# Presentation: 357537

## 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 IV**** **

**Facility III**** **

## 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

**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**

## Tracking of Proteins with Affinity Reagents

***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***

## Technologies

**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**

## Pilots and 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**

**Pilots and Challenges**

## Molecular Crowding: The Cell is Full of Molecular Machines

*Trends in Biochemical Sciences*

**Molecular Crowding:**** The Cell is Full of Molecular Machines**

**Biomolecules inside cells are very concentrated, ~400 mg/ml**

## 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**

**Harnessing Enzymes:**** An Application of Proteins Produced in Facility I**