Transient gene expression: recombinant protein production with suspension-adapted HEK293-EBNA cells
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.
- URL: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11536142
Keywords: 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/genetics ; Time Factors ; Transfection/*methods ; Receptors
Laboratory of Cellular Biotechnology, Department of Chemistry, Swiss Federal Institute of Technology, Lausanne, CH-1015 Switzerland.
Record created on 2007-06-05, modified on 2016-08-08