Transcript Protein-Protein Binding Kit of SUMO Conjugation Cascade by

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
•Gene expression/stability, protein activation/deactivation
FRET
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Försters Resonance Energy Transfer
http://www.zmb.uzh.ch/resources/protocols/FRET.html?version=simple
Purpose
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Develop an in vitro high throughput FRETbased assay kit for SUMO1 and UBC9
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Screening for protein-protein interactions
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Test for inhibitors
Developing a production procedure to
develop a kit and a user manual for
consumer purposes
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Optimization and quality control for
production
Objectives
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Optimization of production
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Protein Expression
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Protein Purification
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To powder, to concentrate
FRET Optimization
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Stringent wash, Stepwise method, Variation of Stepwise
Dialysis/Lyophilization
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Vary Temperature, length of expression
Determine optimal amount of Cypet-SUMO1/Ypet-UBC9 to
gain maximum FRET(FRET does not necessarily depend on
Kd)
Compound Optimization
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Introduce inhibitor (UBC9)
Objectives
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Quality Control
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Stability Testing
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Oxidation tests
Design and Assemble Kit
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Manual
Secondary Objectives
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Add Secretion factors to proteins of
interest to streamline production process
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Current Process:
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Proteins are produced within the cells
If secondary objective met:
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Proteins will be secreted outside the cells
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
to
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powder: Dialysis in buffer, lyophilize, resuspend in water
powder: Dialysis in water, lyophilize, resuspend in buffer
concentrate: Dialysis in buffer, lyophilize, resuspend in water
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
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37 degrees celsius for 3 days tube open
25 degrees celius for 3 days tube open
4 deg celsius for 3 days tube open
37 degrees celsius for 3 days tube closed
25 degrees celius for 3 days tube closed
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
Conclusion
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Learn production process of kit design
Develop the kit