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. 2003 May;42(1):47–55. doi: 10.1023/A:1026125016602

SEAP expression in transiently transfected mammalian cells grown in serum-free suspension culture

Ernst-Jürgen Schlaeger 1, Eric A Kitas 1, Arnulf Dorn 1
PMCID: PMC3449504  PMID: 19002927

Abstract

A transient transfection process was established using a novel 'in-house' developed transfection reagent, Ro-1539. It allows rapid production of large quantities of various recombinant proteins. Here we describe the transient expression of the secreted human placental alkaline phosphatase (SEAP) by HEK293EBNA and CHO cells in serum-free suspension culture. Unexpectedly, high expression levels of SEAP (150 μg/ml) were found 3–4 days post-transfection when placental alkaline phosphatase (AP) was used as the reference enzyme. To confirm these data, an SDS–PAGE analysis was performed and the visible SEAP protein band (MW of 65 kDa) was compared with co-migrated purified placental AP protein as reference. The scanning analysis of the gel showed that SEAP, a truncated form of AP, has a higher specific activity than the purified placental AP. A correction factor was introduced permitting a direct comparison of placental AP activity with the expression levels of SEAP. Scale-up of the transfection system from spinner flask to bioreactor was simple and straightforward, resulting in similar yields of SEAP. Finally, the effectiveness of Ro-1539 was compared to that of other transfection reagents.

Keywords: Chemiluminescence assay, Large scale culture, Ro-1539 transfection, SEAP, Serum-free, Transient gene expression

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References

  1. Baker E.A., Vaughn M.W., Haviland D.L. Choices in transfection methodologies. Focus. 2000;22:31–33. [Google Scholar]
  2. Berger J., Hauber J., Hauber R., Gelger R., Cullen B.R. Secreted placental alkaline phosphatase: a powerful new quantitative indicator of gene expression in eukaryotic cells. Gene. 1988;66:1–10. doi: 10.1016/0378-1119(88)90219-3. [DOI] [PubMed] [Google Scholar]
  3. Boulikas T. Status of gene therapy in 1997: molecular mechanisms, disease targets, and clinical applications. Gene Ther. Mol. Biol. 1998;1:1–172. [Google Scholar]
  4. Bronstein I., Fortin J., Stanley P.E., Stewart G.S.A.B., Kricka L.J. Chemiluminescent and bioluminescent reporter gene assays. Anal. Biochem. 1994;219:169–181. doi: 10.1006/abio.1994.1254. [DOI] [PubMed] [Google Scholar]
  5. Chadd H.E., Chamow S.T. Therapeutic antibody expression technology. Curr. Opin. Biotechnol. 2001;12:188–194. doi: 10.1016/S0958-1669(00)00198-1. [DOI] [PubMed] [Google Scholar]
  6. Cullen B.R., Malim M.H. Secreted placental alkaline phosphatase as a eukaryotic reporter gene. Methods Enzymol. 1992;216:362–368. doi: 10.1016/0076-6879(92)16033-G. [DOI] [PubMed] [Google Scholar]
  7. Davis C.G., Gallo M.L., Corvalan J.R.F. Transgenic mice as a source of fully human antibodies for the treatment of cancer. Cancer Metastasis Rev. 1999;18:421–425. doi: 10.1023/A:1006321231510. [DOI] [PubMed] [Google Scholar]
  8. Doebeli H., Gentz R., Jucker W., Garotta G., Hartmann W.D., Hochuli E. Role of the carboxy-terminal sequence immune interferon (IFN-γ) on the biological activity of human. J. Biotechnol. 1988;7:199–211. doi: 10.1016/0168-1656(88)90052-1. [DOI] [Google Scholar]
  9. Durocher Y., Perret S., Thibaudeau E., Gaumond M.H., Kamen A., Stocco R., et al. A reporter gene assay for high-throughput screening of G-protein-coupled receptors stably or transiently expressed in HEK293EBNA cells grown in suspension culture. Anal. Biochem. 2000;284:316–326. doi: 10.1006/abio.2000.4698. [DOI] [PubMed] [Google Scholar]
  10. Kain S.R., Ganguly S. Methods in Molecular Biology Vol. Totowa, NJ: Humana Press; 1996. Use of secreted alkaline phosphatase as a reporter of gene expression in mammalian cells. [DOI] [PubMed] [Google Scholar]
  11. Ledley F.D. Nonviral gene therapy: the promise of genes as pharmaceutical products. Hum. Gene Ther. 1995;6:1129–1144. doi: 10.1089/hum.1995.6.9-1129. [DOI] [PubMed] [Google Scholar]
  12. Legendre J.Y., Trzeciak A., Bohrmann B., Deuschle U., Kitas E.A., Supersaxo A. Dioleoylmelittin as a novel serum insensitive reagent for efficient transfection of mammalian cells. Bioconjug. Chem. 1996;8:57–63. doi: 10.1021/bc960076d. [DOI] [PubMed] [Google Scholar]
  13. Mutel V., Ellis G.J., Adam G., Klingelschmidt A., Messer A., Schlaeger E.-J., et al. Characterization of [3H]-quisqualate binding to recombinant rat metabotropic glutamate 1a and 5a receptors as well as rat and human brain sections. J. Neurochem. 2000;75:2590–2601. doi: 10.1046/j.1471-4159.2000.0752590.x. [DOI] [PubMed] [Google Scholar]
  14. Padmaja J., Satyanarayana V., Merrifield R.B. Synthetic mellitin, its enantio, retro, and retroenantio isomers, and selected chimeric analogs: their antibacterial, hemolytic, and lipid bilayer action. J. Am. Chem. Soc. 1996;118:8989–8997. doi: 10.1021/ja9542911. [DOI] [Google Scholar]
  15. Schlaeger E.-J. The protein hydrolysate, Primatone RL, is a cost-effective multiple growth promotor of mammalian cell culture in serum-containing and in serum-free media and displays anti-apoptosis properties. J. Immunol. Methods. 1996;194:191–199. doi: 10.1016/0022-1759(96)00080-4. [DOI] [PubMed] [Google Scholar]
  16. Schlaeger E.-J., Christensen K. Transient gene expression in mammalian cells grown in serum-free suspension culture. Cytotechnology. 1999;30:71–83. doi: 10.1023/A:1008000327766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Schlaeger E.-J., Christensen K. and Kitas E.A. 1999. (tiScale-up of transient gene expression in mammalian cells grown in serum free suspension culture. 4th Gene Delivery and Cellular Protein Expression Conference. October 17–21. Lake Tahoe, California.
  18. Schlaeger E.-J. 2000. (tiOptimal transfection complex formation depends on the medium compositions. First European Technology Workshop. October 1–3. Karthause Ittingen, Switzerland.
  19. Schumpp B., Schlaeger E.-J. Optimization of culture conditions for high cell density proliferation of HL60 human promyelocytic leukemia cells. J. Cell Sci. 1990;97:639–647. doi: 10.1242/jcs.97.4.639. [DOI] [PubMed] [Google Scholar]
  20. Yoon K., Thiede M.A., Rodan G.A. Alkaline phosphatase as a reporter enzyme. Gene. 1988;66:11–17. doi: 10.1016/0378-1119(88)90220-X. [DOI] [PubMed] [Google Scholar]

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