A genetically programmable protein module as intracellularly deliverable QD-based FRET probes for viral protease detecti

1. A genetically programmable protein module as intracellularly deliverable QD-based FRET probes for viral protease detection Nikola Finneran Divya Sivaraman, Payal Biswas, and Wilfred Chen Department of Chemical and Environmental Engineering, University of California, Riverside, CA, 92521 August 20, 2009

2. Viral Proteases • Enzymes that catalyze the hydrolysis of peptide bonds • Very specific and highly expressed early on during infection • Cleave polyproteins into functional enzymes necessary for infection and disease progression

3. FRET • Fluorescence Resonance Energy Transfer • Energy transfer from excited donor to acceptor molecule • Spectral overlap of donor emission and acceptor absorption Nature Reviews Molecular Cell Biology 4; 579-586

4. Fluorescent Protein-Based FRET • Fluorophores bound by specific linker protein • Disadvantages of using organic fluorophores and fluorescent proteins include: • narrow excitation bands • broad emission bands • low resistance to photo degradation Hwang et al., AEM. 72(5): 3710–3715 (2006)

5. Quantum Dot-Based FRET • Advantages of using a quantum dot donor: • broad excitation bands • narrow emission bands • higher resistance to photo bleaching • Inability for intracellular delivery of the conjugated protein into cells Nature Materials 5, 581 - 589 (2006)

6. QD-Based Engineered Protein Molecule • Modular protein design CYS

7. Elastin-Like Protein (ELP) • Repeating sequence {(VPGVG)2 (VPGKG) (VPGVG)2}20 • Reversible temperature dependent precipitation

8. TAT Peptide • Allows QD-protein to penetrate mammalian cell walls • HIV-1 TAT peptide • Cluster of basic amino acids made up of 6 arginine and 2 lysineresidues within a linear sequence of 9 amino acids (YGRKKRRQRRR) • Little to no cell toxicity

9. Protein Expression ELP precipitation and centrifuging 48kD CYS Pure unconjugated protein molecule

10. QD and Alexa Dye Conjugatoin • Alexa 568 maleimide dye conjugation with protein module • 2 hour incubation of protein with Alexa 568 maleimide followed by thermal ELP purification

11. QD-Alexa Protein FRET Pair QD : Alexa

12. Conjugated Protein Functionality

13. QD Conclusions and Future Work • Modular peptide design – detect wide range of proteases • Low toxicity for in-vivo protease monitoring • Rapid protease detection • High throughput protease inhibitor screening QD emission His6 cleavage site for PV2Apro ELP QD after cleavage TAT CYS

14. Acknowledgements • National Science Foundation (NSF) • Dr. Victor Rogers, Denise Sanders, and Jun Wang of the BRITE REU Program • Ph.D. candidates Divya Sivaraman, Payal Biswas, and Shen-long Tsai • Professor Wilfred Chen

15. References Hwang, Yu-Chen, Chen, Wilfred, Yates, Marylynn V. Use of Fluorescence Resonance Energy Transfer for Rapid Detection of Enteroviral Infection In Vivo Appl. Environ. Microbiol. 2006 72: 3710-3715 Igor L. Medintz et al., Proteolytic activity monitored by fluorescence resonance energy transfer through quantum-dot–peptide conjugatesNature Materials 5, 581 - 589 (2006) Rüdiger Rudolf, Marco Mongillo, Rosario Rizzuto & Tullio Pozzan. Looking forward to seeing calciumNature Reviews Molecular Cell Biology 4, 579-586 (July 2003) Mahmoud Reza Banki, Liang Feng & David W Wood, Simple bioseparations using self-cleaving elastin-like polypeptide tagsNATURE METHODS | VOL.2 NO.9 | SEPTEMBER 2005 | 659

16. Questions?

17. Virus Cell-Wall Cell-Wall How it Works Viral Protease