a genetically programmable protein module as intracellularly deliverable qd-based fret probes for...
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A genetically programmable protein module as intracellularly deliverable
QD-based FRET probes for viral protease detection
Nikola FinneranNikola Finneran
Divya Sivaraman, Payal Biswas, and Wilfred ChenDivya Sivaraman, Payal Biswas, and Wilfred ChenDepartment of Chemical and Environmental Engineering, Department of Chemical and Environmental Engineering,
University of California, Riverside, CA, 92521University of California, Riverside, CA, 92521
August 20, 2009August 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
FRETFluorescence 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
Fluorescent Protein-Based FRETFluorophores 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 FRETAdvantages 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)
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
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 ExpressionELP precipitation and centrifuging
48kD
CYS
Pure unconjugated protein molecule
QD and Alexa Dye ConjugatoinAlexa 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 WorkModular peptide design – detect wide range of proteases
Low toxicity for in-vivo protease monitoring
Rapid protease detection
High throughput protease inhibitor screening
QDTAT
His6
CYS
cleavage site for PV2Apro
ELP QD
QD emission
after cleavage
AcknowledgementsNational Science Foundation (NSF)
Dr. Victor Rogers, Denise Sanders, and Jun Wang of the BRITE REU Program
Ph.D. candidates Divya Sivaraman, Payal Biswas, Divya Sivaraman, Payal Biswas, and Shen-long Tsaiand Shen-long Tsai
Professor Wilfred ChenProfessor Wilfred Chen
ReferencesHwang, 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
Viral Protease
Virus
Cell-Wall
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