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/v2015 and NFX-50-900 quality managment certification, 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 : Lionel Imbert.

Platform engineer : Lionel Imbert, Céline Juillan-Binard

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

• Protein Analysis On Line (PAOL)

• 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 :

Data produced by the platforms remain the property of the users. The methods used by the platforms remain the property of the platform, but users can request help in writing the “Materials & Methods” sections of publications.
Nevertheless every communication, oral and written, using totally or partially data from ISBG platforms, must acknowledge as follows

"This work used the platforms of the Grenoble Instruct-ERIC center (ISBG ; UAR 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB), supported by FRISBI (ANR-10-INBS-0005-02) and GRAL, financed within the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003)."

By including platform people in the author list if their implication in the project goes further than a basic service.

Examples of publications

• Enabling site-specific NMR investigations of therapeutic Fab using a cell-free based isotopic labeling approach : application to anti-LAMP1 Fab.
  Giraud A, Imbert L, Favier A, Henot F, Duffieux F, Samson C, Frances O, Crublet E, Boisbouvier J.
  J Biomol NMR. 2024
  Pubmed
• Structural Perspective into the Interaction of an Oncogenesis-Relevant pre-miRNA G-Quadruplex Ligand Carrier with the Protein Nucleolin.
  Santos T, Silva M, Imbert L, Campello MPC, Paulo A, Amrane S, Salgado GF, Cruz C, Cabrita EJ.
  Chemistry. 2023 Pubmed
• The structure of the high-affinity nickel-binding site in the Ni,Zn-HypA UreE2 complex.
  Zambelli B, Basak P, Hu H, Piccioli M, Musiani F, Broll V, Imbert L, Boisbouvier J, Maroney MJ, Ciurli S.
  Metallomics. 2023
  Pubmed
• Pre-miRNA-149 G-quadruplex as a molecular agent to capture nucleolin1 Santos T, Miranda A, Imbert L, Jardim A, Caneira CRF, Chu V, Conde JP,
  Campello MPC, Paulo A, Salgado G, Cabrita EJ, Cruz C. .
  Eur J Pharm Sci. 2022 Feb
  Pubmed
• Targeting a G-quadruplex from let-7e pre-miRNA with small molecules and nucleolin.
  Santos T, Miranda A, Imbert L, Monchaud D, Salgado GF, Cabrita EJ, Cruz C.
  J Pharm Biomed Anal. 2022
  Pubmed
• Self-association of MreC as a regulatory signal in bacterial cell wall elongation.
  Martins A, Contreras-Martel C, Janet-Maitre M, Miyachiro M, Estrozi L, Maragno Trindade D, Malospirito C, Rodrigues-Costa F, Imbert L, Job V, Schoehn G, Attrée I and Dessen A.
  Nature Communication 2021
  Pubmed

• In Vitro Production of Perdeuterated Proteins in H2O for Biomolecular NMR Studies.
  Imbert L, Lenoir-Capello R, Crublet E, Vallet A, Awad R, Ayala I, Juillan-Binard C, Mayerhofer H, Kerfah R, Gans P, Miclet E, Boisbouvier J.
  Methods Mol Biol. 2021
  Pubmed

• Exolysin (ExlA) from Pseudomonas aeruginosa Punctures Holes into Target Membranes Using a Molten Globule Domain.
  Bertrand Q, Job V, Maillard AP, Imbert L, Teulon JM, Favier A, Pellequer JL, Huber P, Attrée I, Dessen A.
  J Mol Biol. 2020
  Pubmed

• The mitochondrial carrier pathway transports non-canonical substrates with an odd number of transmembrane segments.
  Rampelt H et al.
  BMC Biol. 2020
  Pubmed

• Structure and assembly of pilotin-dependent and -independent secretins of the type II secretion system.
  Howard SP et al.
  PLoS Pathog. (2019)
  Pubmed

• Structural Basis of Membrane Protein Chaperoning through the Mitochondrial Intermembrane Space.
  Weinhäupl K et al.
  Cell (2018)
  Pubmed

• Transcription factor dimerization activates the p300 acetyltransferase. Ortega E et al.
  Nature (2018)
  Pubmed

• Pore-forming activity of the Pseudomonas aeruginosa type III secretion system translocon alters the host epigenome.
  Dortet et al.
  Nature Microbiology (2018)
  Pubmed

• microRNA-122 amplifies hepatitis C virus translation by shaping the structure of the internal ribosomal entry site.
  P Schult, H Roth, R L. Adams, C Mas, L Imbert, C Orlik, A Ruggieri, A M. Pyle & V Lohmann
  Nature Communications (2018)
  Pubmed

• How Detergent Impacts Membrane Proteins : Atomic-Level Views of Mitochondrial Carriers in Dodecylphosphocholine.
  Kurauskas V, Hessel A, Ma P, Lunetti P, Weinhäupl K, Imbert L, Brutscher B, King MS, Sounier R, Dolce V, Kunji ERS, Capobianco L, Chipot C, Dehez F, Bersch B, Schanda P.
  J Phys Chem Lett (2018)
  Pubmed

• A General Strategy to Access Structural Information at Atomic Resolution in Polyglutamine Homorepeats.
  Urbanek A et al.
  Angew Chem Int Ed (2018)
  Pubmed

• A putative RNA binding protein from Plasmodium vivax apicoplast. S. M. Garcıa-Maurino et al.
  FEBS (2017)
  Pubmed

• Cell-free production, purification and characterization of human mitochondrial ADP/ATP carriers. Woznicka-Misaila A, Juillan-Binard C, Baud D, Pebay-Peyroula E, Ravaud S.
  Protein Expr Purif. (2017)
  Pubmed

• Functional reconstitution of cell-free synthesized purified Kv channels.
  Renauld S et al.
  Biochim Biophys Acta. (2017)
  Pubmed

• Structural Basis of Mitochondrial Dysfunction in Response to Cytochrome c Phosphorylation at Tyrosine 48. Moreno-Beltrán B et al.
  PNAS USA (2017)
  Pubmed

• Autocatalytic association of proteins by covalent bond formation : a Bio Molecular Welding toolbox derived from a bacterial adhesin. Bonnet J et al.
  Scientific Reports. (2017)
  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)
  Pubmed

• Recombinant expression of the precursor of the hemorrhagic metalloproteinase HF3 and its non-catalytic domains using a cell-free synthesis system
  Menezes MC, Imbert L, Kitano ES et al.
  Amino Acids (2016)
  Pubmed

• Induced folding in RNA recognition by Arabidopsis thaliana DCL1.
  Suarez IP et al.
  Nucleic Acids Res. (2015)
  Pubmed

• Structural similarity of secretins from type II and type III secretion systems.
  Tosi T et al.
  Structure. (2014)
  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)
  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)
  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)
  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 et al.
  Biochim Biophys Acta. (2010)
  Pubmed