All-in-one biofabrication and loading of recombinant vaults in human cells

Martín, Fernando; Carreño, Aida; Mendoza, Rosa; Caruana, Pablo; Rodríguez, Francisco; Bravo, Marlon; Benito, Antoni; Ferrer-Miralles, Neus; Céspedes, María Virtudes; Corchero, José Luís

doi:10.1088/1758-5090/ac584d

Cited by 8 publications

(12 citation statements)

References 53 publications

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“…130 nm. This discrepancy between the estimation of NP size by different techniques was already observed for recombinant vaults produced in a human cell line [ 37 ], and it will need further elucidation. However, since NP size according to electron microscopy images (around 70–80 nm) is in agreement with that previously described for native and recombinant vaults, we suggest that it is a more realistic estimation, and it corresponds (as described for other recombinant vaults) to the real NP size.…”

Section: Resultsmentioning

confidence: 96%

“…The particulate nature of vaults hampers the use of standard packed chromatography columns. In this context, we recently described a protocol to purify vault NPs by means of magnetic separation techniques combined with IMAC chemistries, which allowed us to obtain small amounts of highly pure recombinant vaults [ 37 ]. In order to optimize the process, we have compared in this work the protein yield and purity obtained with both magnetic particles and a cobalt-based IMAC resin.…”

Section: Resultsmentioning

confidence: 99%

“…The obtained electron microscopy images showed the presence of pseudo-spherical NPs, confirming that the MVP-H6 monomer produced in bacterial cells maintains its self-assembling properties and is able to form vault-like NPs. It is noticeable that these NPs display a spherical shape, different from natural or recombinant vaults produced in other expression systems, such as mammalian cells [ 37 ]. The NP size was estimated to be around 70–80 nm.…”

Section: Resultsmentioning

confidence: 99%

“…Details of synthetic genes and recombinant plasmids used in this work have been previously described [ 37 ]. Briefly, the sequence of the human major vault protein (MVP, UniProt Q14764; MVP_HUMAN) was modified by adding, at its 3′ end, extra triplets encoding a histidine tag (6xHis).…”

Section: Methodsmentioning

confidence: 99%

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Escherichia coli as a New Platform for the Fast Production of Vault-like Nanoparticles: An Optimized Protocol

Fernández

Carreño

Mendoza

et al. 2022

IJMS

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Vaults are protein nanoparticles that are found in almost all eukaryotic cells but are absent in prokaryotic ones. Due to their properties (nanometric size, biodegradability, biocompatibility, and lack of immunogenicity), vaults show enormous potential as a bio-inspired, self-assembled drug-delivery system (DDS). Vault architecture is directed by self-assembly of the “major vault protein” (MVP), the main component of this nanoparticle. Recombinant expression (in different eukaryotic systems) of the MVP resulted in the formation of nanoparticles that were indistinguishable from native vaults. Nowadays, recombinant vaults for different applications are routinely produced in insect cells and purified by successive ultracentrifugations, which are both tedious and time-consuming strategies. To offer cost-efficient and faster protocols for nanoparticle production, we propose the production of vault-like nanoparticles in Escherichia coli cells, which are still one of the most widely used prokaryotic cell factories for recombinant protein production. The strategy proposed allowed for the spontaneous encapsulation of the engineered cargo protein within the self-assembled vault-like nanoparticles by simply mixing the clarified lysates of the producing cells. Combined with well-established affinity chromatography purification methods, our approach contains faster, cost-efficient procedures for biofabrication in a well-known microbial cell factory and the purification of “ready-to-use” loaded protein nanoparticles, thereby opening the way to faster and easier engineering and production of vault-based DDSs.

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Section: Resultsmentioning

confidence: 96%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Escherichia coli as a New Platform for the Fast Production of Vault-like Nanoparticles: An Optimized Protocol

Fernández

Carreño

Mendoza

et al. 2022

IJMS

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“…However, vault purification requires a two-step process: dialysis followed by size-exclusion chromatography. Martin et al explored the possibility of developing faster and more efficient strategies for vault production and purification [ 32 ]. Instead of the current tedious and time-consuming protocol using insect cells [ 62 ], the researchers employed the expression of His-tagged MVP (MVP-H6) in a human cell line, in which the expressed protein could be purified using one-step immobilized metal affinity chromatography (IMAC).…”

Section: Engineered Design Of Naturally Occurring Self-assembled Prot...mentioning

confidence: 99%

Tailored Functionalized Protein Nanocarriers for Cancer Therapy: Recent Developments and Prospects

Abdel‐Hamid

El-Hafeez

et al. 2023

Pharmaceutics

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Recently, the potential use of nanoparticles for the targeted delivery of therapeutic and diagnostic agents has garnered increased interest. Several nanoparticle drug delivery systems have been developed for cancer treatment. Typically, protein-based nanocarriers offer several advantages, including biodegradability and biocompatibility. Using genetic engineering or chemical conjugation approaches, well-known naturally occurring protein nanoparticles can be further prepared, engineered, and functionalized in their self-assembly to meet the demands of clinical production efficiency. Accordingly, promising protein nanoparticles have been developed with outstanding tumor-targeting capabilities, ultimately overcoming multidrug resistance issues, in vivo delivery barriers, and mimicking the tumor microenvironment. Bioinspired by natural nanoparticles, advanced computational techniques have been harnessed for the programmable design of highly homogenous protein nanoparticles, which could open new routes for the rational design of vaccines and drug formulations. The current review aims to present several significant advancements made in protein nanoparticle technology, and their use in cancer therapy. Additionally, tailored construction methods and therapeutic applications of engineered protein-based nanoparticles are discussed.

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Tuning plasmid DNA amounts for cost-effective transfections of mammalian cells: when less is more

Carreño,

Guerrero-Yagüe,

Casal

et al. 2024

Appl Microbiol Biotechnol

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Transient gene expression (TGE) in mammalian cells is a well-known approach to the fast expression of recombinant proteins. The human cell line HEK (human embryonic kidney) 293F is widely used in this field, due to its adaptability to grow in suspension to high cell densities in serum-free media, amenability to transfection, and production of recombinant proteins in satisfactory quantities for functional and structural analysis. Amounts of plasmid DNA (pDNA) required in transfections for TGE remain high (usually 1 µg pDNA/mL, or even higher), representing a noticeable proportion of the overall cost. Thus, there is an economic need to reduce amounts of coding pDNA in TGE processes. In this work, amounts of both pDNA and transfecting agent used for TGE in HEK 293F cells have been explored in order to reduce them without compromising (or even improving) the productivity of the process in terms of protein yield. In our hands, minimal polyethyleneimine (PEI) cytotoxicity and optimum protein yields were obtained when transfecting at 0.5 µg pDNA/mL (equal to 0.5 µg pDNA/million cells) and a DNA-to-PEI ratio of 1:3, a trend confirmed for several unrelated recombinant proteins. Thus, carefully tuning pDNA and transfecting agent amounts not only reduces the economic costs but also results in higher recombinant protein yields. These results surely have a direct application and interest for the biopharmaceutical industry, always concerned in increasing productivity while decreasing economic costs. Key points • Mammalian cells are widely used to produce recombinant proteins in short times. • Tuning DNA and transfecting agent are of great interest to optimize economic costs. • Reducing DNA and transfecting agent amounts result in higher protein yields. Graphical Abstract

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All-in-one biofabrication and loading of recombinant vaults in human cells

Cited by 8 publications

References 53 publications

Escherichia coli as a New Platform for the Fast Production of Vault-like Nanoparticles: An Optimized Protocol

Escherichia coli as a New Platform for the Fast Production of Vault-like Nanoparticles: An Optimized Protocol

Tailored Functionalized Protein Nanocarriers for Cancer Therapy: Recent Developments and Prospects

Tuning plasmid DNA amounts for cost-effective transfections of mammalian cells: when less is more

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