RIKEN Structural Genomics/Proteomics Initiative (RSGI)

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RIKEN Structural Genomics/Proteomics
Initiative(RSGI)

• Shigeyuki Yokoyama
• RIKEN Genomic Sciences Center and
• Harima Institute at SPring-8

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Cell-Free Protein Synthesis System

• Easy manipulation
• Adding/removing components
• No cloning necessary
• PCR amplified template
• High-throughput

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Cell-Free Synthesis with Dialysis
Dispo/Dialyzer 1ml (Spectra/Por)
f5 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
External solution (3000 µl) Buffer, salts,
etc. ATP, GTP, etc. Amino acids NaN3
Reaction mixture S30 extract Template DNA T7
RNA polymerase Buffer, salts, etc. ATP, GTP,
etc. Amino acids
Dialysis System
Batch System
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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
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Cell-Free Protein Synthesis Escherichia coli
coupled transcription-translation system (Kigawa
Yokoyama) Wheat germ translation system
(Yaeta Endo)
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Wheat Germ Cell-Free Protein Synthesis
System Prof. Yaeta Endo Ehime University RIKEN
GSC
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Cell-free protein synthesis workshop
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

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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
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Template DNA Construction
FW
RV
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Large-scale cell-free protein synthesis from
PCR-amplified template (I)
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Domain identification(Computational)

• Sequence homology
• Neural network

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g-B crystallin
Cyclodextran glucanotransferase
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(No Transcript)
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Protein Structure Determination

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

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Stable-isotope Labeling
Ras(Y32W)GDP (Dialysis cell-free)
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15N (ppm)
120
130
10
9
8
7
1H (ppm)
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Selenomethionyl RasGDPprepared by the Cell-Free
System
P 65 2 2

• 0.1M Sodium cacodylate (6.5)
• 0.2M Calcium acetate
• 18 PEG8000

• SeMet Incorporation gt 95 (MS)

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Selenomethionyl-RasGDPprepared by the Cell-Free
System
MAD Phasing (SPring-8 BL44B2)
(PDB1IOZ)
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Molecular Function
Structure analysis
Function analysis

• Identification of interacting partner(s)
• Protein-protein
• Protein-peptide
• Protein-DNA
• Protein-RNA
• Protein-ligand

• Proper folding
• Complex formation

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Molecular Function

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

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Membrane Proteins Cellular and Extracellular
Domains Integral Membrane Proteins (GPCR, etc.)
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(No Transcript)
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Nature Biotech., in press.
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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)

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Protein Overroduction Expression Plasmids
PCR, TA cloning
ligation
Constructed 1106 plasmids
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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
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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)

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cDNA Collections
Protein Expression
Target Prioritization
Functional Studies
Structural Studies
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Sample screening
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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
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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

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

• 22 AKTAprime system
• 13 AKTAexplorer system

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Preparation of 15N-labeled proteins for HSQC
screening
On/24 samples
4h/24 samples
4-6h/24 samples
1h/24 samples
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NMR screening
Middle-scale production
Concentration
Unfolded
NMR measurements
Folded
Broad signals
Partially unfolded
Excellent
Large-scale preparation
Fine-tuning
Reconstruction
Tag-removal Buffer condition Additives
Structure Determination
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Sample screening