FRET BASED HTS ASSAY KIT FOR SUMO1-UBC9 INTERACTION

David BuiRandall Mello

Richard LauheadMichelle Tran

Introduction

Goal: Development of FRET based kit to screen compounds that could alter binding between SUMO1 and UBC9.

•Why is it important to have this kit?Disregulation of the SUMO pathway has been linked to diseases including ovarian carcinoma, melanoma, and lung adenocarcinoma. (Mo and Moschos 2005)

http://www.biochem.mpg.de/jentsch/Mueller.html

Project Flow Chart

Sensitivity of Flexstation II

Using highly pure proteins, serial dilutions were done to make solutions at different protein concentrations

Values range from 1 ng to 100 µg of protein per well

0 50000 100000 150000-10000000

0

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70000000

f(x) = 626.011334927943 xR² = 0.987703357462313

Ypet-Ubc9 Em530 01/28/10 blank subtracted

Fluorescence(RFU)Linear (Fluores-cence(RFU))

Protein Amount (ng)

RFU

0 50000 1000000

5000000

10000000

15000000

20000000

25000000f(x) = 265.138880047491 xR² = 0.995148847051858

Cypet-Sumo1 Em475 01/29/10 blank subtracted

Fluorescence(RFU)Linear (Fluores-cence(RFU))

Protein amount (ng)

RFU

Sensitivity of Flexstation II- A general HTS development instrument

For accurate fluorescence measurements of single proteins and conjugations, an assay must have values away from background noise

500 ng is the lowest amount for usable assay conditions.

Ypet-Ubc9 (ng)

RFU without blank

RFU without blank

RFU without blank

100000 58734866.98 55943707.39 61961881.1850000 36425642.98 36053579.39 39276933.1825000 20232692.98 20081895.39 23343669.1810000 7194076.48 7613824.887 9610900.176

5000 4313948.98 3672442.637 4147272.6762500 1905796.605 1675790.887 1826577.1761000 432355.386 416138.231 433805.832

500 118134.269 199872.325 247566.004100 8604.365 15551.569 13429.223

50 2701.488 17229.912 11205.07225 371.426 3043.334 8485.24910 -1422.633 -372.635 3125.487

5 -1634.192 1552.071 1672.9862.5 -1682.931 2221.778 2313.75

1 -1428.963 -136.699 965.3610 0 0 0

Cypet-Sumo1 (ng)

RFU without blank

RFU without blank

RFU without blank

91500 22667111.59 23441949.39 23460794.7350000 9461644.594 13937961.39 15112699.7325000 5334212.594 8311063.387 9603005.72910000 2223233.344 2547688.137 4388946.729

5000 1010283.219 1294915.387 2053190.6042500 493361.313 681590.324 730195.9791000 135762.875 196490.34 217703.057

500 58105.11 72562.403 69732.471100 6996.274 6824.616 7213.549

50 1396.175 7010.968 3055.90625 5140.756 3566.683 773.44110 -1246.045 2060.112 739.592

5 -1626.112 3063.877 168.9472.5 -1058.78 2058.266 429.742

1 -465.929 13409.479 -50.3090 0 0 0

Purification Optimization using Ni-His Purification

Ingredients Concentration of Solutions(M)Wash1 Protocol 1 Protocol 2 Protocol 3NaCL 0.3 0.5 0.4

Tris HCL pH 7.4 0.02 0.02 0.02

Wash2 NaCL 0.3 2 1.2

Tris HCL pH 7.4 0.2 0.02 0.02Triton 0.50% 2.00% 1.25%

Wash3 NaCL 0.3 2 1.2

Tris HCL pH 7.4 0.2 0.02 0.02Immidazole 0.02 0.05 0.035

Elution NaCL 0.3 0.3 0.3

Tris HCL pH 7.4 0.02 0.02 0.02Immidazole 0.15 0.25 0.2

Resuspension Buffer Concentration(M)

NaCl 0.5

Tris-HCl pH 7.4 0.2

Immidazole 0.005

•Cell Lysate obtained from 1 Liter of E. coli solution and resuspended in 30 mL of Resuspension buffer• Column purification protocol involves 10mL of lysate poured into a column with 500 µL of agarose nickel bead solution with subsequent 10 mL washes. •Elution took place 500 µL at a time and continued until the beads showed no fluorescence.

Wash solutions adapted from Qiagen Ni-NTA agarose beads purification booklet

Method to Determine Protein Concentration

Bradford Assay: Total protein

concentrations of solution can be calculated using the equation obtained from graph.

0 500 1000 1500 2000 25000

0.2

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1

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f(x) = 2.1414533E-10 x³ − 0.000000986701505 x² + 0.00177445508 x − 0.0070935426R² = 0.999125712990113

Absorbance vs Protein amount

absLogarithmic (abs)Logarithmic (abs)Polynomial (abs)

Protein amount(ng/uL)

Ab

sorb

an

ce(R

FU

)

Purification Optimization using Ni-His Purification

Using the Bradford Assay to determine total protein concentration and the fluorescence curves generated from the sensitivity tests, the purity was calculated for each protocol

Ypet-UBC9Purification protocol

Bradford concentrations(ng/uL)

fluorescent concentration (ng/uL) Purity

Protocol1 7710 5086.89 0.66Protocol2 955 617.27 0.65Protocol3 5500 2783.92 0.51

Cypet-SUMO1Purification protocol

Bradford concentrations(ng/uL)

fluorescent concentration (ng/uL) Purity

Protocol1 4844.94 4708.36 0.97Protocol2 3191.38 1678.81 0.53Protocol3 4642.16 3229.12 0.70

)Pr(

)Pr(

oteinTotalBradford

oteintFluorescenySensitivitPurity

0 50000 100000150000-100000000

10000000200000003000000040000000500000006000000070000000

f(x) = 626.011334927943 xR² = 0.987703357462313

Ypet-Ubc9 Em530 01/28/10 blank

subtractedFluorescence(RFU)Linear (Fluo-rescence(RFU))

Protein Amount (ng)

RFU

Purification Optimization using Ni-His Purification

•Ypet-UBC9 could be around 70% pure•Cypet-SUMO1 is unlikely to be 97% pure

Ypet-UBC9 Cypet-SUMO1

Prot.1 Prot.2 Prot.3

Prot.1 Prot.2 Prot.3

Purity effects on fluorescence

450 470 490 510 530 550 5700

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Purity effects on Cypet-SUMO1 1ug 100%

90%80%70%60%50%40%30%20%10%

Wavelength(nm)

RFU

490 510 530 550 570 590 6100

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Purity effects on Ypet-UBC9 1ug 100%

90%80%70%60%50%40%30%20%10%

Wavelength(nm)

RFU

•Keeping a constant fluorescent protein amount at 1 µg.•BL21 cell lysate proteins were added to change percent purity.•Results show that purity has little effect on fluorescence at 1µg of fluorescent protein.Other Proteins do not interfere with

fluorescent intensity

Purity effects on FRET

450 470 490 510 530 550 570 590 6100

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Purity effects on Fret 500ng100%90%80%70%60%50%40%30%20%10%

Wavlength(nm)R

FU

0% 20% 40% 60% 80% 100% 120%0

0.2

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Purity effect on Fret 500ng

Series1

Purity

530/4

75 fl

uore

scent

rati

on

•Tested purity effects on FRET with each protein at a constant amount of 500 ng. •Results demonstrate that purity of fluorescent proteins and in FRET has no effect at low concentration(10ng/µl).

•Emission max of Ypet-Ubc9 over Cypet-SUMO1 to obtain FRET ratio.•Results demonstrate little to no change in FRET ratio when purity is varied.

Purity does not have significant effect on FRET ratio

Lyophilization Effects

Experimental Design Lyophilize 1mL of protein solution in 1.5mL

tubes for Cypet-SUMO1 and Ypet-UBC9 for the following tests Lyophilized and stored as powder at RT Lyophilized and stored as powder at 4 degree Lyophilized and stored as powder at -20 degree Lyophilized and stored as powder at -80 degree Lyophilized then stored as solution at RT Lyophilized then stored as solution at 4 degree Lyophilized then stored as solution -20 degree Lyophilized then stored as solution -80 degree

Lyophilization Effects

Obtained purity before lyophilization Weighed tube before and after

lyophilization to obtain protein amount and fluorescent protein amounts

Diluted all solutions to have the same protein concentrations, and same total protein amount per well.

Performed FRET analysis of each solution to determine FRET effeciency

Lyophilization Effects

450 470 490 510 530 550 5700

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Cypet-SUMO1 stability after 3 days 2 ug of pro-

tein

non-lyophilized 4 degresuspended RTResuspended 4 degResuspended -80 degresuspended -20 degpowder rtpowder 4 degpowder -20 degpowder -80 degblank

Wavelength(nm)

Flu

ore

scence

490 510 530 550 570 590 6100

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Ypet-UBC9 stability after 3 days 2 ug of protein

resuspended RTResuspended 4 degResuspended -80 degresuspended -20 degpowder rtpowder 4 degpowder -20 degpowder -80 degblanknon-lyophilized 4 deg

Wavlength(nm)

Flu

ore

scence

Lyophilization Effects

450 500 550 600 6500

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FRET Lyophilization stabil-ity after 3 days 1ug per

each proteinyu csnormal 4 degreeresuspended RTResuspended 4 degResuspended -20 degresuspended -80 degpowder rtpowder 4 degpowder -20 degpowder -80 degblank

Wavelength(nm)

Flu

ore

scence

yu cs

norm

al 4

deg

ree

resu

spen

ded

RT

Resus

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ed 4

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Resus

pend

ed -2

0 de

g

resu

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-80

deg

powde

r rt

powde

r 4 d

eg

powde

r -20

deg

powde

r -80

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FRET Ratio 530/475 1 ug per each protein

Series1

FR

ET R

ati

o

Gantt ChartSUMO ASSAY KIT

1/18/2010

1/25/2010

2/1/2010

2/8/2010

2/15/2010

2/22/2010

3/1/2010

3/8/2010

3/15/2010

3/22/2010

3/29/2010

4/5/2010

4/12/2010

4/19/2010

4/26/2010

5/3/2010

5/10/2010

5/17/2010

5/24/2010

5/31/2010

6/7/2010

Tasks Start End%complete

Purification optimization 1/18/2010 2/8/2010 100%

Flexstation 2 fluorescence sensitivity test 1/18/2010 2/8/2010 100%

Dialysis/Lyophilization 2/1/2010 2/22/2010 100%

Protein purity effects 2/1/2010 2/22/2010 50%

Expression optimization 3/1/2010 3/15/2010

Fret sensitivity 3/1/2010 3/15/2010 10%

compound screening sensitivity 3/22/2010 4/5/2010

Stability testing 3/22/2010 4/5/2010 10%

Oxidation testing 4/12/2010 4/26/2010

Kit assembly 4/12/2010 4/26/2010

Add secretion factors to proteins 5/3/2010 5/31/2010

Questions?