1. Protein-Protein Binding Kit of SUMO Conjugation Cascade by FRET Technology David Bui Richard Lauhead Randall Mello Michelle Tran

2. Background SUMO-small ubiquitin-like modifier Like phosphorylation, SUMO can alter the function of proteins Gene expression/stability, protein activation/deactivation

3. FRET Försters Resonance Energy Transfer Försters Resonance Energy Transfer http://www.zmb.uzh.ch/resources/protocols/FRET.html?version=simple

4. Purpose Develop an in vitro high throughput FRET- based assay kit for SUMO1 and UBC9 Develop an in vitro high throughput FRET- based assay kit for SUMO1 and UBC9 Screening for protein-protein interactions Screening for protein-protein interactions Test for inhibitors Test for inhibitors Developing a production procedure to develop a kit and a user manual for consumer purposes Developing a production procedure to develop a kit and a user manual for consumer purposes Optimization and quality control for production Optimization and quality control for production

5. Objectives Optimization of production Optimization of production Protein Expression Protein Expression Vary Temperature, length of expression Vary Temperature, length of expression Protein Purification Protein Purification Stringent wash, Stepwise method, Variation of Stepwise Stringent wash, Stepwise method, Variation of Stepwise Dialysis/Lyophilization Dialysis/Lyophilization To powder, to concentrate To powder, to concentrate FRET Optimization FRET Optimization Determine optimal amount of Cypet-SUMO1/Ypet-UBC9 to gain maximum FRET(FRET does not necessarily depend on K d ) Determine optimal amount of Cypet-SUMO1/Ypet-UBC9 to gain maximum FRET(FRET does not necessarily depend on K d ) Compound Optimization Compound Optimization Introduce inhibitor (UBC9) Introduce inhibitor (UBC9)

6. Objectives Quality Control Quality Control Stability Testing Stability Testing Oxidation tests Oxidation tests Design and Assemble Kit Design and Assemble Kit Manual Manual

7. Secondary Objectives Add Secretion factors to proteins of interest to streamline production process Add Secretion factors to proteins of interest to streamline production process Current Process: Current Process: Proteins are produced within the cells Proteins are produced within the cells If secondary objective met: If secondary objective met: Proteins will be secreted outside the cells Proteins will be secreted outside the cells

8. Progress Chart protein expression single day expression1 single day expression2 overnight expression fermentor protein purification Suggested literature method lab use variation of current lab use Dialysis/lyophilization to powder: Dialysis in buffer, lyophilize, resuspend in water to powder: Dialysis in water, lyophilize, resuspend in buffer to concentrate: Dialysis in buffer, lyophilize, resuspend in water to concentrate: Dialysis in water, lyophilize, resuspend in buffer FRET optimization this involves running fret with different amounts of protein in each well. Suggestion involves running the x axis as amount cypetsumo1 y axis as ypet UBC9. Graph the results to get max fluorescence. stability testing/oxidation leave at 37 degrees celsius for 3 days tube open leave at 25 degrees celius for 3 days tube open leave at 4 deg celsius for 3 days tube open leave at 37 degrees celsius for 3 days tube closed leave at 25 degrees celius for 3 days tube closed leave at 4 deg celsius for 3 days tube closed put kit together optimize protein production method add secretion factors to genes compound screening amount above lyophiliyzed completely to powder above lyophilized just to concentrate

9. Conclusion Learn production process of kit design Learn production process of kit design Develop the kit Develop the kit