FRISBI

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FRENCH INFRASTRUCTURE FOR INTEGRATED STRUCTURAL BIOLOGY

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SOUTH PARIS

MAMMALIAN EXPRESSION

Expression and purification of proteins produced in mammalian cells

We provide the cell line, plasmids, protocols to interested researchers in the framework of a research collaboration.

Two methods can be used:

- Transient transfection of adherent 293T cells. For some human proteins that are difficult to produce in bacteria, this method allowed us to purify mg amount of protein (for a dozen large Petri dishes with a mg of plasmid using calcium phosphate transfection)

- Establishment of a stable Flp-In 293 cell line (1 month). Stable cell lines expressing the tagged protein are then cultured in spinner bottles (6 L , 2 weeks). Stable transfections produce less protein than transient transfections but this approach is required to purify multiprotein complexes assembled around the tagged recombinant protein. This method allows to discover new multiprotein complexes by proteomics or to produce sufficient complexes for in vitro assays.