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