Transient gene expression: recombinant protein production with suspension-adapted HEK293-EBNA cells

Biotechnol Bioeng. 2001 Oct 20;75(2):197-203. doi: 10.1002/bit.1179.


Transient gene expression (TGE) in mammalian cells at the reactor scale is becoming increasingly important for the rapid production of recombinant proteins. We improved a process for transient calcium phosphate-based transfection of HEK293-EBNA cells in a 1-3 L bioreactor volume. Cells were adapted to suspension culture using a commercially available medium (BioWhittaker, Walkersville, MD). Process parameters were optimized using a plasmid reporter vector encoding the enhanced green fluorescent protein (EGFP/CLONTECH, Palo Alto, CA, USA). Using GFP as a marker-protein, we observed by microscopic examination transfection efficiencies between 70-100%. Three different recombinant proteins were synthesized within a timeframe of 7 days from time of transfection to harvest. The first, a human recombinant IgG(1)-type antibody, was secreted into the supernatant of the cell culture and achieved a final concentration of >20 mg/L. An E. coli-derived DNA-binding protein remained intracellular, as expected, but accumulated to such a concentration that the lysate of cells, taken up into the entire culture volume, gave a concentration of 18 mg/L. The third protein, a transmembrane receptor, was expressed at 3-6 x 10(6) molecules/cell.

MeSH terms

  • Bioreactors
  • Blotting, Western
  • Cell Line
  • Culture Media
  • DNA-Binding Proteins
  • Electrophoresis, Polyacrylamide Gel
  • Epstein-Barr Virus Nuclear Antigens / genetics
  • Escherichia coli / genetics
  • Gene Expression
  • Genes, Reporter
  • Genetic Markers
  • Genetic Vectors
  • Green Fluorescent Proteins
  • Humans
  • Immunoglobulin G / genetics
  • Luminescent Proteins
  • Microscopy, Confocal
  • Plasmids
  • Receptors, Serotonin / genetics
  • Receptors, Serotonin, 5-HT3
  • Recombinant Proteins / biosynthesis*
  • Recombinant Proteins / genetics
  • Time Factors
  • Transfection / methods*


  • Culture Media
  • DNA-Binding Proteins
  • Epstein-Barr Virus Nuclear Antigens
  • Genetic Markers
  • Immunoglobulin G
  • Luminescent Proteins
  • Receptors, Serotonin
  • Receptors, Serotonin, 5-HT3
  • Recombinant Proteins
  • Green Fluorescent Proteins