Two-step purification of tag-free norovirus-like particles from silkworm larvae (Bombyx mori)

Boonyakida, Jirayu; Utomo, Doddy Irawan Setyo; Soma, Fahmida Nasrin; Park, Enoch Y.

doi:10.1016/j.pep.2021.106010

Cited by 4 publications

(12 citation statements)

References 57 publications

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“…The VP1-SpyTag variant was purified according to our established protocol . Briefly, VLPs were collected from the fat body lysate via sucrose gradient centrifugation.…”

Section: Materials and Methodsmentioning

confidence: 99%

“…The template for the PCR was pFastBac, carrying the VP1 gene of norovirus GII.4 Tokyo strain (GenBank accession number: BAV93798.1). The cloned plasmids were verified using Sanger sequencing and then transformed into E. coli BmDH10Bac (CP – /Chi – ) for B.…”

Section: Materials and Methodsmentioning

confidence: 99%

“…The template for the PCR was pFastBac, carrying the VP1 gene of norovirus GII.4 Tokyo strain (GenBank accession number: BAV93798.1). 39 The cloned plasmids were verified using Sanger sequencing and then transformed into E. coli BmDH10Bac (CP − /Chi − ) for B. mori nucleopolyhedrovirus (BmNPV) bacmid generation. As described in our previous reports, the successful transformants were isolated as white colonies via blue/ white colony selection.…”

Section: Construction Of Vp1-spytag Variantsmentioning

confidence: 99%

“…In our previous report, the silkworm-derived NoV-LPs exhibited an average hydrodynamic diameter of 34.5 nm, measured using DLS. 39 Therefore, here, we determined the sizes of VLPs using DLS to observe changes in the diameters of the SpyTag variants and decorated VLPs. NoV-(c-SpT) exhibited an average hydrodynamic diameter of 35 nm, similar to our previous observation in a NoV-LP.…”

Section: Design Expression and Purification Of The Vp1-spytag Variantsmentioning

confidence: 99%

“…To date, several expression platforms have been established to produce NoV-LPs, including Escherichia coli, Pichia pastoris, plants, insect cells, and cell-free protein synthesis. − Recently, we demonstrated the use of silkworms (Bombyx mori) and exceptional yield for NoV-LP production . Given the use of a silkworm-based expression system, this platform provides several advantages over the other expression platforms, such as a more flexible workflow, ease of upscaling, simplicity, low risk of contamination, and not requiring aseptic conditions.…”

Section: Introductionmentioning

confidence: 99%

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Improvement of Modular Protein Display Efficiency in SpyTag-Implemented Norovirus-like Particles

et al. 2022

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Genetic fusion and chemical conjugation are the most common approaches for displaying a foreign protein on the surface of virus-like particles (VLPs); however, these methods may negatively affect the formation and stability of VLPs. Here, we aimed to develop a modular display platform for protein decoration on norovirus-like particles (NoV-LPs) by combining the NoV-LP scaffold with the SpyTag/SpyCatcher bioconjugation system, as the NoV-LP is an attractive protein nanoparticle to carry foreign proteins for various applications. The SpyTagged-NoV-LPs were prepared by introducing SpyTag peptide into the C-terminus of the norovirus VP1 protein. To increase surface exposure of the SpyTag peptide on the NoV-LPs, two or three repeated extension linkers (EAAAK) were inserted between the SpyTag peptide and VP1 protein. Fluorescence proteins, EGFP and mCherry, were fused to SpyCatcher and employed as SpyTag conjugation partners. These VP1-SpyTag variants and SpyCatcher-fused EGFP and mCherry were separately expressed in silkworm fat bodies and purified. This study reveals that adding an extension linker did not disrupt the VLP formation; instead, it increased the particle size by 4–6 nm. The conjugation efficiency of the VP1-SpyTag variants with the extended linker improved from ∼15–35 to ∼50–63% based on the densitometric analysis, while it was up to 77% based on an optical quantification of EGFP and mCherry. Results indicate that the linker causes the SpyTag peptides to be positioned further away from the C-termini of VP1 and potentially increases the exposure of the SpyTag to the outer surface of the NoV-LPs, allowing more SpyTag/SpyCatcher complex formation on the VLP surface. Our study provides a strategy for enhancing the conjugation efficiency of NoV-LP and demonstrates the platform’s utility for developing vaccines or functional nanoparticles.

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“…The VP1-SpyTag variant was purified according to our established protocol . Briefly, VLPs were collected from the fat body lysate via sucrose gradient centrifugation.…”

Section: Materials and Methodsmentioning

confidence: 99%

Section: Materials and Methodsmentioning

confidence: 99%

Section: Construction Of Vp1-spytag Variantsmentioning

confidence: 99%

Section: Design Expression and Purification Of The Vp1-spytag Variantsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Improvement of Modular Protein Display Efficiency in SpyTag-Implemented Norovirus-like Particles

et al. 2022

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Virus-like Particles as Antiviral Vaccine: Mechanism, Design, and Application

Zhang

et al. 2023

Biotechnol Bioproc E

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Virus-like particles (VLPs) are viral structural protein that are noninfectious as they do not contain viral genetic materials. They are safe and effective immune stimulators and play important roles in vaccine development because of their intrinsic immunogenicity to induce cellular and humoral immune responses. In the design of antiviral vaccine, VLPs based vaccines are appealing multifunctional candidates with the advantages such as self-assembling nanoscaled structures, repetitive surface epitopes, ease of genetic and chemical modifications, versatility as antigen presenting platforms, intrinsic immunogenicity, higher safety profile in comparison with live-attenuated vaccines and inactivated vaccines. In this review, we discuss the mechanism of VLPs vaccine inducing cellular and humoral immune responses. We outline the impact of size, shape, surface charge, antigen presentation, genetic and chemical modification, and expression systems when constructing effective VLPs based vaccines. Recent applications of antiviral VLPs vaccines and their clinical trials are summarized.

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Simple, versatile, and practical impedimetric immunosensor based on gold nanoparticle-polyaniline nanocomposite for clinical dengue virus detection

Muttaqien

Khoris

Widayanti³

et al. 2023

Biochemical Engineering Journal

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Two-step purification of tag-free norovirus-like particles from silkworm larvae (Bombyx mori)

Cited by 4 publications

References 57 publications

Improvement of Modular Protein Display Efficiency in SpyTag-Implemented Norovirus-like Particles

Improvement of Modular Protein Display Efficiency in SpyTag-Implemented Norovirus-like Particles

Virus-like Particles as Antiviral Vaccine: Mechanism, Design, and Application

Simple, versatile, and practical impedimetric immunosensor based on gold nanoparticle-polyaniline nanocomposite for clinical dengue virus detection

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