PROTENGENCTRANS · Protein engineering for encapsulation and intracellular transport

The main goal of this project is to develop a system for effective encapsulation and transport of negatively charged guests into living cells using an enginnered protein capsid. The research in prof. Hilvert's group demonstrated that it is possible to engineer the inner surface of a capsid formed by lumazine synthase from Aquifex aeolicus (AaLS) to accept positively charged guests while retaining the icosahedral structure. In my project I will be addressing the following issues: - Engineering the inner surface of the AaLS to express positively charged aminoacid residues maintaining the ability of the mutant to form an icosahedral capsid - Investigating stability and potential selectivity of guest encapsulation by the modified capsid - Attaching a delivery tag to the outer surface of the capsid in order to make it cell-permeable - Enabling efficient release of the cargo after cellular uptake, by attaching a degradation tag. The proposed project will provide us with a complete system for coating, intracellular transport and release of negatively charged biomolecules. It will be used to internalize membrane-impermeable molecules like siRNA, negatively charged GFP, and in perspective other proteins or oligonucleotide-based molecules. Based on our results, novel systems for regulating gene expression and investigating mechanisms of RNA interference may be developed. The use of a protein as the encapsulating agent will help to overcome shortages of existing systems for gene therapy using viral vectors, like mutagenesis or immune response. It raises hope for future medical applications of our results.