Protein-Protein Binding Kit of Protein-Protein Binding Kit of SUMO Conjugation Cascade SUMO Conjugation Cascade

by FRET Technologyby FRET Technology

David BuiDavid Bui

Richard LauheadRichard Lauhead

Randall MelloRandall Mello

Michelle TranMichelle Tran

BackgroundBackground•SUMO-small ubiquitin-like modifier•Like phosphorylation, SUMO can alter the function of proteins

•Gene expression/stability, protein activation/deactivation

FRETFRET

Försters Resonance Energy TransferFörsters Resonance Energy Transfer

http://www.zmb.uzh.ch/resources/protocols/FRET.html?version=simple

PurposePurpose

Develop an in vitro high throughput FRET-Develop an in vitro high throughput FRET-based assay kit for SUMO1 and UBC9based assay kit for SUMO1 and UBC9 Screening for protein-protein interactionsScreening for protein-protein interactions

Test for inhibitorsTest for inhibitors

Developing a production procedure to Developing a production procedure to develop a kit and a user manual for develop a kit and a user manual for consumer purposesconsumer purposes Optimization and quality control for productionOptimization and quality control for production

ObjectivesObjectives

Optimization of productionOptimization of production Protein ExpressionProtein Expression

Vary Temperature, length of expressionVary Temperature, length of expression Protein PurificationProtein Purification

Stringent wash, Stepwise method, Variation of StepwiseStringent wash, Stepwise method, Variation of Stepwise Dialysis/LyophilizationDialysis/Lyophilization

To powder, to concentrateTo powder, to concentrate FRET OptimizationFRET Optimization

Determine optimal amount of Cypet-SUMO1/Ypet-UBC9 to Determine optimal amount of Cypet-SUMO1/Ypet-UBC9 to gain maximum FRET(FRET does not necessarily depend on Kgain maximum FRET(FRET does not necessarily depend on Kdd))

Compound OptimizationCompound Optimization Introduce inhibitor (UBC9)Introduce inhibitor (UBC9)

ObjectivesObjectives

Quality ControlQuality Control Stability TestingStability Testing

Oxidation testsOxidation tests Design and Assemble KitDesign and Assemble Kit

ManualManual

Secondary ObjectivesSecondary Objectives

Add Secretion factors to proteins of Add Secretion factors to proteins of interest to streamline production interest to streamline production processprocess Current Process:Current Process:

Proteins are produced within the cellsProteins are produced within the cells If secondary objective met:If secondary objective met:

Proteins will be secreted outside the cellsProteins will be secreted outside the cells

Progress ChartProgress Chartprotein expressionprotein expression

single day expression1single day expression1single day expression2single day expression2overnight expressionovernight expressionfermentorfermentor

protein purificationprotein purificationSuggested literature methodSuggested literature methodlab uselab usevariation of current lab usevariation of current lab use

Dialysis/lyophilizationDialysis/lyophilizationto powder: Dialysis in buffer, lyophilize, resuspend in waterto powder: Dialysis in buffer, lyophilize, resuspend in waterto powder: Dialysis in water, lyophilize, resuspend in bufferto powder: Dialysis in water, lyophilize, resuspend in bufferto concentrate: Dialysis in buffer, lyophilize, resuspend in waterto concentrate: Dialysis in buffer, lyophilize, resuspend in waterto concentrate: Dialysis in water, lyophilize, resuspend in bufferto concentrate: Dialysis in water, lyophilize, resuspend in buffer

FRET optimizationFRET optimizationthis involves running fret with different amounts of protein in each well. this involves running fret with different amounts of protein in each well. Suggestion involves running the x axis as amount cypetsumo1Suggestion involves running the x axis as amount cypetsumo1y axis as ypet UBC9. Graph the results to get max fluorescence. y axis as ypet UBC9. Graph the results to get max fluorescence.

stability testing/oxidationstability testing/oxidationleave at 37 degrees celsius for 3 days tube openleave at 37 degrees celsius for 3 days tube openleave at 25 degrees celius for 3 days tube openleave at 25 degrees celius for 3 days tube openleave at 4 deg celsius for 3 days tube openleave at 4 deg celsius for 3 days tube openleave at 37 degrees celsius for 3 days tube closedleave at 37 degrees celsius for 3 days tube closedleave at 25 degrees celius for 3 days tube closedleave at 25 degrees celius for 3 days tube closedleave at 4 deg celsius for 3 days tube closedleave at 4 deg celsius for 3 days tube closed

put kit togetherput kit togetheroptimize protein production methodoptimize protein production method

add secretion factors to genesadd secretion factors to genes

compound screening amountcompound screening amount

above lyophiliyzed completely to powderabove lyophiliyzed completely to powderabove lyophilized just to concentrateabove lyophilized just to concentrate

ConclusionConclusion

Learn production process of kit Learn production process of kit designdesign

Develop the kitDevelop the kit