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Bacterial Structural Genomics Based on Escherichia coli Genome

M. Cygler
S. Raymond
A. Matte
J. Sivaraman
Y. Li
A. R. G. Barbosa
V. Sauvé
F. Oouelle
R. Larocque
J. Jia
G. Michel
J. D. Schrag
MC

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M. Cygler

Résumé du colloque

We have initiated a structural genomics project based on selected protein families, with the representatives for structural studies obtained from the genome of E. coli. An essential task is the development of effective methods to express, purify and crystallize large numbers of proteins efficiently. We are currently applying robotics methods allowing cloning in an automated manner. Over 100 genes have been cloned to date, and more than 75 overexpressed as soluble proteins in at least one fusion system. Target genes have been cloned as N-terminal fusions with GST and (His)8 affinity tags, which are proteolytically removed after affinity purification. Using this approach, 45 proteins have so far been purified and characterized using a combination of dynamic light scattering, electrophoresis, mass spectrometry and limited proteolysis. Proteins are screened for initial crystallization conditions using a sparse-matrix approach. Specialized database with web-based interface has been developed to follow all relevant experimental data. To date, crystals for 28 proteins have been obtained, with 16 giving diffraction quality crystals. Structures of 12 of these proteins have been determined by SeMet MAD phasing. Examples of solved structures will be given.

Contexte

Section :
Structure et fonction des protéines : aujourd'hui une protéine, demain un protéome
news icon Thème du colloque :
Structure et fonction des protéines : aujourd'hui une protéine, demain un protéome
manager icon Responsables :
Stéphane Gagné
host icon Hôte : Université Laval

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Thème du colloque :

Structure et fonction des protéines : aujourd'hui une protéine, demain un protéome
Bacterial Structural Genomics Based on Escherichia coli Genome
M. Cygler
Bacterial Structural Genomics Based on Escherichia coli Genome
M. Cygler
Bacterial Structural Genomics Based on Escherichia coli Genome
M. Cygler