FRET BASED HTS ASSAY KIT FOR SUMO1-UBC9 INTERACTION

David BuiRichard Lauhead

Randall MelloMichelle Tran

Team E

Introduction• 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

• Forster Resonance Energy Transfer• Energy transfer

between donor and acceptor fluorophores at the distance of 2-10 nm(Dos Remedios and Moens 1995)

• Engineered CFP (Cypet) and YFP (Ypet) genetically engineered to proteins SUMO-1 and E2 Conjugating enzyme Ubc9 of the Sumoylation pathway

Goals• Develop a high throughput

screening kit based on FRET that allows for detection of protein-protein (SUMO1-UBC9) interactions• Allows for basic

screening of inhibitors that alter binding between target proteins

• Combine two target proteins at a specific concentration ratio• If proteins do not bind

FRET does not occur• If proteins do bind

energy transfer will occur

FRET picture adapted from www.nature.com/.../v4/n7/fig_tab/nrm1153_F2.html

• Develop a process to optimize a HTS kit

• Meet NIH standards

Project Flow ChartProtein Expression

Cypet-SUMO1, Ypet-Ubc9, Ubc9

2 weeks

Protein PurificationNi-NTA Column

Chromatography/Dialysis

2 weeks

Protein CharacterizationBradford assay, fluorescence characterization, SDS PAGE

Protein Gel etc..3 weeks

Protein Expression Optimization

IPTG, 7-16 hour growth/expression

2 weeks

Kit Design/ Quality Control Lyophilization, Oxidation, and Stability studies with

urea3 weeks

Z Factor Studies for HTS FRET Ratio optimization Cypet-SUMO1/Ypet-Ubc9

Concentration optimization 3 weeks

Ubc9 Mock Inhibitor Studies Ubc9+Ypet-Ubc9/

Ubc9+Cypet-SUMO1/Ypet-Ubc9+Cypet-SUMO1

concentration experiments 2 weeks

Inhibitor Kd Modeling Effects of Inhibitor Kd

on Bound Protein, Kdapp equations 2 weeks

Last Quarter Summary Determine sensitivity of the FlexStation II

RESULTS: As low as 25 ng, but useable around 500 ng of fluorescent protein

Determine method for characterizing Fluorescent protein concentration RESULTS: Bradford Assay and Fluorescent

Standard Curves Determine if purification affects the assay and an

optimal purification protocol RESULTS: Purity has no effect and Protocol 1 was

chosen Lyophilization Studies

RESULTS: Does not effect protein stability

Work Done Prior to Senior Design

CYpet and Ypet were engineered as FRET pairs

UBC9 was identified as a protein that binds to SUMO1 in the SUMO pathway.

Kd between SUMO1 and UBC9 was determined by BIACORE(SPR) as 0.75

FRET was determined to work between CYpet-SUMO1(CS1) and Ypet-UBC9(YU9)

450 470 490 510 530 550 570020000400006000080000

100000120000140000160000

Fluorophore Spectrums: CYpet and Ypet

FRET Spectrum RawYpet Spectrum ex: 414 nmBackgroundFRET Spectrum processedCypet SpectrumCypet without background

Wavelength (nm)

Fluo

resc

ence

(RFU

)

Expression OptimizationInnoculate 1:50

Protocol 1

Protocol 2

Protocol 3

Protocol 4

IPTG added when OD = .4

.5 mM .1 mM 1mM .5 mM

Time Incubated

3 hours 16 hours

3 hours 16 hours

Incubation Temp. oC

37 25 37 25

Protocol 1

Protocol 2

Protocol 3

Protocol 4

0

2000

4000

6000

8000

10000

12000

14000

Expression optimization

Cypet-SUMO1 Concen-tration producedYpet-UBC9 Concentra-tion Produced

Protocol

Fluo

resc

ent

Prot

ein

conc

entr

atio

n(ng

/uL)

Protocol 2 works the best

Introduction to Z’ Factor The Z’ Factor is a characteristic

parameter for the quality of the assay, without intervention of test compounds

sC+, mC+ :the mean and standard deviation of positive hits sC-, mC- :the mean and standard deviation of negative hits

Well #

Test

Val

ue

Plot of Positive hits and negative hits

Z < 0.0 ; Not usable Assay0.0 < Z < 0.5 ; A Doable Assay0.5 < Z < 1.0 ; An Excellent AssayZ= 1.0 ; Ideal Assay

Ji-Hu Zhang et. al.

Z’ Factor Ratio Ratio:

Use 10 wells for each test of positive and negative hits

Run the tests at different ratios of CS1 to YU9

1:1.2 1:2 1:2.8 1:3.6 1:4.4 1:5.2

-0.4-0.3-0.2-0.1

00.10.20.30.40.50.60.70.80.9

Z' Factor with Each Well hav-ing 0.21 uM Cypet-Sumo1 in

60 uL DB

[CyPet-SUMO1] : [YPet-Ubc9] ratio

Z' F

acto

r

Ratio of 1:2.8 is useable ratio of Cypet-SUMO1 to Ypet-UBC9

Z’ Factor Protein Amount Protein amount

Use ten wells for each run of positive and negative hits

Run the test with differing amount of CS1 and YU9 and a constant ratio of 1 : 2.8

0.15 0.2 0.25 0.3 0.35 0.4-0.1

00.10.20.30.40.50.60.70.8

Z' factor for determining a useable amount of protein

Z' Factor for Protein Amount

Amount of CYpet uM

Z' F

acto

r

Useable protein amount selected is 0.25 mM CS1 and 0.7 mM YU9

Z’ Factor Time Stability How long does the

assay need to incubate before it is measureable?

5 10 20 30 40 50 600

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Z' Factor Time Stability

Z' Factor with 0.25 uM CS1 and ratio 1:2.8

Time(min)

Z' F

acto

r

This shows that this assay is usable after incubating only 5 minutes

Modeling Equations for Kd

in presence of an Inhibitor:

]91[]9[*]1[

YUCSYUCSKdapp

)][1(inh

ddapp KInhibitorkK

)][1(

]9[*]1[]91[

inhd K

InhibitorK

YUCSYUCS

75.0500][

7.0]9[25.0]1[

dkMinhibitor

MYUMCS

mmm

Kd of inhibitor

[CS1

YU9]

mM

Effects of Inhibitor Kd on Bound Protein

max

Thanks to high [inhibitor] this assay will detect inhibitors with high Kd values

Design Conclusions Designed a set of experiments to

optimize the steps from protein expression to binding assays in a HTS format

Demonstrated that purity of proteins has no effect on the FRET ratio

Demonstrated that lyophilization is not necessary in kit design over non-lyophilized protein

Z’-Factor Conclusions FRET-based HTS binding assay meets Z’-Factor signal requirements of

>0.5 at a FRET Ratio of 1:2.8, at concentrations of 0.25 and 0.70 uM per well for Cypet-SUMO1 and Ypet-Ubc9, and over a 60 minute period

BRET Technology: Luciferase reporter enzyme used at 3 uM per well in one 384-well HTS study to reach a Z’-Factor over 0.5 (Molecular Biology in Medicinal Chemistry Volume 21,pg.20. Wiley-VCH 2004)

5 10 20 30 40 50 6000.10.20.30.40.50.60.70.8

Z'-factor Stability at FRET Ratio 1:2.8 and concentra-

tions of CYpet-SUMO1: Ypet-Ubc9 at 0.25:0.70 uM

Z'-Factor

Time(min)

Z'- f

acto

r

Kocan, Martina, Heng B. See, and Ruth M. Seeber. "Demonstration of Improvements to the Bioluminescence Resonance Energy Transfer (BRET) Technology for the Monitoring of G Protein–Coupled Receptors in Live Cells." Journal of Biomolecular Screening (2008). Society of Biomolcecular Sciences.

Future Work• Investigate mock inhibitor to incorporate with kit

• Allows for kit users to compare and analyze other positive hits

• Optimize protein expression• Engineer CYpet-SUMO1 and Ypet-Ubc9 to have an E. coli

secretion sequence

• Package materials and create an instructions manual to bring kit to a commercial level

Acknowledgements

Dr. Jiayu Liao

Vipul Madahar

Yang Song

Gokul Upadhyayula

Thank you!!!!

Jerome Schultz

Questions? www.engr.ucr.edu/~mitran

appendix

0 0.5 1 1.5 2 2.5 3 3.50.3

0.4

0.5

0.6

0.7

0.8

0.9f(x) = − 0.0928184628352445 ln(x) + 0.772608308994995R² = 0.990307680907584

Average FRET ratios with different concentrations of Urea

Average FRET ratios with dif -ferent concentrations of UreaLogarithmic (Average FRET ra-tios with different concentra-tions of Urea)

Urea Concentration M

FRET

Rat

io 5

30/4

75

Appendix-2

450 470 490 510 530 550 5700

20000

40000

60000

80000

100000

120000

140000

160000

180000

200000

Z Factor Run for 1: 2.8

Wavlength(nm)

Fluo

resc

enc(

RFU

)

Appendix 3