The fact that levels of translation can be altered by mutations in the 5' UTR of delRNA-2 suggests how this could be achieved. We will therefore construct a variety of delRNA-2 cassettes which permit varied levels of protein expression. (3) To date, all delRNA-2 plant expression studies have concerned Nicotiana benthamiana. While this is a useful host, we wish to investigate whether the delRNA-2 system can also be used in other plant species, such as Arabidopsis thaliana. (4) While delRNA-2 has proved to be a very effective way of achieving transient expression, there are certain applications for which stable genetic transformation will be required. Recent research has indicated that this may be possible provided a modified, rather than a wild-type, version of a silencing suppressor is used. We will investigate this possibility by transforming N. benthamiana with delRNA-2 constructs expressing GFP and a mutant version of the suppressor P19. (5) As the translational machinery is conserved among eukaryotes, it is quite possible that the delRNA-2 expression system will be effective in organisms other than plants

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