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FRENCH INFRASTRUCTURE FOR INTEGRATED STRUCTURAL BIOLOGY

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INSTRUCT CENTER - FRANCE 1 - IGBMC

MAMMALIAN-EXPRESSION

IGBMC-CERBM Strasbourg provides expression in mammalian cells.

Over expression of proteins in mammalian cells (hamster BHK21 cells) is achieved using an attenuated vaccinia virus vector (Modified Vaccinia Virus Ankara-MVA) that is user safe and may be handled under BSL1 conditions. Genes of interest are cloned downstream of a bacteriophage T7 promoter in a first step to check for feasibility. Protein expression is then examined after plasmid transfection into BHK21 cells and co-infection with a specialized viral vector encoding the bacteriophage T7 RNA polymerase. The IGBMC facility perform this feasibility step with any gene of interest cloned downstream of a T7 promoter and acquire results within a few days. 

To obtain high level protein expression, genes that perform well in the feasibility test are cloned into specialized plasmids that enable their site directed transfer into the viral genome downstream of the bacteriophage T7 promoter. Recombinants viruses encoding genes of interest are isolated in a few weeks under non permissive conditions for recombinant protein expression. High level expression is then accomplished in mammalian cells in the presence of inducer (IPTG). Up to 10 mg of purified protein may be consistently produced from 5 litre suspension cell cultures. A general outline of methods used may be found in the following references: Anal Biochem. 2012; 426(2): 106-8; Anal Biochem. 2010 Sep 1; 404(1): 103-5; Protein Expr Purif. 2007; 56(2): 269-78. More recent improvements improving the speed of operations and protein productivity are unpublished.

Support includes small scale expression tests with plasmids provided by users (needs the T7 promoter). Easy gene assembly using the “Biobrick” system based on compatible restriction sites (EcoRI, XbaI, SpeI, PstI) as described in (Ho-Shing et al., 2012) with a variety of N-terminal tags in a Gateway  entry vector for subsequent construction of recombinants virus is available. Participation of users is encouraged for training in the isolation of viral expression vectors and protein production runs at the IGBMC. Users will be provided with specialized materials and the most recent methods available.

https://www.structuralbiology.eu/update/platform/expression-in-mammalian-cells-at-igbmc-cerbm-strasbourg