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

Abstract

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.

SpyTag/SpyCatcher display of influenza M2e peptide on norovirus-like particle provides stronger immunization than direct genetic fusion

“…The C-termini of noro-VLPs are located in valleys between the surrounding protruding domains, which positions the M2e peptides (equipped with only a short linker in our construct) between noro-VLP protrusions, potentially limiting the access of large proteins. In future studies, this could be potentially circumvented by fusing multiple peptides in succession to a VLP carrier, as described in (Kim et al, 2013;Lee et al, 2018;Lee et al, 2019) or by separating M2e from noro-VP1 Cterminus by an enlengthened linker like the one introduced recently (Boonyakida et al, 2023). Boonyakida et al used the linker to allow more space between noro-VP1 C-terminus and SpyTag, enhancing conjugation rates for SpyCatcher-fused proteins.…”

Research progress of multi-enzyme complexes based on the design of scaffold protein

“…Boonyakida et al inserted a linker (EAAAK)n (n = 2,3) between the SpyTag and the VP1 protein to increase surface exposure of the SpyTag on the NoV-LPs. They discovered that the conjugation efficiency of the VP1–SpyTag with the linker (EAAAK)n (n = 2,3) improved from ∼15–35 to ∼50–63% based on the densitometric analysis (Boonyakida et al 2023 ). Cai et al redesigned albumin-fusion cocaine hydrolase CocH1 (TV-1380) to extend its biological half-life.…”

Preparation Strategies, Functional Regulation, and Applications of Multifunctional Nanomaterials‐Based DNA Hydrogels

“…assembled photothermal reagents AuNP, peptide drugs, and cancer‐cell‐targeting peptides in DNA hydrogels to construct a powerful cancer‐specific peptide drug delivery system. [ 92 ] Herein, the targeted peptide can specifically deliver the nanocomplex to the cancer cells, and after light illumination activated the AuNP, the light‐absorbing thermal conversion effectively deformed the DNA hydrogel, resulting in the release of peptide drugs. The cell viability and Bradford study have confirmed the high efficiency and safety of this photothermal‐triggered mode.…”