Native vaults (72.5 × 41 × 41 nm) are self-assembled, ribonucleoprotein nanocapsules, that consist of multiple copies of 3 proteins (MVP, VPARP, and TEP1) and a small untranslated RNA. Although vaults exist in most eukaryotic cells, their function remains unknown. However, this naturally occurring “organelle” has significant potential as a drug delivery system due to its biocompatibility, large and accessible lumen, and ability to be taken up by mammalian cell lines. The 96 copies of major vault protein (MVP) account for about 70% by mass of the entire vault particle, and stable vault particles can self-assemble from recombinant MVP alone in solution. Our goal is to discover methods to control vault conformational change and structural stability in solution, and to successfully encapsulate material, thereby enabling application of these nanocapsules in drug delivery and nanomaterials synthesis.

Previous studies suggest that the vault has a leaky dynamic structure in solution, which must be overcome if these nanocapsules are to be useful drug delivery vehicles. To resolve this issue, we are investigating the use of an internally synthesized polymer to sequester molecules of interest in a stable state for subsequent delivery. In order to synthesize an encapsulated polymer, horseradish peroxidase (HRP), which catalyzes the polymerization of tyramine, was fused to a protein (INT) that is targeted specifically to the vault interior. The fusion protein (HRP-INT) was expressed using a baculovirus expression system in Sf9 insect cells and then packaged inside of vaults at the crude extract stage. The HRP-INT protein was found to co-purify with vaults and to retain HRP activity, which suggests that the HRP harbored inside the vault remains functional. Current studies include efforts to increase protein expression, and to analyze polymer synthesis using vaults containing entrapped HRP activity.