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

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

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

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

Intra and Inter-Laboratory Coordination is Maintained by the SPINE Database AR81 HSQC

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

96 Well Primer Design Everett et al., J Struct Funct Genomics. In Press

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

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 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

PCR of Fly and Human Targets PolyT priming of PolyA RNA Drosophila embryo, larva & adult Homo sapiens, adult brain adenocarcinoma, & testes. E. coli PCR Homo sapiens RT-PCR bp HR556 HR558 HR559HR560 HR561 HR562 HR563 HR564 HR566 HR569 HR570 HR565 bp

Plate Reader Transformation Harvest Plate Centrifuge Concentration Overnight Culture 96 Well Transfer (MJ9) 96-Well Culture IPTG 24-well blocks 96 to 24 Well Transfer o C 96-Well Ni-NTA Purified Protein A6B8C7E6E9F4G4 D10 B4 SDS-PAGE Induction 37 o C 96-Well Plate 24-Well Block 2.2 ml S-Block 37 o C BL21 LB 96-Well Plate Transfer Protein Bradford-96 Welll 96-Well Expression Screening Plate Sonicate

Cloning/Expression Summary 2 colonies % Cloned/Correct PCR Product E. coli91 B. subtilis98 D. melanogaster85 H. sapiens93 Clones vs Time Automation

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

HR969 HSQC Screening Amenability to Structural Determination by NMR

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

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

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

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

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

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