Expansion of the assembly of cowpea chlorotic mottle virus towards non-native and physiological conditions

Schoonen, Lise; Maas, Roel; Nolte, Roeland J. M.; Hest, Jan C. M. van

doi:10.1016/j.tet.2017.04.038

Cited by 18 publications

(24 citation statements)

References 11 publications

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“…Interestingly, this native ELP‐CCMV showed surprisingly high stability up to 50 °C in pH 5.0 buffer ( T= 3 particles by pH‐induced assembly); however, over an extended incubation period under these conditions, a large fraction of the protein aggregated. In pH 7.5 buffer containing 2.5 m NaCl ( T= 1 particles by ELP‐induced assembly), the protein also demonstrated high stability; however, at more physiologically relevant salt concentrations (500 m m NaCl), capsid assembly did not occur, as we reported previously; this indicated the need for more hydrophobic variants …”

Section: Resultsmentioning

confidence: 99%

“…In pH 7.5 buffer containing 2.5 m NaCl (T = 1p articles by ELP-induceda ssembly), the protein also demonstrated high stability; however,a tm ore physiologically relevant salt concentrations( 500 mm NaCl), capsid assembly did not occur,a sw er eportedp reviously;t his indicated the need for more hydrophobic variants. [36] Next, the assembly behavioro fb oth hydrophobic variants was determined (see Figure S7 in the Supporting Information). DLS experiments showed that the capsids of these variants remained assembled in pH 5.0 buffer.I nterestingly,i np H7.5 buffer containing 500 mm NaCl, the capsids did not completely disassemble;t his indicatest he higher stability of these hydrophobic variants relative to that of the native ELP-CCMV.B ecause ELP-CCMV assembly behavior and stability are dependent on many variables, namely,s alt, capsid protein concentration, pH, temperature, and time, [35] am ore extensive dialysis was performed overnight at 4 8Ct oa chievef ull disassembly of these more hydrophobic variants.…”

Section: Assemblyb Ehavior Of the Elp-ccmv Variantsmentioning

confidence: 99%

“…The pET-15b-H 6 -ELP-CCMV(DN26), pET-15b-H 6 -VY1-VY8-ELP-CCMV(DN26), and pET-15b-H 6 -VW1-VW8-ELP-CCMV(DN26) vectors encoding for the three ELP-CCMV variants used herein have previously been constructed. [34,36] Escherichia coli BLR(DE3)pLysS containing any of these vectors was cultured overnight at 37 8Ci nl ysogeny broth (LB;5 0mL) medium containing ampicillin (100 mg L À1 ) and chloramphenicol (50 mg L À1 ). The overnight culture was used to inoculate 2xTY medium (1 L), containing ampicillin (100 mg L À1 ).…”

Section: Generalexpression and Purification Protocolmentioning

confidence: 99%

“…Although 2 m NaCl was still needed to achieve assembly, this assembly could, at least, be realized at a physiologically relevant pH. Further studies were aimed at introducing more hydrophobic guest residues into the ELP domain, with the objective of lowering the amount of NaCl needed to assemble the capsids at physiological concentrations . It was shown that changing only one or two out of the nine guest residues in the ELP fragment was enough to observe a significant decrease in the so‐called “transition salt concentration”.…”

Section: Introductionmentioning

confidence: 99%

“…Further studies werea imed at introducing more hydrophobic guest residues into the ELP domain,w ith the objective of lowering the amount of NaCl neededt oa ssemble the capsids at physiological concentrations. [36] It was shown that changing only one or two out of the nine guest residues in the ELP fragment was enough to observe as ignificant decrease in the so-called "transition salt concentration". In particular,t he two most hydrophobic variants caught our attention because these remarkably assembled at 150 mm NaCl, which was close to the physiological salt concentration; this indicates the potential of these ELP-modified CCMVc apsids as nanocarriers for in vivo studies.…”

Section: Introductionmentioning

confidence: 99%

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Self‐Assembly and Stabilization of Hybrid Cowpea Chlorotic Mottle Virus Particles under Nearly Physiological Conditions

Timmermans

Vervoort

Schoonen

et al. 2018

Chemistry An Asian Journal

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Capsids of the cowpea chlorotic mottle virus (CCMV) hold great promise for use as nanocarriers in vivo. A major drawback, however, is the lack of stability of the empty wild-type virus particles under physiological conditions. Herein, the assembly behavior and stability under nearly physiological conditions of protein-based block copolymers composed of the CCMV capsid protein and two hydrophobic elastin-like polypeptides are reported. UV/Vis spectroscopy studies, dynamic light-scattering analysis, and TEM measurements demonstrate that both hybrid variants form stable capsids at pH 7.5, physiological NaCl concentration, and 37 °C. The more hydrophobic variant also remains stable in a cell culture medium. These engineered, hybrid CCMV capsid particles can therefore be regarded as suitable candidates for in vivo applications.

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Section: Resultsmentioning

confidence: 99%

Section: Assemblyb Ehavior Of the Elp-ccmv Variantsmentioning

confidence: 99%

Section: Generalexpression and Purification Protocolmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Self‐Assembly and Stabilization of Hybrid Cowpea Chlorotic Mottle Virus Particles under Nearly Physiological Conditions

Timmermans

Vervoort

Schoonen

et al. 2018

Chemistry An Asian Journal

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Artificial Organelles: Towards Adding or Restoring Intracellular Activity

2021

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Compartmentalization is one of the main characteristics that define living systems. Creating a physically separated microenvironment allows nature a better control over biological processes, as is clearly specified by the role of organelles in living cells. Inspired by this phenomenon, researchers have developed a range of different approaches to create artificial organelles: compartments with catalytic activity that add new function to living cells. In this review we will discuss three complementary lines of investigation. First, orthogonal chemistry approaches are discussed, which are based on the incorporation of catalytically active transition metal‐containing nanoparticles in living cells. The second approach involves the use of premade hybrid nanoreactors, which show transient function when taken up by living cells. The third approach utilizes mostly genetic engineering methods to create bio‐based structures that can be ultimately integrated with the cell's genome to make them constitutively active. The current state of the art and the scope and limitations of the field will be highlighted with selected examples from the three approaches.

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Plant molecular farming of virus‐like nanoparticles as vaccines and reagents

Rybicki

2019

WIREs Nanomed Nanobiotechnol

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The use of plants for the production of virus‐like nanoparticles (VNPs) dates back to separating natural empty capsids of plant viruses from whole virions nearly 70 years ago, through to the present use of transgenic plants or recombinant Agrobacterium tumefaciens and/or plant virus‐derived vectors for the transient expression of engineered viral or other structural proteins in plants—a production system also known as molecular farming. Plant production of heterologous proteins has major advantages in terms of convenience—whole plants are generally used, and processes do not need to be sterile—and cost, as bulk biomass production is significantly cheaper than by any other method. Plant‐made VNPs in current use for nanotechnology include whole virions and naturally occurring empty capsids of plant viruses, and particles made by reassembly of coat protein (CP) purified from virions or by recombinant expression. Engineered VNP‐forming animal or human virus CPs expressed in plants include L1 protein from human papillomaviruses, human norovirus CP, hepatitis B surface and core antigens, influenza virus HA protein and HIV Gag polyprotein forming large enveloped particles by budding, orbi‐ and rotavirus particles that require assembly of four co‐expressed proteins, and polio‐ and foot and mouth disease viruses which require proteolytic processing of a polyprotein precursor to form 4‐component VNPs. Both plant and animal virus‐derived plant‐made VNPs can be used for surface and internal display of heterologous peptides or even whole proteins. A significant recent development has been the production of pseudovirions in plants, comprising plant or animal virus CPs and RNA or DNA pseudogenomes that can be used to deliver nucleic acid payloads into cultured cells or specific tissues or tumors in whole animals. This article is characterized under: Biology‐Inspired Nanomaterials > Protein and Virus‐Based Structures Therapeutic Approaches and Drug Discovery > Emerging Technologies Diagnostic Tools > in vivo Nanodiagnostics and Imaging

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Expansion of the assembly of cowpea chlorotic mottle virus towards non-native and physiological conditions

Cited by 18 publications

References 11 publications

Self‐Assembly and Stabilization of Hybrid Cowpea Chlorotic Mottle Virus Particles under Nearly Physiological Conditions

Self‐Assembly and Stabilization of Hybrid Cowpea Chlorotic Mottle Virus Particles under Nearly Physiological Conditions

Artificial Organelles: Towards Adding or Restoring Intracellular Activity

Plant molecular farming of virus‐like nanoparticles as vaccines and reagents

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