Addressing Critical Issues Related to Storage and Stability of the Vault Nanoparticle Expressed and Purified from Komagataella phaffi

Abstract

The vault nanoparticle is a eukaryotic assembly consisting of 78 copies of the 99-kDa major vault protein. They generate two cup-shaped symmetrical halves, which in vivo enclose protein and RNA molecules. Overall, this assembly is mainly involved in pro-survival and cytoprotective functions. It also holds a remarkable biotechnological potential for drug/gene delivery, thanks to its huge internal cavity and the absence of toxicity/immunogenicity. The available purification protocols are complex, partly because they use higher eukaryotes as expression systems. Here, we report a simplified procedure that combines human vault expression in the yeast Komagataella phaffii, as described in a recent report, and a purification process we have developed. This consists of RNase pretreatment followed by size-exclusion chromatography, which is far simpler than any other reported to date. Protein identity and purity was confirmed by SDS-PAGE, Western blot and transmission electron microscopy. We also found that the protein displayed a significant propensity to aggregate. We thus investigated this phenomenon and the related structural changes by Fourier-transform spectroscopy and dynamic light scattering, which led us to determine the most suitable storage conditions. In particular, the addition of either trehalose or Tween-20 ensured the best preservation of the protein in native, soluble form.

An Efficient Method for Vault Nanoparticle Conjugation with Finely Adjustable Amounts of Antibodies and Small Molecules

“…The protocol for the purification of the authentic vault (i.e., devoid of the Z peptide) from K. phaffi and in-depth characterization of its purity and properties have been previously described [ 25 ]. In particular, the purification procedure relies upon an RNase pretreatment of cell-free extracts, followed by size exclusion chromatography (SEC) using Sepharose CL-6B as the matrix.…”

“…The MVP gene variant encoding the Z peptide at the C-terminus (MVP-Z) was cloned, as follows. The C-terminal sequence of MVP was removed from the previously constructed [ 25 ] pGAPZ B MVP vector by digestion with Sac I and Not I. Electrophoresis of the digestion mix on 1% agarose gel using TAE as a running buffer was run for 45 min at 90 V and gel-stained with EtBr, which confirmed the digestion of the plasmid. The Not I/ Sac I-digested pGAPZB carrying the N-terminal sequence of MVP was purified from gel using the QIAquick ® Gel Extraction Kit (QIAGEN, Germantown, MD, USA) and quantified with NanoDrop (NanoDrop 2000c Spectrophotomer, ThermoFischer Scientific).…”

“…Authentic human MVP gene cloning and selection of the best-producing clones has been previously described [ 25 ]. The MVP gene variant encoding the Z peptide at the C-terminus (MVP-Z) was cloned, as follows.…”

“…With a view to exploring the vault’s potential as a platform to be variously harnessed to optimize drug delivery, we formerly expressed authentic human vault (i.e., devoid of the Z peptide) in the methylotropic yeast Komagataella phaffii (formerly Pichia pastoris ) [ 25 ] following a previously described protocol [ 26 ]. Then, we purified it by RNase pretreatment of cell-free extracts followed by size exclusion chromatography (SEC) [ 25 ], a procedure representing a modification of the one that we formerly developed starting from transfected insect cell extracts [ 27 ].…”

Vault, viral, and virus-like nanoparticles for targeted cancer therapy

“…By targeting specific cell surface receptors or penetrating the cell membrane, these NPs transfer drugs/biomolecules to the cytoplasm of targeted cells, potentially improving the efficacy of cancer therapy. 70,71 However, there are several challenges associated with the deployment of vault NPs for cancer therapy, namely the lack of clinical trials, limited in vitro/in vivo studies, biological barriers, specificity, and regulation. Vault NPs must be engineered to target cancer cells specifically, while avoiding healthy cells.…”