Baldi, L.Muller, N.Picasso, S.Jacquet, R.Girard, P.Thanh, H. P.Derow, E.Wurm, F. M.2007-06-052007-06-052007-06-05200510.1021/bp049830xhttps://infoscience.epfl.ch/handle/20.500.14299/7668WOS:000226934800020619115903252Recent 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.BioreactorsCell Culture Techniques/methodsCell LineCellsCulturedCulture MediaSerum-FreeDNA/genetics*Gene Expression Regulation*Gene Transfer TechniquesGenetic Vectors/geneticsHumansPlasmids/geneticsProteins/genetics/isolation & purification/*metabolismSuspensionsTransfectiontext::journal::journal article::research article