Transcript 48x36 Poster Template - Bourns College of Engineering

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
Abstract
Proteases are enzymes that are used in various
cellular processes such as blood coagulation, hormone
maturation and apoptosis. They are also used as the
key virulence factor for pathogenic infection. These
properties make proteases a prime target for detailed
investigation to better understand the disease
development process and can be potentially used to
study various therapeutic agents. One of the most
promising methods for probing protease activity is
based on the principle of fluorescence resonance
energy transfer (FRET). In this study, we develop a
genetically programmable protein module that is easily
adaptable for screening inhibitors for a wide range of
proteases. The specific approach was to generate a
quantum dot (QD)-modified, protease-specific protein
module that can be used as a FRET substrate for
probing protease activity. Intracellular delivery of the
probes was facilitated by the use of a flanking TAT
peptide and the site-specific incorporation of an
acceptor fluorescent dye was accomplished using a
unique cysteine residue. Presence of an elastin domain
within the module enabled the simple purification of the
QD-modified FRET substrate. For the initial testing, we
developed a substrate peptide sequence that contains
the cleavage site which is recognized by the polio viral
protease PV2Apro. Utility of these new probes for
monitoring viral activity and to screen for protease
inhibitors will be discussed.
Experimentation
Fluorescent Protein-Based FRET Pair
TAT Peptide
• Disadvantages of using organic fluorophores and
fluorescent proteins include:
• narrow excitation bands
• broad emission bands
• low resistance to photo degradation
• Allows our QD protein to penetrate through cell
membranes with minimal cell toxicity
QD-Alexa Protein FRET Pair
• Cluster of basic amino acids mad up of 6 arginine
and 2 lysine residues within a linear sequence of 9
amino acids (YGRKKRRQRRR)
QD : Alexa
Protein Expression
ELP precipitation and centrifuging
Figure 10: Conjugated protein’s fluorescent emissions at
different QD : Alexa ratios. Blue curve shows the emissions
without conjugating the Alexa to the protein
Nature Materials 5, 581 - 589 (2006)
Figure 3: Quantum Dot-Based FRET Pair before and after the protease’s
cleavage of the linker sequence
Quantum Dot-Based FRET Pair
Conjugated Protein Functionality
48kD
• Advantages of using a quantum dot donor:
• broad excitation bands
• narrow emission bands
• higher resistance to photo bleaching
•Before Cleavage (blue): The QD is excited
and its emissions are absorbed by the Alexa
dye
pure unconjugated protein module
• A disadvantage however lies in the inability for
intracellular delivery of the conjugated protein into
cells
Methods
Figure 5: Process of protein expression from cells containing the
expression vector to the purified unconjugated protein
QD and Alexa Dye Conjugation
• Alexa 568 maleimide dye conjugation with protein
module
•As the protein is cleaved (red) and FRET is
disrupted, there is an increase in QD
emissions and a decrease in Alexa emissions
Before Cleavage
QD-Based Genetically Engineered Protein Module
Background
• 2 hour incubation of protein module with thiol-reactive
dye, Alexa 568 maleimide, followed by thermal ELP
purification of conjugated protein modules
Protease
• Proteases are enzymes that catalyze the
hydrolysis of peptide bonds, breaking down proteins
Figure 11: Emissions after 3.5 hours of protease activity
CYS
• Viral proteases are very important virulence factors
in infection as they catalyze the hydrolysis of the
longer polyprotein into functional enzymes for
continuation of the viral lifecycle and infection
•Proteolysis is very specific and the viral proteases
are highly expressed early on allowing for more
rapid detection
FRET
References
Figure 6: Unconjugated Alexa Figure 7: Supernatant after three ELP
568 in the supernatant after
cycles shows no unconjugated Alexa
three ELP purification cycles dye (left). Proteins conjugated to the
Alexa dye pelleted down and
suspended in 10mM Hepe’s Buffer
(middle and right respectively)
Elastin-Like Protein (ELP) Domain
• Repeating sequence {(VPGVG)2 (VPGKG)
(VPGVG)2} 20
Figure 1: Donor and acceptor FRET-pair
T>Tt
T<Tt
Nature Reviews Molecular Cell Biology 4; 579-586
NATURE METHODS | VOL.2 NO.9 | SEPTEMBER 2005 | 659.
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Hwang et al., AEM. 72(5): 3710–3715 (2006)
Figure 4: Comparison of heated ELP-protein solution (left) with cool dissolved
protein (right)
1.2
QD emission
Alexa568 absorption
1
1.2
1
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0
450
500
550
600
650
Normalized absorbance (a.u.)
ELP
Normalized fluorescence. (a.u.)
• Reversible temperature dependent precipitation
• Fluorescence Resonance Energy Transfer
Figure 2: Protein-based FRET
pair. Protease cleavage results
in the emission of CFP rather
than YFP
After Cleavage
0
700
wavelength (nm)
Figure 8: Spectral overlap of QD emissions on Alexa 568 absorption
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 quantumdot–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
Acknowledgements
We would like to thank the National Science Foundation,
Dr. Victor Rogers, Denise Sanders, Jun Wang, and
Shen-Long Tsai