Emerald bio nollert_pegs_draft_v4.3

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Emerald bio nollert_pegs_draft_v4.3

  1. 1. Parallel protein purification at the mg scale• Introduction Emerald Bio• Concepts & workflow• Multi-target parallel processing• Automated parallel protein purification Peter Nollert, Ph.D. Chief Technology Officer Emerald Bio pnollert@embios.com May 3, 2012 8th Annual Protein Engineering Summit - PEGS 11/16/2012 Copyright 2012 Slide 1
  2. 2. Our Protein X-ray Crystallography Structure Output April 15: 482 SSGCID.org Emerald is depositing 1 new structure every 1.7 days11/16/2012 PEGS 2012 Slide 2
  3. 3. SSGCID: the Most ProductiveStructural Genomics Center in 2011 1.5 2.0 2.5 3.0 Resolution (Å)3/20/2012 PEGS 2012
  4. 4. MTPP: Multi-Target Parallel Processing >100 structures delivered, >60 ligand bound 13 different targets crystallized MTPP and Quality Average Resolution: 2.3Å Average MolProbity: 94 percentile11/16/2012 PEGS 2012 Slide 4
  5. 5. Hundreds of Structures Require Thousands of Proteins 684 vector constructs 220 different proteins purified 9.7g = total protein produced 5g11/16/2012 PEGS 2012 Slide 5
  6. 6. MPTT: Multi Target Parallel Processing 16 targets selected 14 targets as protein reagent 9 unique target structures Construct Expression Protein X-ray Structure Crystals Generation Testing Samples 269 131 E.coli 231 Purifications Constructs 132 Baculovirus 6 HEK29311/16/2012 PEGS 2012 6
  7. 7. Emerald Bio’s : Protein Resource Operating SystemTARGET SPECIFICPROCEDURE Expression Recombinant SAMPLES & Formulations PurificationDEVELOPMENT Constructs Expression Systems INSIGHT Pure Proteins Genes Protein Crystals Compounds Protein / ligand 3D co-structures Target Info Fragment Based Drug DiscoveryBIOLOGY &CHEMISTRY Ligand Optimization Mechanism of Action Intellectual Property TOOLS Gene Process & Data Opti E-screen Protein Composer Management Matrix Builder Maker™ ™ System Maker 7
  8. 8. High-throughput parallel protein purificationwith the Protein MakerApplications: Antibody Purification from up to 1 Liter load • Biomarker discovery (immuno depletion) • Parallel expression and purification testingParallel Protein Chromatography • Carries out up to 24 purifications / run • One step purification or two step purification (up to 12 samples) • Uses standard 1-5 mL chromatography columns in parallel • Unattended operation, up to 4 runs / day • Small (1mL) to large (1L) load volumes 11/16/2012 PEGS 2012 8 US Pat.# 6,818,060
  9. 9. Protein Maker: Automated Parallel Protein PurificationThroughput:Multiple targets & variants are Target variation:provided in batches Purification of internal deletion variants; His-taggedApproach: Parallelchromatographic IMAC purification Analysis:of 24 target protein • SDS-PAGE • Crystallization Lysates Eluates Crystals 11/16/2012 PEGS 2012 9
  10. 10. Protein Maker: Parallel lysis scouting Problem: Standard lysis buffer may not Example render all protein targets soluble. Sample: Cytochrome P450 Solution: Test 12 different cell-lysis buffers insoluble in standard lysis buffer followed by small scale (1 mL) IMAC Result: of 12 lysis buffers 2 yield purification soluble CYP51A (CHAPS/bOG + 500 mM NaCl) Lyse E.coli Ni-IMAC cells in 12 standard SDS-PAGE Scale-up different elution analysis cell lysis buffers protocolSmith, E., et al. (2011). Acta Cryst.F , 67 (9), 1015-1021 PEGS 2012 10 11/16/2012
  11. 11. Protein Resources Operating SystemTARGET SPECIFICPROCEDURE Expression Recombinant SAMPLES & Formulations PurificationDEVELOPMENT Constructs Expression Systems INSIGHT Pure Proteins Genes Protein Crystals Compounds Protein / ligand 3D co-structures Target Info Fragment Based Drug DiscoveryBIOLOGY &CHEMISTRY Ligand Optimization Mechanism of Action Intellectual Property TOOLS Process & Data Opti Gene E-screen Protein Management Matrix Composer Builder Maker 11/16/2012 System Maker 11
  12. 12. More Results:• Emerald Bio @ PEGS trade show floor: booth# 62/63• Email: pnollert@embios.com11/16/2012 PEGS 2012 12

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