Transient expression of rabies virus glycoprotein (RVGP) in Drosophila melanogaster Schneider 2 (S2) cells

Autor(es)

Suarez-Patino, Sandra Fernanda ; Mancini, Renato Astray ; Pereira, Carlos Augusto ; Torres Suazo, Claudio Alberto ; Mendonca, Ronaldo Zucatelli ; Calil Jorge, Soraia Attie

• Tipo de documento: Artigo Científico
• Fonte: Journal of Biotechnology; v. 192, p. 8-pg., 2014-12-20.

Resumo

The transient transfection process has been developed to allow rapid production of recombinant proteins. In this paper, we describe the transient expression of recombinant rabies virus glycoprotein (RVGP) in Drosophila melanogaster Schneider 2 (S2) cells. Different cell transfection reagents were evaluated, together with the effects of different cell cultivation procedures on RVGP expression. Yields of RVGP in the range 50-90 ng/10(7) cells were obtained in multi-well plate transfection experiments, where it was observed that RVGP expression was linked to the DNA concentration. RVGP expression was 1.3 times higher using 10 mu g rather than 5 mu g of DNA. Inhibition of RVGP expression was observed at higher concentrations of DNA, with DNA concentrations above 15 mu g decreasing RVGP expression 1.5-fold for cells transfected with polyethylenimine (PEI) and 1.6-fold for cells transfected with cationic lipid. The results of shake flask transfection indicated that S2 cells were more effectively transfected in suspension than under static conditions. RVGP yields of 182.2 ng/10(7) cells (PEI), 201 ng/10(7) cells (calcium phosphate), and 215 ng/10(7) cells (cationic lipid) were obtained for S2 cell suspension cultures. The highest volumetric RVGP concentration (309 ng/mL) was found for cells transfected with cationic lipid. This value was 1.21 and 1.16 times higher, respectively, than for cells transfected with PEI (253.4 ng/mL) and calcium phosphate (237.2 ng/mL). There was little effect of transfection on the kinetics of cell growth, with growth rates of 1.12 and 1.19 d(-1) for transfected and control cells, respectively. In spinner flasks, the expression of RVGP was 150 and 138 ng/10(7) cells for transfection using PEI and calcium phosphate, respectively. A comparison of the different transfection reagents (calcium phosphate, cationic lipid, and cationic polymer) showed no significant differences in RVGP expression when shake flasks were used. Overall, the data indicated that transient expression in D. melanogaster S2 cells is a practical way of synthesizing RVGP for use in structural and functional studies. (C) 2014 Elsevier B.V. All rights reserved. (AU)

• Processo FAPESP: 11/08331-0 - Estudo da dinâmica quantitativa de transcrição de gene heterólogo em células animais transfectadas para otimização de bioprocesso
• Beneficiário: Carlos Augusto Pereira
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 10/08742-8 - Avaliação da resposta imunológica de camundongos à glicoproteína do vírus rábico (RVGP) sintetizada in vivo pela imunização com Semliki Forest Vírus (SFV) recombinante
• Beneficiário: Renato Mancini Astray
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 02/09482-3 - Expressão de genes heterólogos em células de dípteros: biologia molecular e engenharia de processos
• Beneficiário: Carlos Augusto Pereira
• Modalidade de apoio: Auxílio à Pesquisa - Temático