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Large-Scale Transfection of Mammalian Cells

  • Lucia BaldiEmail author
  • David L. Hacker
  • Carine Meerschman
  • Florian M. Wurm
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 801)

Abstract

The large-scale transfection of mammalian cells allows moderate (milligram to gram) amounts of recombinant proteins (r-proteins) to be obtained for fundamental or clinical research. In this article, we describe a one-liter transfection using polyethyleneimine (PEI) for DNA delivery into human embryonic kidney (HEK-293) cells cultivated in serum-free suspension to produce a recombinant human monoclonal antibody that yields up to about 1 g/L in a 10-day process. The method is based on a DNA delivery step performed at high cell density (20×106 cells/mL) by direct addition of DNA and PEI to the culture. Subsequently, the cells are diluted 20-fold for the 10-day production phase in the presence of valproic acid (VPA), a histone deacetylase inhibitor. The methods for plasmid purification, antibody quantification by enzyme-linked immunosorbent assay (ELISA), and affinity purification with protein A are also described.

Key words

HEK-293 Transfection ELISA Protein A Suspension culture Recombinant protein Polyethyleneimine 

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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Lucia Baldi
    • 1
    Email author
  • David L. Hacker
    • 2
  • Carine Meerschman
    • 3
  • Florian M. Wurm
    • 1
  1. 1.Laboratory of Cellular BiotechnologyÉcole Polytechnique Fédérale de LausanneLausanneSwitzerland
  2. 2.Laboratory of Cellular Biotechnology and Protein Expression Core FacilityÉcole Polytechnique Fédérale de LausanneLausanneSwitzerland
  3. 3.Protein Expression Core FacilityÉcole Polytechnique Fédérale de LausanneLausanneSwitzerland

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