Engineering of vault nanocapsules with enzymatic and fluorescent properties

Abstract

One of the central issues facing the emerging field of nanotechnology is cellular compatibility. Nanoparticles have been proposed for diagnostic and therapeutic applications, including drug delivery, gene therapy, biological sensors, and controlled catalysis. Viruses, liposomes, peptides, and synthetic and natural polymers have been engineered for these applications, yet significant limitations continue to prevent their use. Avoidance of the body's natural immune system, lack of targeting specificity, and the inability to control packaging and release are remaining obstacles. We have explored the use of a naturally occurring cellular nanoparticle known as the vault, which is named for its morphology with multiple arches reminiscent of cathedral ceilings. Vaults are 13-MDa ribonucleoprotein particles with an internal cavity large enough to sequester hundreds of proteins. Here, we report a strategy to target and sequester biologically active materials within the vault cavity. Attachment of a vault-targeting peptide to two proteins, luciferase and a variant of GFP, resulted in their sequestration within the vault cavity. The targeted proteins confer enzymatic and fluorescent properties on the recombinant vaults, both of which can be detected by their emission of light. The modified vaults are compatible with living cells. The ability to engineer vault particles with designed properties and functionalities represents an important step toward development of a biocompatible nanocapsule.capsule ͉ nanoparticle

The Vault Exterior Shell Is a Dynamic Structure that Allows Incorporation of Vault-Associated Proteins into Its Interior

“…Thus far, the highest resolution reconstruction of vaults (16 Å) is from the CP-MVP construct used to form vaults in this study (10). The reconstruction of the CP-MVP recombinant vault appeared virtually identical on the exterior to reconstructions of vaults isolated from rat tissue (10,23), and spectroscopic results from this and previous studies using luciferase assays and fluorescence quenching indicate that the vault interior is only accessible over time (24). Therefore, some mechanism must allow for a significant modification of the vault structure that is not readily apparent in the static 3-D image that enables entry of large proteins into its interior.…”

“…Using cryoEM it has been previously shown that co-infection of a single insect cell culture with baculoviruses expressing MVP and either VPARP, TEP1, or both proteins leads to the formation of vaults containing the expected protein components in the vault interior (10). The recombinant MVP-only vaults have also been shown to sequester co-expressed luciferase-INT or GL-INT described above (24). Furthermore, the activity of these two proteins could be shielded from the external environment by the thin protein shell of vaults.…”

“…Construction of the VPARP, TEP1, GL-INT and CP-MVP (cysteine rich N-terminal epitope tag-MVP)-expressing baculoviruses are described elsewhere (10,24). The C-terminal MVPinteraction domain of VPARP (aa 1563-1724, M-INT) was inserted into the E.coli expression vector, pET28a (Novagen).…”

“…Quenching of purified GL-INT vaults and measurement of fluorescence intensity were carried out as previously described (24).…”

“…Co-infection of insect cells with baculoviruses coding for MVP and either TEP1, VPARP, or both leads to the formation of recombinant vaults containing these proteins (10). In addition, when an MVP-interaction domain (INT -the C-terminus of VPARP, aa 1471-1724) is fused to heterologous proteins, either luciferase or a green fluorescent protein variant -green lantern (GL), the fusion proteins have been shown to assemble into MVP recombinant vaults (13,24). CryoEM reconstructions indicate that all of the vault-interacting components are bound to the inner surface of the vault particle (10,24).…”