PROTENGENCTRANS · Protein engineering for encapsulation and intracellular transport

Periodic Report Summary - PROTENGENCTRANS (Protein engineering for encapsulation and intracellular transport)

Numerous proteins in nature form compartments that serve many purposes such as storage and transport of minerals (ferritin), protein folding (chaperones), regulation of enzyme catalysis (enzymatic complexes, e.g. fatty acid synthases), or nucleic acid delivery (viruses). Increasing numbers of natural protein containers (mainly capsid-forming viruses) or their modified versions are being used for nanotechnology, with detailed applications ranging from controlled synthesis of novel materials, catalysis, bioimaging, to drug delivery and gene therapy. In our group, we decided to explore the enzyme lumazine synthase (LS) as a potentially versatile non-viral protein container. In certain species, LS forms an enzyme complex with riboflavin synthase and together they catalyse two last steps in the biosynthesis of vitamin B2. Due to its biocompatibility, spontaneous self-assembly into structures of defined size, and tolerance to structural changes that can be easily introduced by genetic methods, this protein constitutes an attractive scaffold for introducing novel functions. Lumazine synthase from a thermophile Aquifex aeolicus (AaLS) is particularly suitable for modification due to its extreme structural stability. Its 60-meric capsids remain stable even above 700 degrees Celsius, so introduction of multiple mutations does not usually affect the structure. In our project, we have decorated the inner surface of the AaLS capsid with positively charged aminoacid residues (up to +900 per capsid) in order to create a container for negatively charged cargo molecules. The modified AaLS* protein was so effective that it spontaneously sequestered oligonucleotides from its surroundings during production inside E.coli cells. However, after isolation and purification it was possible to remove cellular RNA molecules by digestion with RNAse A followed with size exclusion and to load the capsids with other cargo. Various oligonucleotides, including plasmids and DNA ladders, negatively charged proteins, and gold nanoparticles coated with negatively charged shells have been efficiently encapsulated. We have also developed methodology to decorate the AaLS* capsids with genetically encoded, tissue-selective protein-transduction domains (PTD) or chemoselectively attached tags of various types (fluorophores, short oligopeptides). Currently, we are exploring conditions for loading PTD-AaLS* with guest molecules, monitoring cellular uptake of the complexes, and inducing subsequent intracellular release of the guest. We anticipate that this novel system may find applications in gene therapy (delivery of oligonucleotides to replace malfunctioning genes or silencing hyperactive ones) and in tissue-specific imaging (delivery of dyes or fluorophores) to enable accurate surgical removal of tumours. Encapsulation of prodrugs based on negatively charged cytotoxic molecules enclosed within the AaLS* capsid and delivery to neoplactic cells in a tissue-selective manner is another attractive option.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз