Version :
Integrated Structural Biology Grenoble

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

Isotopic labelling

Presentation

The Isotopic Labeling Platform of the IBS is devoted to the large-scale production of proteins uniformly or specifically enriched in stable isotopes (15N, 13C and 2H) for biomolecular NMR spectroscopy studies. The platform is offering access to innovative labeling schemes, developed locally on the platform by the IBS NMR group. The platform benefits from extensive scientific contacts with the IBS high field NMR Spectroscopy and Cell-Free platforms. The Isotopic Labeling Platform obtained an ISO 9001/v2008 quality managment certification in July 2011, a key label and a warranty for all users of well-managed activities.

Activities proposed by the platform:

Users have access to the facility for large scale expression of labeled proteins in E.coli, under the supervision of a qualified platform engineer. Dedicated benches, optimised protocols and adequate isotopically labelled chemicals are available for the following services:

• Training in large-scale production of isotopically labelled proteins (practical experience in protein biochemistry is required).

• Feasibility studies of protein over-expression in perdeuterated media.

• Large-scale over-expression of [15N, 13C and 2H]-proteins, and [13C1H3]-methyl specifically labeled proteins in highly (>98%) perdeuterated protein background.

• Development of innovative labeling scheme (collaboration only)

Key words

Proteins, Advanced isotopic labeling, Perdeuteration, Methyl specific labeling, NMR.

Staff

Head of the Platform : Jérôme Boisbouvier

Staff in charge : Isabel Ayala

Localisation and equipment

In the IBS 1rst floor, we have 70 m2 of wet lab dedicated to the over-expression of proteins in E.coli. One bench is full-time devoted to the platforme users.

Access mode

All required adaptations, scale-up and productions of labeled materials are performed on the platform by the user under supervision of a qualified engineer. (Load the acceptance conditions sheet for acces)

How to make a request ?

A request form should be sent to the platform by email (Isabel Ayala). Following receipt of the form, we will study the feasibility of your request within 1month. If the request is accepted, we will contact you in order to propose available periods for the access to the platform.

Click here(English version), or here (French version) to download the request form, and explain briefly your project, the staff in charge of the platform will contact you upon reception of the form.

Cost and duration

Academics : The cost of isotopic materials and consumables used for the production of proteins will be refunded by the users to the IBS platform.

Approved Instruct Users : Access costs (Consumables, travel and accommodation) can be covered by Instruct (for more information click here).

Private companies : quotation under request.

Duration : Days (protocol transfer) to few weeks (large scale production of labeled proteins).

Additional information

Samples:

• Only security level 1 samples are accepted.

• The plasmid used for the expression of the protein must be kanamycin-resistant.

• The level of expression in rich-media of the target protein has to be above 10mg/L.

Contact Isabel Ayala for more information.

Follow up / acknowledgments

• Providing access mode: Acknowledgement in publications and oral presentations

Acknowledgements in publication:«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)».

• Collaboration: Co-authors in publications and oral presentations.

Link with other platforms and services:

-  IBS high field NMR Platform (link)
-  Cell-free Platform for in vitro expression of labelled RNA and proteins (link)
-  RoBioMol Platform for preparation of libraries of mutants or constructs (link)
-  Our innovative precursors are now commercially available. For more information, please consult the dedicated web page : www.nmr-bio.com

Examples of recent publications :

Innovative isotopic labelling approaches for NMR:

- Specific Labeling and Assignment Strategies of Valine Methyl Groups for NMR Studies of High Molecular Weight Proteins.
Mas G, Crublet E, Hamelin O, Gans P, Boisbouvier J. Biomol NMR 57: 251-262 (2013).

- Selective isotopic unlabeling of proteins using metabolic precursors: application to NMR assignment of intrinsically disordered proteins.
Rasia RM, Brutscher B, Plevin M. ChemBioChem 13(5):732-9 (2012).

- An Optimized Isotopic Labelling Strategy of Isoleucine-γ2 Methyl Groups for solution NMR Study of High Molecular Weight Proteins.
Ayala I, Hamelin O., Amero C, Pessey O., Plevin M.J., Gans P., Boisbouvier J. Chem. Comm. 48,1434-1436 (2012).

- A Systematic Mutagenesis-driven Strategy for Site-Resolved NMR Studies of Supramolecular Assemblies. Amero CD, Durá MA, Noirclerc-Savoye M, Perollier A, Gallet B, Plevin MJ, Vernet T, Franzetti B, Boisbouvier J. J. Biomol NMR. 50, 229-236 (2011).

- A simple biosynthetic method for stereospecific resonance assignment of prochiral methyl groups in proteins Plevin MJ, Hamelin O, Boisbouvier J., Gans P J. Biomol NMR 49, 61-67 (2011).

- Stereospecific isotopic labeling of methyl groups for NMR spectroscopic studies of high-molecular-weight proteins.
Gans P, Hamelin O, Sounier R, Ayala I, Durá MA, Amero CD, Noirclerc-Savoye M, Franzetti B, Plevin MJ, Boisbouvier J.
Angew Chem Int Ed Engl. 49:1958-62 (2010)

- Parallel screening and optimization of protein constructs for structural studies.
Rasia RM, Noirclerc-Savoye M, Bologna NG, Gallet B, Plevin MJ, Blanchard L, Palatnik JF, Brutscher B, Vernet T, Boisbouvier J.
Protein Sci. 8:434-9 (2010).

- An efficient protocol for the complete incorporation of methyl-protonated alanine in perdeuterated protein.
Ayala I, Sounier R, Usé N, Gans P, Boisbouvier J. J Biomol NMR. 43:111-9 (2009).

For more information on the technologies developped on the platform:

- A cost-effective protocol for the parallel production of libraries of 13CH3-specfically labeled mutants for NMR studies of high molecular weight proteins Crublet E, Kerfah R, Mas G., Noirclerc-Savoye M., Lantez V., Vernet T., Boisbouvier J. Structural Genomics, Methods in Molecular Biology, Springer, vol 1091, chapter 17, ISBN 978-1-62703-690-0 (2014).

- Isotope-Labelling of Methyl Groups for NMR Studies of Large Proteins. Plevin MJ, Boisbouvier J. Book chapter in Recent Developments in Biomolecular NMR, Royal Society of Chemistry, Biomolecular Science Series. DOI:10.1039/9781849735391 (2012).

- Le marquage isotopique spécifique : Un outil pour repousser les frontières de la RMN biomoléculaire. Gans P, Boisbouvier J. Actualité Chimique 347, 5-11 (2010).