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The Cell Free expression platform of IBS is devoted to large scale production (milligrams quantities) of soluble proteins, membrane proteins and RNAs for structural studies (X-ray, NMR, EM,...). The platform benefits from scientific input of the Membrane Transporters and Membrane and Immunity teams for expression and solubilisation of membrane proteins and from the Biomolecular NMR group for isotopic labelling of proteins and RNA. The Cell Free platform has implemented a quality management process for ISO 9001: 2015 certification, a key label and a warranty for all users of well-managed activities.
SummaryThe Cell Free expression platform of IBS is devoted to large scale production (milligrams quantities) of soluble proteins, membrane proteins and RNAs for structural studies (X-ray, NMR...).The platform benefits from scientific input of the Membrane Transporters and Membrane and Immunity teams for expression and solubilisation of membrane proteins and from the NMR group for isotopic labelling of RNA and proteins.The Cell Free platform is setting up a quality process aimed at an ISO 9001 labelling. We propose the following services:• Small scale protein expression screening for optimisation of protein constructs. Optimisation of reaction conditions for large scale expression.• Small scale RNA expression screening. Optimisation of reaction conditions for large scale expression• Training courses in RNAse Free wet-lab are proposed to new Cell Free platform users.• Access to the facility for protein/RNA large scale Cell Free expression, under supervision of qualified platform engineer. Dedicated bench, optimised protocols, home made cell free extracts/enzymes and consumables are available to users.
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.
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):
Training course:
Large scale expression:
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.
