GTL User Facilities

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GTL User Facilities

• Facility I Production and Characterization of
  Proteins

Eric J. Ackerman
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Genomes to Life Facilities for 21st Century
Biology

• Facility IProduction and Characterization of
  Proteins
• Use genome data to generate and characterize
  proteins and affinity reagents.

Facility I
Facility II
Facility IV
Facility III
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Facility I Production and Characterization of
Proteins

• High-throughput production of proteins on
  genome-wide scale
• Produce affinity reagents for each protein
• Biophysical characterizations
• Reagents, databases, and computational tools
  accessible to the broad scientific community
• Overcomes a principal roadblock to whole-system
  analysis

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Exported Products from Facility I

• What will Facility I deliver?
• Validated clones and expression protocols for
  proteins affinity reagents
• Proteins (but not generally exported themselves)
• Affinity reagents
• Detailed, standardized biophysical
  characterizations

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Functions of Facility I

• High-Throughput Production of
• Each gene in formats suitable for protein
  expression
• Active, full-length, purified proteins (2 mg
  quantities each, 10-25,000 proteins/year, 6
  genomes/year)
• Protein variants or mutants
• Economical affinity reagents for each protein
• Biophysical characterizations e.g. solubility,
  disorder the structure-function paradigm
• Accessible databases computational tools

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Tracking of Proteins with Affinity Reagents
Library of antibody-like molecules
target
Affinity selection
Affinity reagent
that watches your target as it moves within live
cells
Select an affinity reagent to the target
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Technologies

• Expression Systems
• Cell-free E. coli wheat germ
• Cellular E. coli, yeast, etc.
• Purification Stabilization
• Affinity Reagents
• Single-chain Ab formats
• Phage display
• Yeast-surface display
• Biophysical Characterizations solubility
  fingerprint dye binding measurements, CD, mass
  spectrometry
• Computation to track improve production, tools

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Pilots and Challenges

• PILOTS
• Cellular Structural Genomics Centers
• Cell-free Japan
• Yeast-surface phage-display antibodies
• CHALLENGES
• Computer tools to make sense of the data, develop
  reliable rules to guide production,
  characterization, labeling sites
• Stability storage
• Membrane intractable proteins
• Disorder
• Refolding
• Affinity reagents

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Molecular Crowding The Cell is Full of Molecular
Machines
Biomolecules inside cells are very concentrated,
400 mg/ml
Trends in Biochemical Sciences
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Harnessing Enzymes An Application of Proteins
Produced in Facility I
Stable enzymes entrapped in nanopores may one day
be routinely used to inactivate
pollutants. Enzymes in this environment are
stable for extended periods of time.
J. Am. Chem. Soc. 2002, 124, 11242-3
Pacific Northwest National Laboratory