# In Vitro Refolding of Vault-like Protein Nanocapsules with a Novel Scaffolding Mechanism

## TL;DR

Scientists successfully refolded vault-like protein nanoparticles in the lab using a new scaffolding method, creating uniform particles that could be useful for drug delivery.

## Contribution

The novel scaffolding mechanism enables scalable and homogeneous assembly of vault-like nanoparticles using DNA or hyaluronic acid-binding tags.

## Key findings

- Vault-like nanoparticles with ~26 nm hydrodynamic radius were formed using DNA or hyaluronic acid-binding tags.
- FRET analysis confirmed nanoparticle assembly only when DNA-binding tags were present.
- This is the first scalable bioengineering method for homogenous vault-like nanoparticles.

## Abstract

We attempted the in vitro scaffold-coordinated refolding of denatured major vault protein monomers into assembled vault-like nanoparticles. DNA or hyaluronic acid-binding tags were added to the MVP monomers, allowing MVP to align rotationally and translationally along these linear molecules. This was proposed to mimic the polyribosome assembly in vivo. Tagged MVP variants were expressed in E. coli and purified under denaturing conditions. Dynamic light scattering showed the formation of nanoparticles with a hydrodynamic radius of ~26 nm, consistent with the formation of vault-like nanoparticles. This was confirmed by transmission electron microscopy, FRET analysis, and cargo loading of CFP-INT fusion. CFP- and YFP-tagged MVP showed FRET only in the presence of MVP with a DNA-binding tag. This is the first successful instance of bioengineering of homogenous and heterogeneous vault-like nanoparticles, and at a potentially much larger scale than current protocols.