1. David Bui Richard Lauhead Randall Mello Michelle Tran Team E

2. 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

3. 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

4. Protein Expression Cypet-SUMO1, Ypet-Ubc9, Ubc9 2 weeks Protein Purification Ni-NTA Column Chromatography/Dialysis 2 weeks Protein Characterization Bradford 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 urea 3 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

5.  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

6.  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)

7. Innoculate 1:50 Protocol 1 Protocol 2 Protocol 3 Protocol 4 IPTG added when OD =.4.5 mM.1 mM1mM.5 mM Time Incubated 3 hours16 hours3 hours16 hours Incubation Temp. o C 37253725 Protocol 2 works the best

8.  The Z’ Factor is a characteristic parameter for the quality of the assay, without intervention of test compounds  C+,  C+ :the mean and standard deviation of positive hits  C-,  C- :the mean and standard deviation of negative hits Well # Test Value Plot of Positive hits and negative hits Z < 0.0 ; Not usable Assay 0.0 < Z < 0.5 ; A Doable Assay 0.5 < Z < 1.0 ; An Excellent Assay Z= 1.0 ; Ideal Assay Ji-Hu Zhang et. al.

9.  Ratio:  Use 10 wells for each test of positive and negative hits  Run the tests at different ratios of CS1 to YU9 Ratio of 1:2.8 is useable ratio of Cypet-SUMO1 to Ypet-UBC9

10.  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 Useable protein amount selected is 0.25  M CS1 and 0.7  M YU9

11.  How long does the assay need to incubate before it is measureable? This shows that this assay is usable after incubating only 5 minutes

12.  Equations for K d in presence of an Inhibitor: K d of inhibitor [CS1YU9]  M Effects of Inhibitor Kd on Bound Protein max Thanks to high [inhibitor] this assay will detect inhibitors with high K d values

13.  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

14.  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) 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.

15. 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

16. Dr. Jiayu Liao Vipul Madahar Yang Song Gokul Upadhyayula Thank you!!!! Jerome Schultz

17.  www.engr.ucr.edu/~mitran