, Volume 54, Issue 1, pp 15–24 | Cite as

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

  • Mariza G. Santos
  • Soraia A. C. Jorge
  • Karl Brillet
  • Carlos A. PereiraEmail author
Original Research


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 ∼90% of S2MtEGFP fluorescent cells at day 5 after copper sulfate (CuSO4) 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 CuSO4 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 × 105 counts/s after 72 h of induction. Induction with 700 μM of CuSO4 performed at the exponential phase of the S2MtEGFP culture (106 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 CuSO4 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.


Schneider Drosophila cells EGFP Cell transfection Gene expression Sodium butyrate 



Enhanced green fluorescent protein gene


Metallothionein promoter


Schneider 2 Drosophila melanogaster cells


S2 cells transfected with Mt inducible plasmid containing the EGFP gene


Sodium butyrate


Copper sulfate


Drosophila expression system





This work was supported in part by grants from the FAPESP (02/09482-3, 05/50565-8), CNPq and Fundação Butantan. We thank Dr. Bergmann Morais (UnB, Brasília, Brazil) for pGie1EGFP plasmid supplying and Dr. Jorge MC Ferreira Jr and Dr. Orlando Ribeiro, for flow cytometry analysis. C.A. Pereira is recipient of CNPq research fellowship. S.A.C. Jorge and M.A.G. Santos had scholarships from FAPESP (01/08914-4, 02/04003-0, 03/08978-8).


  1. Angelichio ML, Beck JA, Johansen HE, Ivey-Hole M (1991) Comparison of several promoters and polyadenylation signals for use in heterologous gene expression in cultured Drosophila cells. Nucleic Acids Res 19:5037–5043PubMedCrossRefGoogle Scholar
  2. Calos MP, Lebkowski JS, Botchan MR (1983) High mutation frequency in DNA transfected into mammalian cells. Proc Natl Acad Sci USA 80:3015–3019PubMedCrossRefADSGoogle Scholar
  3. Chang KH, Yang JM, Chun HOK, Chung IS (2005) Enhance activity of recombinant β-secretase from Drosophila melanogaster S2 cells transformed with cDNAs encoding human β1,4-galactosyltransferase and Gal β1,4-GlcNac α2,6-sialyltransferase. J Biotechnol 116:359–367PubMedCrossRefGoogle Scholar
  4. Chen T, Sun H, Lu J, Zhao Y, Tao D, Li X, Huang B (2002) Histone acetylation is involved in hsp70 gene transcription regulation in Drosophila melanogaster. Archi. Biochem Biophys 408:171–176CrossRefGoogle Scholar
  5. Culp JS, Johansen H, Hellmig B, Beck J, Matthews TJ, Delers A, Rosemberg M (1991) Regulated expression allows high level production and secretion of HIV-1 gp 120 envelope glycoprotein in Drosophila Schneider cells. Bio/technology 9:173–178PubMedCrossRefGoogle Scholar
  6. Delm L, Wolf H, Wagner R (1999) High level expression of hepatitis B virus surface antigen in stably transfected in Drosophila Schneider-2 cells. J Virol Methods 79:191–203CrossRefGoogle Scholar
  7. Dorner AJ, Wasley LC, Kaufman RJ (1989) Increased synthesis of secreted proteins induces expression of glucose-regulated proteins in butyrate-treated Chinese hamster ovary cells. J Biol Chem 264:20602–20607PubMedGoogle Scholar
  8. Han K (1996) An efficient DDAB-mediated transfection of Drosophila S2 cells. Nucl Acids Res 24:4362–4363PubMedCrossRefGoogle Scholar
  9. Hazelrigg T, Mansfield JH (2006) Green fluorescent protein applications in Drosophila. Methods Biochem Anal 47:227–257PubMedGoogle Scholar
  10. Hill RM, Brennan SO, Birch NP (2001) Expression, purification and functional characterization of the serine protease inhibitor neuroserpin expressed in Drosophila S2 cells. Protein Expr Purif 22:406–413PubMedCrossRefGoogle Scholar
  11. Huang EP, Marquis CP, Gray PP (2004) Process development for recombinant chinese hamster ovary (CHO) cell line utilizing a metal induced and amplified metallotionein expression system. Biotechnol Bioeng 88:438–450CrossRefGoogle Scholar
  12. Lee JM, Park JH, Park JO, Chang KH, Chung IS (2000) Expression of recombinant erythropoietin in stably transformed Drosophila melanogaster S2 cells. In Vitro Cell Dev Biol Anim 36:348–350PubMedCrossRefGoogle Scholar
  13. Li B, Tising S, Kosaka AH, Nguyen B, Osen EG, Bach C, Chan H, Barnett J (1996) Expression of Human Dopamine β Hydroxylase in Drosophila Schneider 2 Cells. Biochem J 313:57–64PubMedGoogle Scholar
  14. Li RW, Li CJ (2006) Butyrate induces profound changes in gene expression related to multiple signal pathways in bovine kidney epithelial cells. BMC genomics 7:234PubMedCrossRefADSGoogle Scholar
  15. Nilsen SL Castellino FJ (1999) Expression of human plasminogen in Drosophila Schneider S2 cells. Protein Expr Purif 16:136–143CrossRefGoogle Scholar
  16. Park HJ, Kim HY, Han KH, Chung SI (1999) Optimization of transfection conditions for expression of green fluorescent protein in Drosophila melanogaster S2 cells. Enzyme Microb Technol 25:558–563CrossRefGoogle Scholar
  17. Perret BG, Wagner R, Lecat S, Brillet K, Rabut G, Bucher B, Pattus F (2003) Expression of EGFP-amino-tagged human mu opioid receptor in Drosophila Schneider 2 cells: a potential expression system for large-scale production of G-protein coupled receptors. Protein Expr Purif 31:123–132PubMedCrossRefGoogle Scholar
  18. Phillips GN Jr (2006) The three-dimensional structure of green fluorescent protein and its implications for function and design. Methods Biochem Anal 47:67–82PubMedGoogle Scholar
  19. Ping W, Ge J, Li S, Zhou H, Wang K, Feng Y, Lou Z (2006) Baculovirus-mediated gene expression in chicken primary cells. Avian Dis 50:59–63PubMedCrossRefGoogle Scholar
  20. Razzaque A, Mizusawa H, Seidman MM (1983) Rearrangement and mutagenesis of a shuttle vector plasmid after passage in mammalian cells. Proc Natl Acad Sci USA 80:3010–3014PubMedCrossRefADSGoogle Scholar
  21. Sambrook J. and Russel D.W (2001) Molecular cloning: a laboratory manual. New York Cold Spring Harbor Laboratory PressGoogle Scholar
  22. Shin HS, Cha HJ (2002) Facile and statistical optimization of transfection conditions for secretion of foreign proteins from insect Drosophila S2 cells using green fluorescent protein reporter. Biotechnol Prog 18:1187–1194PubMedCrossRefGoogle Scholar
  23. Shin HS, Lim HJ, Cha HJ (2003) Quantitative monitoring for secreted production of human interleukin-2 in stable insect Drosophila S2 cells using a green fluorescent protein fusion partner. Biotechnol Prog 19:152–157PubMedCrossRefGoogle Scholar
  24. Song J, Liang C, Chen X (2006) Transduction of avian cells with recombinant baculovirus. J Virol Methods 135:157–162PubMedCrossRefGoogle Scholar
  25. Van der Straten A, Johansen H, Rosenberg M, Sweet R (1989) Introduction and constitutive expression of gene products in cultured Drosophila cells using hygromicin B selection. Curr Methods Mol Cell Biol 1:1–8Google Scholar
  26. Ward WW (2006) Biochemical and physical properties of green fluorescent protein. Methods Biochem Anal 47:39–65PubMedCrossRefGoogle Scholar
  27. Ward TH, Lippincott-Schwartz J (2006) The use of green fluorescent protein in mammalian cells. Methods Biochem Anal 47:305–337PubMedGoogle Scholar
  28. Zhao YM, Chen X, Sun H, Yuan ZG, Ren GL, Li XX, Lu J, Huang BQ (2006) Effects of histone deacetylase inhibitors on transcriptional regulation of the hsp70 gene in Drosophila. Cell Res 16:566–576PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2007

Authors and Affiliations

  • Mariza G. Santos
    • 1
  • Soraia A. C. Jorge
    • 1
  • Karl Brillet
    • 2
  • Carlos A. Pereira
    • 1
    Email author
  1. 1.Laboratório de Imunologia ViralInstituto ButantanSao PauloBrazil
  2. 2.Département Récepteurs et Protéines Membranaires, Ecole Supérieure de Biotechnologie de StrasbourgInstitut Gilbert-Laustriat, UMR7175/Université-Strasbourg IIllkirchFrance

Personalised recommendations