Structural Biology and Genomics Platform Didier Busso - April 26, 2007 Platform’s Technical Coordinator [email protected]

Structural Biology and Genomics Platform

Didier Busso - April 26, 2007

Platform’s Technical [email protected]://lbgs.u-strasbg.fr/sbgp/

Structural Biology and Genomics DepartmentIGBMC1, rue Laurent Fries67404 Illkirch cedex

Structural Biology and Genomics

Didier Busso

Platform presentation



General introduction



The Bio-crystallography

The protein of interest has to be isolated from a living organism (human, plant or animal).

Usually, the protein is overproduce artificially: the DNA sequence encoding the protein is inserted into an expression vector which is transferred into an expression host that will produce the protein.

Thus, the soluble protein is purified by chromatography and concentrated.

The purified protein is transformed in crystal (which may contain up to 1 billion of billion of the protein).

Crystals of TTNPB-SRC-1-hRARb LBD. Kammerer et al., Acta Cryst. (2004) D60: 2048-2050.




At the Synchrotron, protein crystal is submitted to X-rays generating diffraction data sets.

It is from this 3-D image that researchers will understand how the protein works. These researches have a primordial role in the development of new drugs.

Diffraction data sets are analyzed by computers to reconstitute a three dimensional picture of the protein.

Structure of RXR-LBD bound to DHA and co-activator peptide.Egea et al., Mol. Endocrinol. (2002) 16: 987-997.



Target selection and domains definition

Cloning of Open Reading Frames

Expression screening (culture conditions, fusion(s), additives, …)

Biomass production

Protein purification and characterization

Protein crystallization

Structure solving

=> Tidious for numerous ORFs




A Platform to ?

Speed-up the discovery process in the new post-genomic context

Tackle more difficult projects related to human health

Miniaturize and parallelize experiments as much as possible to increase yield



The Platform’s missions

To be a center of technology and expertise

To be a site of exchange and support

To be an innovative center for the scientific community



Cancéropole

Génopole

Contracts

Programs linking european Structural Genomics centers of excellence.

Struct

ural B

iolo

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d Gen

omic

s Pla

tform

Its position

Inventoried as operational RIO Platform since 2003

Academic users

Industrial users



Its structure within IGBMC facilities

Struct

ural B

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d Gen

omic

s Pla

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Proteomic Platform

Bioinformatic Platform

Imaging Platform

Tight interaction with other Platforms and services

Transcriptomic PlatformMouse Clinical Institute

IGBMC common services



Cartesian RobotGreiner CrystalQuick plate

Leica macroscope wit x-y automated stage

Examples

Crystallization and Imaging

ValidationElectron Microscopy

Its structure

Cloning

Gateway®: a powerful strategy

Tecan Genesis Robot GScope: cloning tools

Validation

Databases

Access tables / SQL server

from cloning to crystals

and Quality Controls

Bar –coding + forms

Analytical ultracentrifugationDLS

NMR

Gel filtration

FPLC systems

Purification and Characterization

Validation

Mass spectrometry

Infors multi-fermentor 100 liters fermentorCulture flask

(E. coli and Insect cells)

Optimization and Production

Validation

SDS-PAGEDeep-well culture plates Microplate sonicator

Small-scale Expression

Structure solving

Bioinformatics



General organization

No Feasability

Yes

Struct

ural B

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gy an

d Gen

omic

s Pla

tform

Know-howProtocolsMaintenanceDevelopmentsDatabaseSupport/service

Platform advisingScientific Commitee

Bench work

Project

Technical support




Project deposition http://lbgs.u-strasbg.fr/sbgp/




Users



The Platform is a bridge between fundamental research and industry.

Representative typical contracts with industry (R&D):

- ASTRAZENECA 17/12/2002 - 17/12/200X (renewable)Structure-function relations for the human nuclear receptor NURR1. - BAYER 21/12/2002 - 17/12/200XCrystallization and X-ray structure analysis of the ligand binding domain (LBD) of the ecdysone receptor (EcR). - SCHERING 2004 – 200X (renewable) - CHUGAI 2004 – 2005 - AVANTIS 2005 – 2006

Some services for industry:

- ALIX 2004 – 200X (renewable)

- SUMITOMO 01 03 2004 – 28 02 2005.Training in protein crystallography of nuclear receptors.

- MOLECULAR ENGINE LABORATORIES 2004-2005Purification, crystallization and structure solving of the TCTP protein.




SBGP Website (restricted access)

Vector information



Protocols and user guides




Vector information




Bibliography




Conclusions

Integrated platforms from bioinformatic to structure solving

Automated cloning and mini-expression screening

Production facility for large scale culture

Purification and characterization systems

Automated crystallization and screen preparation

Integrated data base from bioinformatic to structural determination

Structure solving and modeling facilities



Statistics

Data from the Structural Biology and Genomics Department



Technical Coordinator: Didier Busso

Edouard TroeschLoubna Salim

Production Module

Matthieu Stierlé

Crystallization Module

Pierre Poussin-Courmontagne

Structural Biology and Genomics Department

IGBMC’s common services

Dino Moras Jean-Claude Thierry

Cloning-expression Module

Documents

Structural Biology and Genomics Platform Didier Busso - April 26, 2007 Platform’s Technical Coordinator [email protected]