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FRENCH INFRASTRUCTURE FOR INTEGRATED STRUCTURAL BIOLOGY

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GENE TAGGING

The protein production platform at the Instruct-France 1 centre also provide access to gene tagging with the CRISPR-Cas9 system for introduction of affinity tags to facilitate the purification of endogenous complexes or for tagging proteins with fluorescent reporters in view of imaging and functional proteomics applications, in the frame of a partnership with the TacGene platform from the Museum d’Histoire Naturelle (Paris, France).

The target protein is produced under the control of its natural promoter and the resulting complexes are authentic (no problems of stoichiometry, PMTs, missing components) but yield is limited by the natural abundance of the complex. The pipeline includes the following steps: (i) establishment of K562 knocked-in cell lines in which a subunit of the target assembly has been fused to the FLAG affinity tag, (ii) validation of selected clones.

The centre offers the possibility to produce selected clones in large scale suspension cultures and also affinity purification of the target complex. For that a second access for Mammalian expression Strasbourg is requested.