Final Activity Report Summary - ARTIFICIAL VIRUSES (Design of artificial viruses by combinatorial protein engineering)
With this research project we aimed at developing a high-throughput screening method for the evolutionary selection of protein-based non-viral gene delivery systems that make use of the cell-entry mechanisms of bacterial protein toxins. The entire project, which was scheduled to last two years, involved generation of gene libraries encoding for chimeric DNA-carrier proteins, expression of these gene libraries inside the aqueous compartments of a water-in-oil emulsion and subsequent selection of protein-DNA complexes for their capacity to transfect mammalian cells in culture. One year after the start of the project, we have managed to design, clone and express the chimeric proteins mentioned in the initial project description. Unfortunately, expression levels of the chimeric proteins inside the w/o emulsions was rather low and needed optimisation. We have spent a great deal of our time optimising the S30 extracts and reaction mixtures used for transcribing and translating the gene constructs inside emulsions, which turned out to be worthwhile. The expression levels we have now reached are high enough to continue the work on selecting chimeric proteins for their capacity to facilitate the cell entry of associated pDNA. The coming year we hope to finalise the selection experiments and demonstrate the in vitro compartmentalisation can be used for the screening and selection of artificial viruses.