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User Guide

E. coli based Cell-free expression 

Cell-free expression is in particular suitable for the production of difficult proteins such as membrane proteins, toxins, complexes or peptides, and for an economical process for NMR samples isotopic labelling (¹⁵N; ¹³C).

Expression is performed in cell-free extracts of E. coli and reactions can be supplemented with a variety of compounds such as ligands, cofactors, inhibitors, chemicals, detergents or lipids. Reaction volumes are small in the range of few ml and incubation times usually do not exceed 16 hrs. With optimized production protocols, several mg of protein per ml of reaction can be obtained.

Over the last years, the Cell-Free platform has been visited by many European scientists who have obtained proteins or RNA to solve their problematic expression and continue they structural studies, by NMR, X-ray and EM.

Access mode

The platform is accessible to academic users.

Completed Request form need to be sent to platform engineer. The most adapted strategy and schedule for samples production (protein/RNA) will be choose after preliminary expression screening by the platform engineer.

Users will be trained and will have access to the cell-free platform in order to produce themselves their samples under platform engineer guidance.

Preparatory work required

Protein:

We need the cDNA of your target protein. Send us an expression vector containing target cDNA under T7 promoter control exclusively (pIVEX vector is requested for optimal yields).

* Only security level 1 samples are accepted

RNA:

Nature of DNA matrix (DNA oligonucleotides or plasmid containing T7 promoter) conditions will be discussed during the first contact in accordance with size of target RNA.

* Only security level 1 samples are accepted

Contact Lionel Imbert for more information.

cDNA/plasmids will be not stored at the end of the feasibility study.

Expected deliverables to user

Sample quality control is performed by SDS-PAGE and/or Western Blot (anti-His-Tag). Results will be send by email and include:

Small-scale expression screening (proteins and RNAs):

• Picture of gels
• Summary and analysis of preliminary screening tests

Training course:

• Training agreement
• Picture of gels
• Expressed protein (purification required)
• Expressed and purified RNA sample.

Large scale expression:

• Expressed protein (purification required)
• Expressed and purified RNA sample.

For more information and to see some of our publications, please see our ISBG webpage

For E. coli protein production for NMR, check our INSTRUCT or ISBG platform and for characterization please see ISBG platform catalog.