Presentation at PEGS2012 Boston, MA

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. Our Protein X-ray Crystallography Structure Output
April 15: 482
SSGCID.org
Emerald is depositing 1 new
structure every 1.7 days
11/16/2012 PEGS 2012 Slide 2

3. SSGCID: the Most Productive
Structural Genomics Center in 2011
1.5 2.0 2.5 3.0
Resolution (Å)
3/20/2012 PEGS 2012

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 percentile
11/16/2012 PEGS 2012 Slide 4

5. Hundreds of Structures Require Thousands of Proteins
684 vector constructs
220 different proteins purified
9.7g = total protein produced
5g
11/16/2012 PEGS 2012 Slide 5

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 HEK293
11/16/2012 PEGS 2012 6

7. Emerald Bio’s : Protein Resource Operating System
TARGET SPECIFIC
PROCEDURE
Expression Recombinant SAMPLES &
Formulations Purification
DEVELOPMENT Constructs Expression Systems INSIGHT
Pure Proteins
Genes Protein Crystals
Compounds Protein / ligand
3D co-structures
Target Info Fragment Based
Drug Discovery
BIOLOGY &
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. High-throughput parallel protein purification
with the Protein Maker
Applications: Antibody Purification
from up to 1 Liter load
• Biomarker discovery (immuno depletion)
• Parallel expression and purification testing
Parallel 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. Protein Maker:
Automated Parallel Protein Purification
Throughput:
Multiple targets & variants are Target variation:
provided in batches Purification of internal deletion
variants; His-tagged
Approach: Parallel
chromatographic IMAC purification Analysis:
of 24 target protein • SDS-PAGE
• Crystallization
Lysates Eluates Crystals
11/16/2012 PEGS 2012 9

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 protocol
Smith, E., et al. (2011). Acta Cryst.F , 67 (9), 1015-1021
PEGS 2012 10
11/16/2012

11. Protein Resources Operating System
TARGET SPECIFIC
PROCEDURE
Expression Recombinant SAMPLES &
Formulations Purification
DEVELOPMENT Constructs Expression Systems INSIGHT
Pure Proteins
Genes Protein Crystals
Compounds Protein / ligand
3D co-structures
Target Info Fragment Based
Drug Discovery
BIOLOGY &
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. More Results:
• Emerald Bio @ PEGS trade show floor: booth# 62/63
• Email: pnollert@embios.com
11/16/2012 PEGS 2012 12