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Integrated Structural Biology Grenoble

Contact person(s) related to this article / BOISBOUVIER Jerome / IMBERT Lionel

Cell free

Summary

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...).
The platform benefits from scientific input of the Membrane Transporters group for expression and solubilisation of membrane proteins and from the NMR group for isotopic labelling of RNA and proteins.
The Cell Free platform confirmed ISO 9001/v2008 and NFX-50-900 quality managment certification in July 2015, a key label and a warranty for all users of well-managed activities.

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.

- 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.

Key words

Cell Free, CECF, in-vitro, Transcription, Traduction, Proteins, Membrane Proteins, RNA, Isotopic labelling, large scale production, NMR, X-Ray Crystallography.

Dedicated staff

Responsibility: Jérôme Boisbouvier.

Platform engineer: Lionel Imbert

Specific equipment

- 1 fully equipped lab space of 20m2 reserved for platform users (see schedule below) dedicated to RNAse free wet-lab.
- 1 qualified engineer, in charge of platform maintenance, protocols optimisation, implementation of new protocols, small scale screening, supervision, advice and help to external platform users.

Access mode

The platform is accessible to local PSB users as well as national (Frisbi) and international (Instruct) users.
Completed Request form need to be sent to platform engineer. Upon platform’s agreement for the project request, the platform engineer will define, in collaboration with external users, the most adapted strategy and schedule for protein/RNA cell free expression. The platform engineer will perform preliminary expression screening. Users will be trained and will have access to the cell free platform in order to produce themselves their samples under platform engineer guidance.

Agenda

Cost

Academics: Participation to the RNAse Free wet-lab consumables.
Industrials: Please contact us for cost information.

Location

The platform is located in the Institut de Biologie Structurale (IBS), Room 221.

How to make a request ?

Press here to send a request

Samples

Only non-pathogenic biological samples are accepted. Decree of the July 18, 1994 establishing the list of biological pathogens, amended by Decrees of April 17, 1997 and June 30, 1998 (Decrees in French). The list of biological pathogens is available here (in French).
Please recovered the "GMO agreement number" of your Institution to send us with your request.

Protein:

We need the cDNA of your target protein. Send us an expression vector (pIVEX vectors are requested for optimal yields), optimized for in-vitro expression and E. coli) containing target cDNA under T7 promoter control exclusively . The concentration of plasmids should be 1µg/µL in RNAse free water.

* 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

DNA will be stored at -20°C until small scale trials and not conserved after them.

Contact Lionel Imbert for more information.

Results

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

Cell free production of the mitochondrial carrier rUCP1: effect of additives on rUCP1 solubility

Solubility optimisation:cell free production of membrane protein with different detergents

First coming:
- User agreement (companionship)
- 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 protein purification or characterization please consult IBS platform catalog.

Link with other platforms and services:

• IBS high field NMR Platform

Isotopic Labelling Platform for proteins in-vivo over-expression in E. coli

RoBioMol Platform for screening of detergents for membrane protein solubilization and affinity purification; detergent exchange on solubilised membrane proteins (i.e. after cell free reaction) and gene cloning

RoBioMol Platform for mutagenesis of your constructs as described here for membrane proteins

• Protein Analysis On Line (PAOL)

• Membrane protein platform purification (MP3)

High Throughput Membran Protein Crystallization

• Our innovative precursors are now commercially available. For more information, please consult the dedicated web page : www.nmr-bio.com

Follow up / acknowledgements

Service mode: Acknowledgements in publications and oral presentations

Acknowledgements in publications:

Please find below the sentence that has to be written in all publications based on our UMS platforms

"This work used the platforms of the Grenoble Instruct centre (ISBG; UMS 3518 CNRS-CEA-UJF-EMBL) with support from FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). We thank Lionel Imbert and/or Jérôme Boisbouvier, for assistance and/or access to the Cell Free platform".

Examples of recent publications

- A putative RNA binding protein from Plasmodium vivax
apicoplast. S. M. Garcıa-Maurino et al. FEBS Open Bio 2017 Dec. 31

- Woznicka-Misaila A, Juillan-Binard C, Baud D, Pebay-Peyroula E, Ravaud S. Cell-free production, purification and characterization of human mitochondrial ADP/ATP carriers. Protein Expr Purif. 2017 Dec 4. pii: S1046-5928(17)30570-3
[https://www.ncbi.nlm.nih.gov/pubmed/?term=29217202]

- Functional reconstitution of cell-free synthesized purified Kv channels.
Renauld S et al. Biochim Biophys Acta. Sep 2017
[https://www.ncbi.nlm.nih.gov/pubmed/28888365]

- Structural Basis of Mitochondrial Dysfunction in Response to Cytochrome c Phosphorylation at Tyrosine 48.
Moreno-Beltrán, Blas et al. PNAS USA 114.15 2017 E3041–E3050. PMC. May 2017.
Pubmed

- Autocatalytic association of proteins by covalent bond formation: a Bio Molecular Welding toolbox derived from a bacterial adhesin.
Bonnet J, Cartannaz J, Tourcier G, et al. Scientific Reports. 2017 7:43564. doi:10.1038/srep43564.
Pubmed

- RNA binding and chaperone activity of the E. coli cold-shock protein CspA.
Rennella R, Sara T, Juen M, Wunderlich C, Imbert L, Solyom S, Favier A, Ayala I, Weinhäulp K, Schanda P, Konrat R, Kreutz and Brutscher B.
Nucleic Acids Res. 2017 gkx044. doi: 10.1093/nar/gkx044
Pubmed

- Recombinant expression of the precursor of the hemorrhagic metalloproteinase HF3 and its non-catalytic domains using a cell-free synthesis system
Menezes, M.C., Imbert, L., Kitano, E.S. et al. Amino Acids 2016 48: 2205. doi:10.1007/s00726-016-2255-7
Pubmed

- Induced folding in RNA recognition by Arabidopsis thaliana DCL1.
Suarez IP, Burdisso P, Benoit MP, Boisbouvier J, Rasia RM
Nucleic Acids Res. 2015 Jul 27;43(13):6607-19.
Pubmed

- Structural similarity of secretins from type II and type III secretion systems.
Tosi T, Estrozi LF, Job V, Guilvout I, Pugsley AP, Schoehn G, Dessen A.
Structure. 2014 Sep 2;22(9):1348-55.
Pubmed

- Small angle neutron scattering for the study of solubilised membrane proteins.
Breyton C, Gabel F, Lethier M, Flayhan A, Durand G, Jault JM, Juillan-Binard C, Imbert L, Moulin M, Ravaud S, Härtlein M, Ebel C.
Eur Phys J E Soft Matter. 2013 Jul;36(7):71.
Pubmed

- The RNA-binding region of human TRBP interacts with microRNA precursors through two independent domains.
Benoit MP, Imbert L, Palencia A, Pérard J, Ebel C, Boisbouvier J, Plevin MJ.
Nucleic Acids Res. 2013 Apr;41(7):4241-52
Pubmed

- Production of UCP1 a membrane protein from the inner mitochondrial membrane using the cell free expression system in the presence of a fluorinated surfactant.
Blesneac I, Ravaud S, Juillan-Binard C, Barret LA, Zoonens M, Polidori A, Miroux B, Pucci B, Pebay-Peyroula E.
Biochim Biophys Acta. 2011 Dec 27; 1818(3):798-805.
Pubmed

- Structure and RNA Interactions of the Plant MicroRNA Processing-Associated Protein HYL1.
Rasia RM, Mateos J, Bologna NG, Burdisso P, Imbert L, Palatnik JF, Boisbouvier J.
Biochemistry. 49:8237-9 (2010).
Pubmed

- Crystallization of the membrane protein hVDAC1 produced in cell-free system.
Deniaud A, Liguori L, Blesneac I, Lenormand JL, Pebay-Peyroula E.
Biochim Biophys Acta. 2010 Aug;1798(8):1540-6.
Pubmed