48x36 Poster Template

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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

• Fluorescent Protein-Based FRET Pair
• Disadvantages of using organic fluorophores and
  fluorescent proteins include
• narrow excitation bands
• broad emission bands
• low resistance to photo degradation

Abstract
Experimentation

• TAT Peptide
• Allows our QD protein to penetrate through cell
  membranes with minimal cell toxicity
• 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 FRET Pair
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.
QD Alexa
Protein Expression
ELP precipitation and centrifuging
Figure 10 Conjugated proteins 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 proteases cleavage of the linker
sequence

• Quantum Dot-Based FRET Pair
• Advantages of using a quantum dot donor
• broad excitation bands
• narrow emission bands
• higher resistance to photo bleaching
• A disadvantage however lies in the inability for
  intracellular delivery of the conjugated protein
  into cells

• Conjugated Protein Functionality
• Before Cleavage (blue) The QD is excited and its
  emissions are absorbed by the Alexa dye
• As the protein is cleaved (red) and FRET is
  disrupted, there is an increase in QD emissions
  and a decrease in Alexa emissions

48kD
pure unconjugated protein module
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
• 2 hour incubation of protein module with
  thiol-reactive dye, Alexa 568 maleimide, followed
  by thermal ELP purification of conjugated protein
  modules

Before Cleavage
Methods
QD-Based Genetically Engineered Protein Module
Background
After Cleavage

• Protease
• Proteases are enzymes that catalyze the
  hydrolysis of peptide bonds, breaking down
  proteins
• 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

Figure 11 Emissions after 3.5 hours of protease
activity
CYS
References
Hwang, Yu-Chen, Chen, Wilfred, Yates, Marylynn V.
Use of Fluorescence Resonance Energy Transfer for
Rapid Detection of Enteroviral Infection In
VivoAppl. Environ. Microbiol. 2006 72 3710-3715
Igor L. Medintz et al., Proteolytic activity
monitored by fluorescence resonance energy
transfer through quantum-dotpeptide 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
Figure 6 Unconjugated Alexa 568 in the
supernatant after three ELP purification cycles
Figure 7 Supernatant after three ELP cycles
shows no unconjugated Alexa dye (left). Proteins
conjugated to the Alexa dye pelleted down and
suspended in 10mM Hepes Buffer (middle and right
respectively)

• Elastin-Like Protein (ELP) Domain
• Repeating sequence (VPGVG)2 (VPGKG) (VPGVG)2
  20
• Reversible temperature dependent precipitation

• FRET
• Fluorescence Resonance Energy Transfer

Figure 1 Donor and acceptor FRET-pair
Nature Reviews Molecular Cell Biology 4 579-586
Acknowledgements
NATURE METHODS VOL.2 NO.9 SEPTEMBER 2005
659.
We would like to thank the National Science
Foundation, Dr. Victor Rogers, Denise Sanders,
Jun Wang, and Shen-Long Tsai
Figure 2 Protein-based FRET pair. Protease
cleavage results in the emission of CFP rather
than YFP
Figure 4 Comparison of heated ELP-protein
solution (left) with cool dissolved protein
(right)
Figure 8 Spectral overlap of QD emissions on
Alexa 568 absorption
Hwang et al., AEM. 72(5) 37103715 (2006)