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Protein-Protein Binding Kit of SUMO Conjugation Cascade by FRET Technology

Protein-Protein Binding Kit of SUMO Conjugation Cascade by FRET Technology. David Bui Richard Lauhead Randall Mello Michelle Tran. Background. SUMO-small ubiquitin-like modifier Like phosphorylation, SUMO can alter the function of proteins

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Protein-Protein Binding Kit of SUMO Conjugation Cascade by FRET Technology

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  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 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 • Screening for protein-protein interactions • Test for inhibitors • Developing a production procedure to develop a kit and a user manual for consumer purposes • Optimization and quality control for production

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

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

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

  8. Progress Chart

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

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