Transient gene expression in suspension HEK-293 cells: application to large-scale protein production.
Recent advances in genomics, proteomics, and structural biology raised the general need for significant amounts of pure recombinant protein (r-protein). Because of the difficulty in obtaining in some cases proper protein folding in bacteria, several methods have been established to obtain large amounts of r-proteins by transgene expression in mammalian cells. We have developed three nonviral DNA transfer protocols for suspension-adapted HEK-293 and CHO cells: (1) a calcium phosphate based method (Ca-Pi), (2) a calcium-mediated method called Calfection, and (3) a polyethylenimine-based method (PEI). The first two methods have already been scaled up to 14 L and 100 L for HEK-293 cells in bioreactors. The third method, entirely serum-free, has been successfully applied to both suspension-adapted CHO and HEK-293 cells. We describe here the application of this technology to the transient expression in suspension cultivated HEK-293 EBNA cells of some out of more than 20 secreted r-proteins, including antibodies, dimeric proteins, and tagged proteins of various complexity. Most of the proteins were expressed from different plasmid vectors within 5-10 days after the availability of the DNA. Transfections were successfully performed from the small scale (1 mL in 12-well microtiter plates) to the 2 L scale. The results reported made it possible to establish an optimized cell culture and transfection protocol that minimizes batch-to-batch variations in protein expression. The work presented here proves the applicability and robustness of transient transfection technology for the expression of a variety of recombinant proteins.
Baldi
Lucia
L
Laboratory of Cellular Biotechnology, Institute for Biological Engineering and Biotechhnology, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland. lucia.baldi@epfl.ch
Muller
Natalie
N
Picasso
Sylviane
S
Jacquet
Raymond
R
Girard
Philippe
P
Thanh
Huy Phan
HP
Derow
Elisabeth
E
Wurm
Florian M
FM
D019149:Bioreactors; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D004247:DNA / Q000235:genetics; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D010957:Plasmids / Q000235:genetics; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013535:Suspensions; D014162:Transfection
D016895:Culture Media, Serum-Free; D011506:Proteins; D013535:Suspensions; D004247:DNA