Comparison of two eukaryotic systems for the expression of VP6 protein of rotavirus specie A: transient gene expression in HEK293-T cells and insect cell-baculovirus system

Abstract

The VP6 protein of rotavirus A (RVA) is a target antigen used for diagnostic assays and also for the development of new RVA vaccines. We have compared the expression of VP6 protein in human embryonic kidney (HEK293-T) cells with results obtained using a well-established insect cell-baculovirus system. The recombinant VP6 (rVP6) expressed in HEK293-T cells did not present degradation and also retained the ability to form trimers. In the insect cell-baculovirus system, rVP6 was expressed at higher levels and with protein degradation as well as partial loss of ability to form trimers was observed. Therefore, HEK293-T cells represent a less laborious alternative system than insect cells for expression of rVP6 from human RVA.

Rotavirus VP6: involvement in immunogenicity, adjuvant activity, and use as a vector for heterologous peptides, drug delivery, and production of nano-biomaterials

“…Various expression systems (Fig. 1 A), from the prokaryotic E. coli [ 2 , 72 , 126 ] to eukaryotes such as yeast (including the baker's yeast Saccharomyces cerevisiae and the methylotrophic yeasts Pichia pastoris and Hansenula polymorpha ), baculovirus-silkworm systems [ 17 ], insect cell/baculovirus (IC-BV) systems [ 38 , 67 ], mammalian cells [ 38 ], herpes simplex virus 1 (HSV-1)-based vectors [ 90 ], and plant cells [ 37 , 89 , 127 ] have been used to produce multimeric VP6 structures that morphologically resemble VP2- and VP6-containing VLPs, as well as tubes.

Fig.

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“…1 The RV protein VP6 is capable of self-assembling into nano-sized spherical and tubular forms when expressed in isolation under defined conditions. ( A ) Different expression systems from prokaryotic E. coli [ 2 , 72 , 126 ] to eukaryotic systems such as yeast [ 17 ], insect cell/baculovirus systems (IC-BV) [ 38 , 67 ], mammalian cells [ 38 ], HSV-1 [ 90 ], and plant cells [ 37 , 89 , 127 ] have been used successfully to produce assembled structures of VP6 tubes/VLPs. ( B ) Applications of VP6 tubes/VLPs, as vaccines (immunogenicity and protection) [ 15 , 44 , 54 , 55 , 64 – 66 , 70 , 95 , 108 ], adjuvants [ 15 , 16 , 55 , 76 – 78 , 108 – 110 ], immunological carriers [ 49 , 53 , 92 , 96 , 102 , 111 ], drug delivery tools [ 5 , 91 , 122 , 126 ], and nano-biomaterials [ 5 , 23 , 24 , 41 , 42 , 50 , 91 , 122 , 126 ] …”

“…Interestingly, the efficacy of the E. coli -derived VP6-VLPs or trimers against RV infection was significantly lower than that of E. coli -derived DLP2/6 [ 70 ]. In addition to baculovirus and E. coli expression systems, mammalian cells [ 38 ] and HSV-1-based vectors [ 90 ] as well as plant cells [ 37 , 89 , 127 ] have also been shown to have the capacity to produce self-assembled RV VP6 structures to induce protective immune responses against virus infection [ 78 , 90 ].…”

“…The first biochemical analyses of the native oligomeric structure of VP6 were performed using infected cells or virus particles, selectively removing VP6 from double-layered particles (DLPs; VP2/6) [ 11 , 51 ]. In parallel, several other researchers attempting heterologous expression of RV VP6 also reported the production of this protein in its native oligomeric state [ 2 , 17 , 37 , 38 , 67 , 72 , 89 , 90 , 126 , 127 ] and suggested the use of nano-tubular (tube)/nano-spherical (VLP) structures of VP6 as an immunological carrier for heterologous and homologous Ags [ 49 , 102 , 111 ] as well as a drug delivery system [ 126 ]. Of note, these structures were shown to react with VP6-specific mAbs, indicating that their native immunoreactive determinants were conserved [ 44 ].…”