1. Facility I: Production and Characterization of Proteins
GTL User Facilities
Facility I: Production and Characterization of Proteins
Eric J. Ackerman

2. Genomes to Life Facilities for 21st Century Biology
Facility I Production and Characterization of Proteins
Use genome data to generate and characterize proteins and affinity reagents.
Facility I
Facility II
Facility III
Facility IV

3. 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

4. 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

5. 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

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

7. 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

8. Pilots and Challenges PILOTS CHALLENGES
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

9. Molecular Crowding: The Cell is Full of Molecular Machines
Biomolecules inside cells are very concentrated, ~400 mg/ml
Trends in Biochemical Sciences

10. 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.
Pacific Northwest National Laboratory
J. Am. Chem. Soc. 2002, 124, 11242−3