Engineering of SV40-based nano-capsules for delivery of heterologous proteins as fusions with the minor capsid proteins VP2/3

Inoue, Takamitsu; Kawano, Masaaki; Takahashi, Ryou‐u; Tsukamoto, Hiroko; Enomoto, Teruya; Imai, Takeshi; Kataoka, Kohsuke; Handa, Hiroshi

doi:10.1016/j.jbiotec.2007.12.006

Cited by 80 publications

(66 citation statements)

References 27 publications

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“…Fusion at the C-terminus of VP2C is expected to position GFP inside the cavity of the VLP (Inoue et al 2008;Pleckaityte et al 2015). This attempt was done by two approaches, the first is to bind VP2C-GFP and VP1 capsomeres in vitro, and the second is to facilitate binding of VP2C-GFP and VP1 capsomeres in vivo.…”

Section: Resultsmentioning

confidence: 99%

“…The minor coat proteins VP2 or VP3 are not required for self-assembly, but they have been shown to enable foreign protein encapsidation inside the MPyV VLP (Abbing et al 2004;Boura et al 2005;Inoue et al 2008). …”

Section: Protein Encapsidation By Mpyvmentioning

confidence: 99%

“…One VP2 (and VP3) protein binds to the hydrophobic inner cavity of one VP1-capsomere via a conserved hydrophobic region close to the C-terminal of VP2, forming a VP1:VP2 capsomere complex (Barouch and Harrison 1994;Chen et al 1998) (Figure 2.3). While the hydrophobic binding Encapsidation has been achieved by either co-expression of VP1 and foreign protein fused VP2/VP2C within insect cells (Boura et al 2005;Inoue et al 2008), or by separate expression in E.coli and separate chromatographic purification before co-assembly in vitro (Abbing et al 2004). The GFP has been fused to VP2/VP2C in most cases to enable chromatographic detection at A488 nm (Abbing et al 2004;Boura et al 2005;Inoue et al 2008) shown to be better processed by the MHC Class I pathway and are more likely to be presented by APCs than short peptides (Berthoud et al 2011).…”

Section: Protein Encapsidation By Mpyvmentioning

confidence: 99%

“…Fusion of GFP protein at the N-terminus of VP2 was attempted to encapsidate GFP inside the MPyV VLP. However, this approach resulted in externalisation of the GFP (Abbing et al 2004;Boura et al 2005;Pleckaityte et al 2015).On the other hand, fusion at the C-terminus of the C-terminal binding fragment of VP2 (VP2C) of SV40 and HPyV allowed encapsidation of GFP inside MPyV VLPs (Inoue et al 2008;Pleckaityte et al 2015).…”

Section: Encapsidation Is Achievable By Utilising a Segment Of The C-mentioning

confidence: 99%

“…Tc-epitope proteasomal processing is intracellular, and unlike recognition of (Abbing et al 2004;Boura et al 2005;Inoue et al 2008). In my research, GFP was fused to the C-terminus of VP2.…”

Section: Optimisation Of Encapsidationmentioning

confidence: 99%

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Murine polyomavirus VLPs as a platform for cytotoxic T cell epitope-based influenza vaccine candidates

Abidin¹

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This research examined the ability of virus-like particles (VLP) assembled from the coat proteins of Murine polyomavirus (MPyV) to carry cytotoxic T cell (Tc) epitopes. MPyV VLPs can be assembled in vitro from purified subunits composed of pentamers of the major coat protein VP1 called capsomeres; and this approach provides fine control over VLP composition and structure. Tc-epitopes are often highly hydrophobic, which poses a significant bioengineering challenge and two distinct approaches were pursued to determine the optimal delivery strategy of Influenza Tc-epitopes by MPyV VLP. Firstly, identified epitopes were externally presented using established sites for peptide insertion on the MPyV major coat protein, VP1. Secondly, polypeptides possessing multiple epitopes were encapsidated within VP1 VLP using precise elements of the minor coat protein VP2/3 that interact with the interior cavity of capsomeres. Hydrophobicity-related capsomere aggregation arising from single Tc epitope presentation was successfully circumvented by engineering charged amino acids (DD) flanking the epitope displayed on a surface-exposed loop of VP1 and by use of an optimised buffer condition. A multiparametric, high-throughput buffer screen was implemented to optimise pH and buffer additives during affinity tag removal. Stable modified capsomere recovered from this reaction were assembly competent in the presence of L-Arginine, and VLP yield was high when modular capsomeres were co-assembled with unmodified VP1 capsomeres forming stable mosaic VLPs. The ability of the MPyV VLP to encapsidate foreign protein was first evaluated and optimised with green fluorescent protein (GFP) as a model protein to enable monitoring and analysis.A minimal VP1-interacting sequence was defined from the carboxy-terminus of VP2/3 (VP2C) and fused to the amino-terminus of GFP, ensuring internalisation of the reporter protein. VP1:VP2C-GFP capsomere complexes were successfully recovered when VP1 was co-expressed with VP2C-GFP, whereas complex formation was not observed when VP1 and VP2C-GFP were mixed in vitro.Recovered capsomere complexes were assembly competent and amount of encapsidated GFP was controllable when VP1:VP2C-GFP capsomere complexes were co-assembled with unmodified VP1 capsomeres in a defined molar ratio. The encapsidation approach was then applied to a subdomain of the Influenza matrix protein M1 by co-expressing VP1 with VP2C-M1-I. M1-I capsomere complexes were assembly competent as indicated by gel electrophoresis, dynamic light scattering, and transmission electron microscopy. To enable recovery of VLPs for analysis and characterisation as well as to confirm encapsidation and the formation of mosaic VLPs, a semi-preparative size exclusion chromatography method was developed. This research was able to address bioengineering challenges posed by hydrophobic epitope presentation and developed MPyV

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Section: Resultsmentioning

confidence: 99%

Section: Protein Encapsidation By Mpyvmentioning

confidence: 99%