Service / Technology Instance

About Protein Production in Mammalian Cells for in-cell NMR, Florence, Italy

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The mammalian expression for in-cell NMR platform at CERM/CIRMMP is devoted to the to the production of mammalian cell samples overexpressing the protein of interest for its characterization 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 performed to determine the expression level and to assess the feasibility of in-cell NMR. Cell samples for NMR are produced in T75 flasks. Different protein labelling strategies are possible, e.g. U-15N labelling; amino acid type-selective 13C,15N labelling. Co-expression of two or more proteins is possible. In-cell NMR experiments are required to be performed at the Solution NMR Facility at CERM.


Instruments Available:

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 performed to determine the expression level and to assess the feasibility of in-cell NMR. Cell samples for NMR are produced in T75 flasks. Different protein labelling strategies are possible, e.g. U-15N labelling; amino acid type-selective 13C,15N  labelling. Co-expression of two or more proteins is possible. In-cell NMR experiments are performed at the Solution NMR Facility at CERM.

User Guide

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 platform is designed to be requested together with access to the Solution NMR platform at CERM/CIRMMP for sample analysis by in-cell NMR.

Proposals must include the requests for both Mammalian Expression for in-cell NMR and Solution NMR platforms.

Gene cloning

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

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.

Sample production for in-cell NMR experiments

Once protein expression at sufficient levels is established, the user will come at 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, variation 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.

Instruct Centre

Instruct Centre IT

CERM/CIRMMP

Via Sacconi 6

50019 Sesto

Florence

Italy

www.cerm.unifi.it

Protein Production in Mammalian Cells for in-cell NMR, Florence, Italy

Contacts:

Enrico Luchinat
Enrico Luchinat
Università degli Studi di Firenze
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Francesca Morelli
Francesca Morelli
CERM/CIRMMP
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