1 / 20

FRET Based HTS Assay Kit for SUMO1-UBC9 Interaction

FRET Based HTS Assay Kit for SUMO1-UBC9 Interaction. David Bui Richard Lauhead Randall Mello Michelle Tran Team E. Introduction. Why is it important to have this kit?

sailor
Download Presentation

FRET Based HTS Assay Kit for SUMO1-UBC9 Interaction

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. FRET Based HTS Assay Kit for SUMO1-UBC9 Interaction David Bui Richard Lauhead Randall Mello Michelle Tran Team E

  2. 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) • 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 http://www.biochem.mpg.de/jentsch/Mueller.html

  3. 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 notbind FRET does not occur • If proteins do bind energy transfer will occur • Develop a process to optimize a HTS kit • Meet NIH standards FRET picture adapted from www.nature.com/.../v4/n7/fig_tab/nrm1153_F2.html

  4. Project Flow Chart

  5. 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

  6. 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)

  7. Expression Optimization Protocol 2 works the best

  8. Introduction to Z’ Factor Plot of Positive hits and negative hits sC+, mC+ :the mean and standard deviation of positive hits sC-, mC- :the mean and standard deviation of negative hits Test Value 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 The Z’ Factor is a characteristic parameter for the quality of the assay, without intervention of test compounds Well # Ji-Hu Zhang et. al.

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

  10. Z’ Factor Protein Amount Useable protein amount selected is 0.25 mM CS1 and 0.7 mM YU9 • 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

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

  12. Modeling Effects of Inhibitor Kd on Bound Protein • Equations for Kd in presence of an Inhibitor: max [CS1YU9] mM Kd of inhibitor Thanks to high [inhibitor] this assay will detect inhibitors with high Kd values

  13. 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

  14. 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) 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. 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

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

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

  18. appendix

  19. Appendix-2

  20. Appendix 3

More Related