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Tricks and improvements in Structural Genomics

Explore microbial genomes such as Mimivirus and new antibiotic targets in structural genomics projects with experimental protocols for protein expression, purification, and biochemical characterization.

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Tricks and improvements in Structural Genomics

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  1. Tricks and improvements in Structural Genomics Chantal Abergel Structural and Genomic Information Laboratory UMR 2589-CNRS, IBSM, 31 chemin J. Aiguier, 13009 Marseille, FRANCE http://www.igs.cnrs-mrs.fr

  2. Exploration of microbial genomes Microbial Genomics Mimivirus - T. whipplei - M. timonensis - A. baumannii R. felis - R. africae - R. belli - R. massiliae, R. conorii - Experiments Bioinformatics Fundamental: surprises Directed: biomedical Structural Genomics Functional Genomics

  3. Structural genomics projects: TargetDB • Escherichia coli • Orfan genes • Conserved genes of unknown function • search for new antibiotic targets • PROFUN : search for new antifungal targets • Saccharomyces cerevisiae: • Essential genes conserved in fungi • absent in vertebrate genomes • Candida albicans: • Conserved in fungal pathogens • absent in vertebrate genomes

  4. MasterDB

  5. ~ 300 Targets

  6. Incomplete Factorial Design: Experimental matrix (SAmBa) Automated protocol, 12 conditions (out of 96 possible) 4 strains 3 temperatures 3 growth media

  7. Dot Blot for recombinant protein detection • Total extracts and soluble extracts (supernatant after centrifugation) deposited on nitrocellulose membranes • Use of anti (his)5 antibody coupled HRP (Qiagen) • ECL detection Rosetta 17°C 2YT Up to 12 proteins tested a day Soluble expression condition Dot blot statistical analysis 90%

  8. Scale up (1L culture) Affinity purification Microdialysis Non ionic ionic Non ionic ionic pI pH • Overnight dialysis • ODtotal • ODsoluble

  9. Absorbance λ nm Best buffer: ODtotal/ODsoluble ~1 Desalting Concentration

  10. Purification optimization • Crystallization Biochemical & Biophysical Characterization • Stability of produced proteins ? • Folding ? • Protein solution monodispersity • Electrophoresis (native, IEF, SDS) • N-terminal Sequencing /Mass Spectrometry • Circular Dichroïsm • Dynamic light scattering • Stability over time

  11. Combinatorial approach • Functional & Structural predictions • Bioinformatics • sequence homologies (BMCD) • binding motif, hypothetical ligand, co-factors… • Electrophoresis (native, IEF…) Set of variables to be tested

  12. SAmBA: For crystallization prospection www.igs.cnrs.fr/samba

  13. Tecan diluting distributing robot 8 needles Greiner crystallization plates 5x96 wells

  14. Promising crystals ...

  15. In house testing ...

  16. Poor diffraction

  17. Soaking/desiccation

  18. YbgL Step 1

  19. YbgL Step 2

  20. YbgL Final

  21. YggV Step 1

  22. YggV Final

  23. CA0996 Step 1

  24. CA0996 Final

  25. What do they have in common ? (1)

  26. What do they have in common ? (2)

  27. Experimental Team Sabine Chenivesse Sophie Cozzani Céline Deregnaucourt Sandra Jeudy Marjorie Varagnol Bioinformatics Team Hiroyuki Ogata Stéphane Audic Olivier Poirot Pr. Jean-Michel Claverie

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