RIKEN Structural Genomics/Proteomics Initiative (RSGI)

1. RIKEN Structural Genomics/Proteomics Initiative(RSGI) Shigeyuki Yokoyama RIKEN Genomic Sciences Center and Harima Institute at SPring-8

2. Optional tRNA A.A. Plasmid DNA Protein RNA Polymerase A.A.-tRNA mRNA EF-Tu Ribosome Cell-Free Protein Synthesis System • Easy manipulation • Adding/removing components • No cloning necessary • PCR amplified template • High-throughput

3. Cell-Free Synthesis with Dialysis Dispo/Dialyzer 1ml (Spectra/Por) φ5 mm x 50 mm CE membrane MW cutoff 50kDa Internal solution (300 µl) S30 extract Template DNA T7 RNA polymerase Buffer, salts, etc. ATP, GTP, etc. Amino acids RNase inhibitor NaN3 Reaction mixture S30 extract Template DNA T7 RNA polymerase Buffer, salts, etc. ATP, GTP, etc. Amino acids External solution (3000 µl) Buffer, salts, etc. ATP, GTP, etc. Amino acids NaN3 Batch System Dialysis System

4. PCR PCR Protein synthesis on 96-well plate Primers cDNAs Selection and Quantification Centrifugation TCA Precipitation Construction of Templates Liquid Scintillation Counting Cell-Free Protein Synthesis Results Incubation Productivity Solubility Protein Products

5. Cell-Free Protein Synthesis Escherichia coli coupled transcription-translation system (Kigawa & Yokoyama) Wheat germ translation system (Yaeta Endo)

6. Wheat Germ Cell-Free Protein Synthesis System Prof. Yaeta Endo Ehime University & RIKEN GSC

7. 1st workshop Joint Center for Structural Genomics, Dr. Ian Wilson, Dr. Thomas Selby 2nd workshop TB Structural Genomics Consortium, Dr. Thomas Terwilliger, Dr. Hong Cai Northeast Structural Genomics Consortium, Dr. Gaetano Montelione, Drs. Li Chung Ma and Takeshi Yoshida 3rd workshop Berkeley Structural Genomics Center, Dr. Sung-Hou Kim, Dr. Didier Busso Proteomics at Clinical Genomics Centre, Drs. C. Arrowsmith A. Edwards and E. Pai, Dr. Dinesh Christendat Southeast Collaboratory for Structural Genomics,Dr. Bi-Cheng Wang, Dr. Peter Horanyi Oxford University group, Dr. Iain Campbell, Dr. David Staunton 4th workshop Laboratory of Dr. Joel Janin, Dr. Bruno Collinet Proteomics at Clinical Genomics Centre, Drs. C. Arrowsmith A. Edwards and E. Pai, Dr. Adelinda Yee Cambridge University group, Dr. Ernest Laue, Dr. Natalia Murzina New York Structural Genomics Research Consortium, Dr. Stephen Burley, Dr. Hua Chen (not participate) 5th workshop Pacific Northwest National Labortory Group, Dr. Eric J. Ackerman Southeast Collaboratory for Structural Genomics, Dr. Bi-Cheng Wang, Dr. LiQing Chen Cell-free protein synthesis workshop

8. PCR PCR Protein synthesis on 96-well plate Primers cDNAs Selection and Quantification Centrifugation TCA Precipitation Construction of Templates Liquid Scintillation Counting Cell-Free Protein Synthesis Results Incubation Productivity Solubility Protein Products

9. Template DNA Construction FW RV

10. Large-scale cell-free protein synthesis from PCR-amplified template (I)

11. Domain identification(Computational) • Sequence homology • Neural network

12. g-B crystallin Cyclodextran glucanotransferase

14. Protein Structure Determination • NMR • Stable-isotope labeling • Multidimensional multinuclear NMR • X-ray crystallography • SeMet incorporation • Multiwavelength anomalous dispersion (MAD) phasing

15. Stable-isotope Labeling Ras(Y32W)•GDP (Dialysis cell-free) 110 15N (ppm) 120 130 10 9 8 7 1H (ppm)

16. XAFS Relative Intensity (deg.) Selenomethionyl Ras•GDPprepared by the Cell-Free System P 65 2 2 • 0.1M Sodium cacodylate (6.5) • 0.2M Calcium acetate • 18% PEG8000 • SeMet Incorporation > 95% (MS)

17. Selenomethionyl-Ras•GDPprepared by the Cell-Free System MAD Phasing (SPring-8 BL44B2) (PDB:1IOZ)

18. Molecular Function Structure analysis • Identification of interacting partner(s) • Protein-protein • Protein-peptide • Protein-DNA • Protein-RNA • Protein-ligand Function analysis • Proper folding • Complex formation

19. Molecular Function • Molecular interaction(Domain-based strategy) • Protein-protein • Protein-ligand • Yeast two-hybrid system • Proteome (2D EP & MS) • Protein chip • Mutant analysis • NMR

20. Membrane Proteins Cellular and Extracellular Domains Integral Membrane Proteins (GPCR, etc.)

22. Nature Biotech., in press.

23. RIKEN Structural Genomics/Proteomics Initiative (RSGI) Target Genomes/Proteomes • Bacteria • Thermus thermophilus HB8: genome (Structurome Research Group, Harima Institute) • Higher eukaryotes • Mus muscus: full-length cDNA collection (Gene Exploration Group, GSC) • Homo sapiens: full-length cDNA collection (Kazusa DNA Research Institute) etc. • Alabidopsis thaliana: full-length cDNA collection (Plant Functional Genomics Res. Group, GSC)

24. Protein Overroduction – Expression Plasmids PCR, TA cloning ligation Constructed: 1106 plasmids

25. Progress Report Proteins (1) Synthesis of PCR primers 1870 (2) Correct amplification by PCR 1153 (3) Plasmids for overexpression 1106 (4) Overproduction in E. coli 583 (5) Purification 346 (6) Crystallization 132 (7) 3D structure 43

26. RIKEN Structural Genomics/Proteomics Initiative (RSGI) Target Genomes/Proteomes • Bacteria • Thermus thermophilus HB8: genome (Structurome Research Group, Harima Institute) • Higher eukaryotes • Mus muscus: full-length cDNA collection (FANTOM, 20,000; FANTOM II, 60,000) (Gene Exploration Group, GSC) • Homo sapiens: full-length cDNA collection (Kazusa DNA Research Institute) etc. • Alabidopsis thaliana: full-length cDNA collection (Plant Functional Genomics Res. Group, GSC) (15,000)

27. cDNA Collections Protein Expression Target Prioritization Structural Studies Functional Studies

28. Cell-free protein synthesis (Small-scale on 96-well plate) Cell-free synthesis (Midi-scale with 15N) Affinity purification Cloning & Sequencing NMR measurement (HSQC) Cell-free synthesis (Small-scale dialysis) Sample screening

29. PCR PCR Protein synthesis on 96-well plate cDNAs Primers Selection and Quantification Centrifugation TCA Precipitation Construction of Templates Liquid Scintillation Counting Cell-Free Protein Synthesis Results Incubation Productivity Solubility Protein Products

30. Robot systems • cDNA & Primer re-array • TECAN Genesis Workstation 150 • BECKMAN COULTER Biomek 2000 & ORCA • PCR setup • TECAN Genmate • Cell-free synthesis • BECKMAN COULTER Biomek 2000 & ORCA • BECKMAN COULTER Biomek FX • Plasmid preparation • BECKMAN COULTER Biomek 2000 & ORCA • BECKMAN COULTER Biomek FX • Affinity purification (His-tagged protein) • QIAGEN BioRobot 8000

31. RIKEN Structural Genomics/Proteomics Initiative (RSGI) cDNA library Cell-free protein synthesis (Large-scale with SI/HA) Target selection & Domain identification (Bioinformatics) Purification Cell-free protein synthesis (Small-scale) GSC NMR facility SPring-8 synchrotron facility Screening (Productivity, Solubility, etc.) Structure analysis (NMR/X-ray)

32. Protein preparation facility (GSC) • 22 AKTAprime system • 13 AKTAexplorer system

33. Preparation of15N-labeled proteins for HSQC screening On/24 samples 4h/24 samples 4-6h/24 samples 1h/24 samples

34. NMR screening Middle-scale production Concentration NMR measurements Unfolded Folded Broad signals Excellent Partially unfolded Large-scale preparation Fine-tuning Reconstruction Tag-removal Buffer condition Additives Structure Determination

35. Cell-free protein synthesis (Small-scale on 96-well plate) Cell-free synthesis (Midi-scale with 15N) Affinity purification Cloning & Sequencing NMR measurement (HSQC) Cell-free synthesis (Small-scale dialysis) Sample screening