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PhD Defence Robin Klem

encapsulins as functional protein nanocages

Robin Klem is a PhD student in the research group Biomolecular Nanotechnology. His supervisor is prof.dr. J.J.L.M. Cornelissen from the Faculty of Science and Technology.

In this thesis applications and structural analyses of encapsulins are described. Encapsulins are protein nanocages of ~20 nm in diameter and are found in various bacteria. The size and biocompatibility make them interesting candidates as, for example, drug transporters.

With cryo-EM, the structure of encapsulins was analysed at near-atomic resolution, providing new insights in the density of the encapsulin shell and the pores which could allow for transport of molecules across the protein shell. This principle was used when the enzyme l-asparaginase was loaded inside the encapsulin cavity and it could still catalyse the reaction of l-asparagine to l-aspartate. However, the high density of the shell reduced the catalysis, especially compared with the more porous CCMV.

This demonstrated the potential of using encapsulins as enzyme delivery system to cells.  The cell uptake was also explored and it was found that non-macrophage cells showed low uptake of encapsulins loaded with fluorescent monomeric Teal Fluorescent Protein. In macrophages, however, there was high uptake of encapsulins but the cargo protein remained intact. Thus, encapsulins have reduced uptake in most cells, which increase the potential for targeted drug delivery, since non specific uptake will be low.

Targeted drug delivery could be realised by modifying the exterior of encapsulins. The potential to modify encapsulins was also explored by coupling a trypsin inhibiting mini-protein to the exterior. This complex inhibited trypsin, proving that encapsulins can be modified on their exterior using click-chemistry.

Thus, in this thesis, fundamental information on encapsulins and potential applications has been explored. Based on this research, encapsulins could be used for medical purposes.