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

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

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

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

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

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

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

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

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

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