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.
- URL: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15903252
Keywords: Bioreactors ; Cell Culture Techniques/methods ; Cell Line ; Cells ; Cultured ; Culture Media ; Serum-Free ; DNA/genetics ; *Gene Expression Regulation ; *Gene Transfer Techniques ; Genetic Vectors/genetics ; Humans ; Plasmids/genetics ; Proteins/genetics/isolation & purification/*metabolism ; Suspensions ; Transfection
Laboratory of Cellular Biotechnology, Institute for Biological Engineering and Biotechhnology, Ecole Polytechnique Federale de Lausanne, CH-1015 Lausanne, Switzerland. firstname.lastname@example.org
Research Support, Non-U.S. Gov't
Record created on 2007-06-05, modified on 2016-08-08