Expression of rotavirus VP6 protein: a comparison amongstEscherichia coli, Pichia pastorisandHansenula polymorpha

Abstract

During this study. we successfully expressed a codon-optimized gene for rotavirus VP6 protein intracellularly in two methylotrophic yeasts, Pichia pastoris and Hansenula polymorpha, during methanol induction. Expressions were performed in shake flasks and subsequently scaled-up to 1.3 L bioreactors. The yields obtained in the yeasts were compared with that observed in Escherichia coli. Despite producing the lowest biomass levels of all the expression systems in shake flasks, the highest VP6 concentration was obtained with E. coli. In shake flasks, P. pastoris yielded higher volumetric levels of VP6 than H. polymorpha, but specific production of VP6 was approximately similar in both yeasts. In the controlled environment of bioreactors, yeast strains attained typical high cell densities, but also increased VP6 production compared to all shake flask cultures. Unlike in shake flask expressions, H. polymorpha outperformed both P. pastoris as well as E. coli during bioreactor cultivation. VP6 production was in all three expression systems growth-associated. In contrast to yeast expressions, bacterial expressed VP6 protein was found to be insoluble upon analysis. This is the first report of VP6 expressed in methylotrophic yeast and holds the promise for the inexpensive production of VP6 as a possible vaccine candidate or drug delivery mechanism.

Activation of PI3K, Akt, and ERK during early rotavirus infection leads to V-ATPase-dependent endosomal acidification required for uncoating

“…RVA GST - VP8* fusion proteins were purified using the Pierce GST spin purification kit (Pierce, IL, USA) according to the manufacturer’s protocol. Recombinant RVA NSP1 and VP6 proteins were also expressed and purified as mentioned before [ 61 – 62 ]. The full genome amplicons of NSP1 and VP6 were cloned into plasmids pET28a and pPROEX HTc, respectively, digested with NcoI and XhoI, and the resulting constructs were verified by sequencing.…”

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.

…”

“…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 ] …”

“…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 ].…”

Advances in Using Hansenula polymorpha as Chassis for Recombinant Protein Production

“…Furthermore, H. polymorpha , has been extensively used for the production of Virus-like particle (VLP), which are viral proteins that can be used for the development of vaccines (Kumar and Kumar, 2019). Only in the last decade, H. polymorpha was used to produce VLPs for the hepatitis B virus (Li et al, 2011; Xu et al, 2014), hepatitis C virus (He et al, 2008), hepatitis E virus (Su et al, 2017), Human papillomavirus (Li et al, 2009; Liu et al, 2015; Bredell et al, 2018), rabies virus (Qian et al, 2013), and rotavirus (Bredell et al, 2016). Recently, a chimeric VLP was development for H. polymorpha (Wetzel et al, 2018).…”