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.
Meissner
P
P
Laboratory of Cellular Biotechnology, Department of Chemistry, Swiss Federal Institute of Technology, Lausanne, CH-1015 Switzerland.
Pick
H
H
Kulangara
A
A
Chatellard
P
P
Friedrich
K
K
Wurm
F M
FM
D019149:Bioreactors; D015153:Blotting, Western; D002460:Cell Line; D003470:Culture Media; D004268:DNA-Binding Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D017930:Genes, Reporter; D005819:Genetic Markers; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics; D008164:Luminescent Proteins; D018613:Microscopy, Confocal; D010957:Plasmids; D011985:Receptors, Serotonin / Q000235:genetics; D044406:Receptors, Serotonin, 5-HT3; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D013997:Time Factors; D014162:Transfection / Q000379:methods*
D003470:Culture Media; D004268:DNA-Binding Proteins; D019309:Epstein-Barr Virus Nuclear Antigens; D005819:Genetic Markers; D007074:Immunoglobulin G; D008164:Luminescent Proteins; D011985:Receptors, Serotonin; D044406:Receptors, Serotonin, 5-HT3; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins