Addressing Critical Issues Related to Storage and Stability of the Vault Nanoparticle Expressed and Purified from Komagataella phaffi
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Published:2023-02-20
Issue:4
Volume:24
Page:4214
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ISSN:1422-0067
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Container-title:International Journal of Molecular Sciences
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language:en
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Short-container-title:IJMS
Author:
Tomaino Giulia12ORCID, Pantaleoni Camilla1, Ami Diletta1, Pellecchia Filomena1, Dutriaux Annie2ORCID, Barbieri Linda1ORCID, Garbujo Stefania1ORCID, Natalello Antonino1ORCID, Tortora Paolo1ORCID, Frascotti Gianni1ORCID
Affiliation:
1. Department of Biotechnology and Biosciences, University of Milano-Bicocca, I-20126 Milano, Italy 2. Université Paris Cité, CNRS, Institut Jacques Monod, F-75013 Paris, France
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.
Funder
Italian Ministry of University and Research CHRONOS University of Milano-Bicocca
Subject
Inorganic Chemistry,Organic Chemistry,Physical and Theoretical Chemistry,Computer Science Applications,Spectroscopy,Molecular Biology,General Medicine,Catalysis
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