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The mammalian expression for in-cell NMR platform at CERM/CIRMMP is devoted to the production of samples of mammalian cells overexpressing the protein of interest for its characterization by in-cell NMR. It relies on transient transfection in HEK293T adherent cells.
Access to this Service has to be requested together with the Solution NMR Service at CERM/CIRMMP.
The facility equipment includes: CO2 incubator for adherent cells, laminar flow hood, centrifuges, autoclave. It also includes a Nikon Eclipse Ts2R-FL optical microscope and a Guava easyCyteTM Flow Cytometer for additional cell sample characterization.
This facility is specifically dedicated to the production of mammalian cell samples overexpressing a protein of interest for characterisation by in-cell NMR. It relies on transient transfection in HEK293T adherent cells. The gene of interest is cloned in a vector optimized for high constitutive cytoplasmic expression. Small scale transfections are first performed to determine the expression level and to assess the feasibility of in-cell NMR. Cell samples for NMR are then typically produced in T75 flasks. Co-expression of two or more proteins is possible. In addition to uniform 15N labelling of the protein, a wide choice of amino acid-type selective labelling strategies are possible, such as: 13C,15N labelling, methyl-13C labelling, both alone and in combination with uniform 15N. Furthermore, fluorinated amino acids can be incorporated in the expressed protein. Successful incorporation has been shown for 3-fluoro-L-tyrosine, 4-fluoro-L-phenylalanine, 5- and 6-fluoro-L-tryptophan.
Access to this Service has to be requested together with the Solution NMR Service at CERM/CIRMMP.
CERM/CIRMMP offers a platform dedicated to test and produce samples from mammalian cells overexpressing a protein of interest for its characterisation by in-cell NMR. Access to the platform allows users to perform small-scale tests to evaluate transfection and expression efficiency of their construct(s), and to produce full-scale cell samples with different labelling strategies for in-cell NMR analysis. Access to this Service is designed to be requested together with access to the Solution NMR Service at CERM/CIRMMP for sample analysis by in-cell NMR.
Proposals must therefore include the requests for both Mammalian Expression for in-cell NMR and Solution NMR Services.
The DNA vector(s) will be provided by the facility to the user, which will clone the construct(s) of interest at his/her home institution, and will check them by gene sequencing.
Protein expression tests are required to assess the feasibility of in-cell NMR experiments. Transfection and expression tests will be performed at CERM/CIRMMP. The positive outcome (i.e. high expression levels in the soluble fraction of at least one construct) is necessary to provide access to the Solution NMR platform for in-cell NMR experiments.
NMR-suitable protein levels are usually detectable by Coomassie-stained SDS-PAGE, however, detection by Western blot can also be performed, if the construct contains a FLAG-tag.
Once protein expression at sufficient levels is established, the user will come to the platform for full-scale sample preparation and acquisition of in-cell NMR experiments at the Solution NMR platform.
Given the highly experimental nature of the technique, variations to the access scheme outlined above are possible, which will be discussed on a case-by case basis. The platform staff will make all efforts before and during the access to support the work of the user and ensure a successful outcome. However, successful protein expression or in-cell NMR data acquisition cannot be guaranteed.
