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High-Throughput Protein Production Platform for the Northeast Structural Genomics Consortium

High-Throughput Protein Production Platform for the Northeast Structural Genomics Consortium. ER82. WR66. Rutgers Protein Production:.

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High-Throughput Protein Production Platform for the Northeast Structural Genomics Consortium

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  1. High-Throughput Protein Production Platform for the Northeast Structural Genomics Consortium ER82 WR66 Rutgers Protein Production: Thomas Acton, Ken Conover, Bonnie Cooper, Yiwen Chiang, Natalia Denissova, Chi Kent Ho, Li-Chung Ma,Ritu Shastry, Lydia Shih, Rong Xiao, Stephen Anderson, Masyori Inouye, Gaetano T. Montelione

  2. 50 2500 Clones / Solubility Testing 12/50 (25%) 600 Large Scale Ferm/Purification 2/12 (15%) 100 Crystal Hits 1/12 (~10%) 50-60 3D Crystal Structures 3/12 (25%) 150 “Good” HSQC >1/12 (~10%) >50-60 3D NMR Structures 600 Soluble Proteins Year per week per year

  3. Rost Clusters: Structural Genomics Targets • Protein domain families / clusters • Full length proteins < 340 amino acids • No member > 30% identity to PDB structures • No regions of low complexity • Not predicted to be membrane associated ~ 20,000 Rost Clusters

  4. Phylogenetic Distribution of NESG Target Proteins Human cytomegalovirus Aeropyrum pernix Lactococcus lactis Aquifex aeolicus M. thermoautotrophicum Arabidopsis thaliana Neisseria meningitidis Archaeglobus fulgidis Other Bacillus subtilis Pyrococcus furiosus Brucella melitensis Pyrococcus horikoshi Caenorhabditis elegans Saccharomyces cerevisiae Campylobacter jejuni Staphylococcus aureus Caulobacter crescentus Streptococcus pyogenes Drosophila melanogaster Streptomyces coelicolor Deinococcus radiodurans Thermoplasma acidophilum Escherichia coli Thermotoga maritima Fusobacterium nucleatum Thermus thermophilus Haemophilus influenzae Vibrio cholerae Helicobacter pylori Homo sapiens S. cerevisiae Arabidopsis thaliana Homo sapiens C. elegans D. melanogaster

  5. Intra and Inter-Laboratory Coordination is Maintained by the SPINE Database http://spine.nesg.org/sum.pl AR81 HSQC

  6. Multiplex Expression System Classical Restriction Endonuclease/Ligase-dependent cloning E. coli Expression Vectors Eukaryotic Expression systems

  7. 96 Well Primer Design http://www-nmr.cabm.rutgers.edu/bioinformatics/index.html Everett et al., 2004. J Struct Funct Genomics. In Press

  8. Auto-Steps with the Biorobot 8000 DNA Mini-preps PCR Reaction Qiaquick Purify Set up-96 well Colony PCR PCR Gel Puri. RestrictionDigest Transform Cycle Sequencing Ligation Big Dye removal

  9. cDNA Synthesis / RT-PCR Bottleneck for cloning Eukaryotic genes cDNA-Template for PCR cDNA Libraries - 1. Availability- Cost etc. • 2. Not fully sequenced-Quality not fully spliced/length, etc. • 3. Transfer - 384 -Well plates • 4. Representative- not all tissue/development etc. A. Extract RNA (total)/PolyA B. Reverse Transcription from mRNA Oligo dT primer Common template for all reactions Similar to Bacterial cloning

  10. Homo sapiens RT-PCR HR570 HR562 HR556 100 bp HR561 HR569 HR558 HR564 HR559 HR560 HR563 HR565 HR566 bp 561 462 462 462 459 462 462 657 417 207 681 390 E. coli PCR PCR of Fly and Human Targets • PolyT priming of PolyA RNA Drosophila embryo, larva & adult • Homo sapiens, adult brain adenocarcinoma, & testes.

  11. Concentration A6 B8 C7 E6 E9 F4 G4 D10 B4 96-Well Expression Screening Plate Sonicate Transformation 96-Well Plate BL21 24-Well Block Plate Reader Harvest 96-Well Culture 96-Well Plate Plate Centrifuge 96-Well Ni-NTA 96-Well Plate 2.2 ml S-Block 37o C LB Overnight Culture Bradford-96 Welll 96 Well Transfer (MJ9) 37o C 2.2 ml S-Block 24-well blocks O/N @17o C 96 to 24 Well Transfer Transfer Protein 37o C Induction Purified Protein IPTG SDS-PAGE 96-Well Plate

  12. Cloning/Expression Summary 2 colonies % Cloned/Correct PCR Product E. coli 91 B. subtilis 98 D. melanogaster 85 H. sapiens 93 Clones vs Time Automation

  13. Fermentation and Purification Taylor Graham ‘04 17o C Room 55 - 2L Positions Step 1: Ni-Affinity Chromatography Akta 3-D

  14. HSQC Screening Amenability to Structural Determination by NMR HR969

  15. Sample Optimization - Buffer Screening Microdialysis Buttons- Optimization for NMR Vary Buffer Conditions - Stability Screen for ppt. Small sample mass (50 ug/button) 100 mM Arginine Bagby S, Tong KI, Liu D, Alattia JR, Ikura M. 1997. J Biomol NMR.

  16. Analytical Gel Filtration with Light Scattering Aggregation Screening - Crystallization LS RI Proterion - 96 Well Less Sample More Conditions Monodisperse Conditions Philip Manor, Roland Satterwhite and John Hunt

  17. Gel Filtration Under Monodisperse Conditions Superdex 75 • Pool Fractions • Concentrate (~10 mg/ml) • Small Aliquots • Flash Freeze • Ship to Columbia/HWI Explorer Purifier Four Akta Primes

  18. ÄKTAxpress™ 4 modules in parallel16 samples AC-GF/DS Affinity Chromatography (AC) HiTrap™ Chelating HP, 1 and 5 ml Gel Filtration (GF) HiLoad 16/60 Superdex 200 pg AC 5 hours AC/GF 12 hours

  19. Distribution of the First 99 NESG Structures Phylogenetic Method Eukaryotic Archea X-ray NMR Eubacteria All targets are members of eukaryotic protein families

  20. Solubility/2004 Stats Solubility vs Organism 2004 Production 2004 HR Success • 8 HR (Human) proteins in advanced stages of NMR • 3 HR Crystal structures *defined as greater than 60% soluble by SDS-PAGE analysis

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