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FRENCH INFRASTRUCTURE FOR INTEGRATED STRUCTURAL BIOLOGY

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AFMB

INSECT CELLS EXPRESSION

The Drosophila S2 cell expression system offers several attractive features such as high cell density growth, regulated expression, high copy number integration, and cell robustness with an easy manipulation. Transient transfection of S2 cells associated with by western blot detection permits a rapid evaluation of the protein expression level. Then polyclonal or monoclonal cell lines are established by cotransfection of the expression vector and a selection vector. The resistant cells are selected using an antibiotic. A first large-scale production is achieved using a polyclonal cell line and pseudoclonal cell lines are selected in the meantime. The cultures are scaled at about one liter of medium to produce reasonable amount of protein. We usually design the expression vectors for a secretion of the target protein into the medium. This latter is aseptically collected and the cells are re-suspended in fresh medium for a novel induction of the expression. The proteins are purified by at least two steps: (1) from the medium by affinity (depending on the tag, which can be His6 or strep) (2) a size exclusion gel filtration. In some cases, before the final purification step, the tag is removed. This strategy generally allows the production of several mg.

For more details please visit: http://www.afmb.univ-mrs.fr/recombinant-protein-production-in