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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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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
Functions of Facility I
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target
Affinity reagent
Library of ‘antibody-like’ molecules
Affinity selection
Select an affinity reagent to the target
that watches your target as it moves within live cells…
Tracking of Proteins with Affinity Reagents
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• 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
Technologies
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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
Pilots and Challenges
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Trends in Biochemical Sciences
Molecular Crowding: The Cell is Full of Molecular Machines
Biomolecules inside cells are very concentrated, ~400 mg/ml
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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.
Pacific Northwest National Laboratory
J. Am. Chem. Soc. 2002, 124, 11242−3
Harnessing Enzymes: An Application of Proteins Produced in Facility I