title: Transient expression of rabies virus glycoprotein (RVGP) in Drosophila melanogaster Schneider 2 (S2) cells.
abstract: 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 50-90ng/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 10μg rather than 5μg of DNA. Inhibition of RVGP expression was observed at higher concentrations of DNA, with DNA concentrations above 15μg 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 182.2ng/10(7) cells (PEI), 201ng/10(7) cells (calcium phosphate), and 215ng/10(7) cells (cationic lipid) were obtained for S2 cell suspension cultures. The highest volumetric RVGP concentration (309ng/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 (253.4ng/mL) and calcium phosphate (237.2ng/mL). There was little effect of transfection on the kinetics of cell growth, with growth rates of 1.12 and 1.19d(-1) for transfected and control cells, respectively. In spinner flasks, the expression of RVGP was 150 and 138ng/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.
authors.affiliation: Laboratório de Imunologia Viral, Instituto Butantan, São Paulo, SP, Brazil; Departamento de Engenharia Química, Universidade Federal de São Carlos, São Carlos, SP, Brazil.
authors.forename: Sandra Fernanda
authors.initials: SF
authors.lastname: Suárez-Patiño
authors.affiliation: Laboratório de Imunologia Viral, Instituto Butantan, São Paulo, SP, Brazil.
authors.forename: Renato Astray
authors.initials: RA
authors.lastname: Mancini
authors.affiliation: Laboratório de Imunologia Viral, Instituto Butantan, São Paulo, SP, Brazil.
authors.forename: Carlos Augusto
authors.initials: CA
authors.lastname: Pereira
authors.affiliation: Departamento de Engenharia Química, Universidade Federal de São Carlos, São Carlos, SP, Brazil.
authors.forename: Claudio Alberto Torres
authors.initials: CA
authors.lastname: Suazo
authors.affiliation: Laboratório de Parasitologia, Instituto Butantan, São Paulo, SP, Brazil.
authors.forename: Ronaldo Zucatelli
authors.initials: RZ
authors.lastname: Mendonça
authors.affiliation: Laboratório de Imunologia Viral, Instituto Butantan, São Paulo, SP, Brazil. Electronic address: soraia.jorge@butantan.gov.br.
authors.forename: Soraia Attie Calil
authors.initials: SA
authors.lastname: Jorge
mesh_terms: D000818:Animals; D002130:Calcium Phosphates; D002460:Cell Line; D004247:DNA; D004331:Drosophila melanogaster / Q000235:genetics*; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011094:Polyethyleneimine; D011820:Rabies virus; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D002130:Calcium Phosphates; D006023:Glycoproteins; D014764:Viral Proteins; D011094:Polyethyleneimine; D004247:DNA; C020243:calcium phosphate
keywords: Drosophila melanogaster S2 cells; RVGP; Rabies virus glycoprotein; Transfection agents