Rabies virus glycoprotein expression in Drosophila S2 cells. I: Design of expression/selection vectors, subpopulations selection and influence of sodium butyrate and culture medium on protein expression

Abstract

The cDNA encoding the rabies virus glycoprotein (RVGP) gene was cloned in expression plasmids under the control of the inductive metallothionein promoter. They were designed in order to bear or not a secretion signal (i) and a cDNA coding for the selection hygromycin. These vectors were transfected into S2 cells, cell populations selected and subpopulations were then obtained by reselection with hygromycin. Cell cultures were examined for kinetics of cell growth, detection of RVGP mRNA and expression of RVGP. All cell populations were shown to express the RVGP mRNA upon induction. S2MtRVGPHy cell population, transfected with one vector that contains RGPV gene and selection gene, was shown to express higher amounts of RVGP as evaluated by flow cytometry ( approximately 52%) and ELISA (0.64 microg/10(7)cells at day 7). Subpopulation selection allowed a higher RVGP expression, specially for the S2MtRVGPHy(+) (5.5 microg/10(7)cells at day 7). NaBu treatment leading to lower cell growth and higher RVGP expression allowed an even higher RVGP synthesis by S2MtRVGPHy(+) (8.4 microg/10(7)cells at day 7). SF900II medium leading to a higher S2MtRVGPHy(+)cell growth allowed a higher final RVGP synthesis in this cell culture. RVGP synthesis may be optimized by the expression/selection vectors design, cell subpopulations selection, chromatin exposure and culture medium employed.

Kinetic studies of recombinant rabies virus glycoprotein (RVGP) cDNA transcription and mRNA translation in Drosophila melanogaster S2 cell populations

“…They showed also better expression levels (Fig. 2) and in general confirmed our previous observations of RVGP productivity (Lemos et al 2009). For this cell population, at the exponential cell growth phase, we observed higher initial levels of RVGP producer cells and P RVGP , accompanied by characteristic levels of RVGP mRNA.…”

“…Drosophila melanogaster S2 cells (DES Ò , InvitrogenLife Technologies, Carlsbad, CA, USA) were transfected with the RVGP cDNA under the control of a constitutive (actin-Ac) or an inducible (metallothionein-Mt) promoter (Yokomizo et al 2007;Lemos et al 2009). Originated S2 cell populations were named S2AcRVGP-2k and S2MtRVGP-Hy, respectively.…”

“…Several complex glycoproteins were already expressed in the S2 cell system, using these promoters (Mallender et al 2001;Zhang et al 2007;Scotter et al 2006;Brillet et al 2006;Kim et al 2005;Johansson et al 2007;Jennings et al 2006;Li et al 2005;Lim et al 2004;Lee et al 2007). Aiming the expression of high levels of RVGP under the control of these promoters in the S2 cell system, for vaccination as well as structure/function evaluation, many studies were already carried out on cell growth and heterologous recombinant protein expression kinetics (Yokomizo et al 2007;Galesi et al 2008;Swiech et al 2008a;Batista et al 2009;Ventini et al 2010;Lemos et al 2009), as well as on metabolism and synthesis of secondary products (Swiech et al 2008b, c) and culture medium formulation and supplementation (Galesi et al 2007;Batista et al 2008Batista et al , 2011Mendonça et al 2008Mendonça et al , 2009. For constitutive RVGP expression using the actin promoter, instead of a gradual and sustained increase of RVGP, we have observed a sharp RVGP increase, at the beginning of the stationary cell growth phase, which could not be associated with the culture system or the cell culture media, pH, oxygen concentration or substrate change (Galesi et al 2008;Ventini et al 2010;Batista et al 2011).…”

Recombinant rabies virus glycoprotein synthesis in bioreactor by transfected Drosophila melanogaster S2 cells carrying a constitutive or an inducible promoter

“…In previous publications we have described the establishment of S2 cell lines expressing the RVGP and metabolic characteristics of these cells in culture (Lemos et al, 2009;Yokomizo et al, 2007;Bovo et al, 2008;Galesi et al, 2008). The Drosophila Expression System (DES) was developed in such a way allowing us a constitutive or an inducible gene expression, based respectively on the use of actin or metallothionein promoters (Invitrogen, Carlsbad, CA).…”

Recombinant respiratory syncytial virus F protein expression is hindered by inefficient nuclear export and mRNA processing

“…These approaches include expression in bacteria [37][38][39], expression in insect cells through the use of baculovirus expression vectors [40][41][42][43], and expression in mammalian cells either through the use of virus vectors [44][45][46][47] or standard mammalian expression vectors [12][13][14][15][16][48][49][50].…”