Improving heterologous protein expression in transfected Drosophila S2 cells as assessed by EGFP expression

Abstract

Drosophila melanogaster S2 cells were co-transfected with plasmid vectors containing the enhanced green fluorescent protein gene (EGFP), under the control of metallothionein promoter (pMt), and the hygromycin selection gene, in view of establishing parameters for optimized gene expression. A protocol of transfection was worked out, leading after hygromycin selection, to approximately 90% of S2MtEGFP fluorescent cells at day 5 after copper sulfate (CuSO(4)) induction. As analyzed by confocal microscopy, S2MtEGFP cell cultures were shown to be quite heterogeneous regarding the intensity and cell localization of fluorescence among the EGFP expressing cells. Spectrofluorimetry kinetic studies of CuSO(4) induced S2MtEGFP cells showed the EGFP expression at 510 nm as soon as 5 h after induction, the fluorescence increasing progressively from this time to attain values of 4.6 x 10(5) counts/s after 72 h of induction. Induction with 700 muM of CuSO(4) performed at the exponential phase of the S2MtEGFP culture (10(6) cells/mL) led to a better performance in terms of cell growth, percent of fluorescent cells and culture intensity of fluorescence. Sodium butyrate (NaBu) treatment of CuSO(4) induced S2MtEGFP cell cultures, although leading to a loss of cell culture viability, increased the percent of EGFP expressing cells and sharply enhanced the cell culture fluorescence intensity. The present study established parameters for improving heterologous protein expression in stably transfected Drosophila S2 cells, as assessed by the EGFP expression.

Fig. 1

Schematic construction of pMtEGFP vector

Fig. 2

Kinetics of cell growth (A), % of fluorescent cells (B) and fluorescence intensity (C) of S2 and S2MtEGFP cells induced with 500 or 700 μM of CuSO₄ at day 3 of culture. •, S2MtEGFP cells + 700 μM of CuSO₄; ■, S2MtEGFP cells + 500 μM of CuSO₄; ♦, S2 cells; ○, S2MtEGFP cells. Arrows indicate the time of induction at day 3. Results are the average of at least three independent experiments. ^*: statistically different

Fig. 3

S2MtEGFP cell culture as evaluated by confocal microscopy (A) and flow cytometry (B). S2MtEGFP cultures were induced with 700 μM of CuSO₄. At day 5 of induction they were observed in a confocal microscopy and at different times after induction they were evaluated by flow cytometry. For confocal microscopy: (I), optical microscopy; (II), fluorescence microscopy; (III) merged image. For flow cytometry they were observed at days 0, 2, 5, 8, 9 and 12 of induction and the % of fluorescent cells is indicated as M1. Data are representative of independent experiments

Fig. 4

Kinetics of EGFP intensity expression by S2MtEGFP cells as evaluated by spectrofluorimetry. S2MtEGFP cell culture was induced with 700 μM of CuSO₄ and at different times the EGFP fluorescence emission was detected at 510 nm (A) and the kinetics curve was recorded (B). Data are representative of independent experiments

Fig. 5

Kinetics of cell growth (A), % of fluorescent cells (B) and fluorescence intensity (C) of S2MtEGFP cells induced with 700 μM of CuSO₄ and treated with 5 mM of NaBu. At day 3, cell cultures were induced with CuSO₄ and/or treated with NaBu. Cell samples were periodically taken for evaluation of viable cell concentration by hematocytometer or fluorescence by flow cytometry. ○, S2MtEGFP cells; •, S2MtEGFP cells + 700 μM of CuSO₄; Δ, S2MtEGFP cells + 5 mM NaBu; ▴, S2MtEGFP cells + 700 μM of CuSO₄ + 5 mM NaBu. Arrows indicate the time of induction at day 3. Results are the average of at least three independent experiments. *: statistically different