Transcript A genetically programmable protein module as

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
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
FRET
Fluorescence Resonance Energy Transfer
Energy transfer from excited donor to acceptor molecule
Nature Reviews Molecular Cell Biology 4; 579-586
Spectral overlap of donor emission and acceptor
absorption
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)
Quantum Dot-Based FRET
Advantages of using a quantum dot donor:
broad excitation bands
narrow emission bands
higher resistance to photo bleaching
Nature Materials 5, 581 - 589 (2006)
Inability for intracellular delivery of the
conjugated protein into cells
QD-Based Engineered Protein Molecule
Modular protein design
CYS
Elastin-Like Protein (ELP)
Repeating sequence {(VPGVG)2 (VPGKG)
(VPGVG)2}20
Reversible temperature dependent precipitation
ELP
T>Tt
T<Tt
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 lysine residues within a linear
sequence of 9 amino acids (YGRKKRRQRRR)
Little to no cell toxicity
Protein Expression
ELP precipitation and centrifuging
48kD
CYS
Pure unconjugated protein molecule
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
QD-Alexa Protein FRET Pair
QD : Alexa
Conjugated Protein Functionality
Conclusions and Future Work
Modular peptide design – detect wide range of
proteases
Low toxicity for in-vivo protease monitoring
Rapid protease detection
QD emission
His6
cleavage site for
PV2Apro
ELP
QD
CYS
TAT
after
cleavage
QD
High throughput protease inhibitor screening
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
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 conjugates
Nature Materials 5, 581 - 589 (2006)
Rüdiger Rudolf, Marco Mongillo, Rosario Rizzuto & Tullio Pozzan. Looking
forward to seeing calcium Nature 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 tags NATURE METHODS
| VOL.2 NO.9 | SEPTEMBER 2005 | 659
Questions?
How it Works
Virus
Viral Protease