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Developing Novel Supported Membrane Interfaces for
SPR Study of Transmembrane Proteins
Heather Ferguson*, Matthew J. Linman† , Quan Cheng†
† Department
of Chemistry - University of California Riverside 92521
*Walla Walla University, WA 99324
Results
Introduction
An important step to the preparation of the sensing layers is successfully biotinylating proteins. These data show that BSA biotinylated with
Amine-PEG3 (Figure 4) gave a much greater SPR signal than the non-biotinylated control response (Figure 3).
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NeutrAvidin
(0.5 mg/ml)
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BSA (0.5 mg/ml)
NeutrAvidin (0.5 mg/ml)
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Biotin-BSA
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Future Work
Figure 4. Sensorgram showing the response between Amine-PEG3Biotinylated BSA and NeutrAvidin
• Determine ideal membrane interface design for
effective and functional EGFR immobilization for
protein binding.
Red = Control CHO Cells
Black = EGFR overexpressed in cells
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Time (min.)
Time (min.)
As molecules bind to
the surface, the
signal increases
proportionally to the
amount of
accumulated mass.
•Detected a slightly higher signal between cells overexpressing EGFR when using polyclonal anti-EGFR TK
compared to control cells. Using a higher concentration
of antibody may increase the analytical signal.
•Current method of combining EGFR and PC vesicles
can be improved.
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Surface plasmon resonance is a label-free biosensing technique
that is capable of real-time monitoring of biological interactions. Ppolarized light excites the electrons in the gold layer, and results in
a minimum of reflected light intensity, marking the surface plasmon
angle. SPR is sensitive to changes in the index of refraction near
the surface (< 200 nm).
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5.5 x 10 cells (CHO or EGFR overexpressed CHO)
•Create an interface based on the calcinated
chip (glassified layer on gold) for direct
immobilization of the EGFR in a membrane. 4
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•Apply best interface design to a microarray
format for high-throughput screening with SPR
imaging.
Signal = 360 mdeg
Signal = 190 mdeg
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Erbitux (0.25 mg/mL)
EDC/NHS
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References
Time (min.)
Figure 5. Sensorgram showing enhanced signal to mAb for EGFR, Erbitux, in
the presence of overexpressed EGFR cells compared to control cells.
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Red = Control CHO Cells
Black = EGFR overexpressing cells
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Control cells
and EGFR cells
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Control cells
and EGFR cells
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Time (min.)
Lipids cannot bind directly to gold, so a protein layer such as bovine
serum albumin is used. NeutrAvidin and biotin bind together very
strongly and serve as linking units to tether antibody and the lipid
bilayer containing EGFR to the surface.
Erbitux
0.25 mg/ml
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Neutravidin
0.25 mg/ml
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Figure 6. Sensorgram showing the binding responses of control cells and cells
overexpressing EGFR to anti-EGFR TK immobilized on a functionalized selfassembled monolayer (SAM). The signal for the EGFR-expressing cells is slightly
higher than the control cells.
1. Hopkins, A. L.; Groom, C. R. Nat.Rev. Drug
Discovery 2002, 1, 727–730.
2. Hubbard, S. R. Cancer Cell 2005, 7, 287-288.
3. Li, Shiqing; et al. Cancer Cell 2005, 7, 301-311.
4. Linman, M. J.; Culver S.P.; Cheng Q. Langmuir
2009, 25, 3075-3082.
PC vesicles +
EGFR cells
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Acknowledgements
Biotin-BSA
0.25 mg/ml
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Anti-EGFR TK (20 g/ml)
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Figure 2. Cartoon
representation of the
desired molecular layers
on gold.
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Figure 1. Schematic of SPR set-up. The analyte in the flow channel are
cells expressing EGFR and the immobilized molecule is anti-EGFR.
Diagram from : Cooper, M.A. Nat. Rev. Drug Discovery 2002, 1, 517.
www.PosterPresentations.com
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Figure 3. Sensorgram showing the response between unbiotinylated
BSA and NeutrAvidin
Methods
POSTER TEMPLATE BY:
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•Biotinylation procedure needs to be perfected until it is
more reliable and gives reproducible results.
•Overexpressed EGFR cells show preferential binding to
mAb for EGFR, Erbitux, compared to control cells.
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Membrane proteins are known key molecules in cellular activities
such as maintaining cell structure, transport, and signalling.
Therefore an understanding of the function, especially of the affinity
property of membrane proteins, is of particular importance to the
pharmaceutical industry where 60% of drug targets are membrane
proteins.1 However, membrane proteins are difficult to study in their
native environment and do not function properly when removed
from a lipid membrane. We set out to mimic a mammalian lipid
membrane environment using phosphatidylcholine (PC) vesicles
that surround the transmembrane protein epidermal growth factor
receptor (EGFR). EGFR is often overexpressed in cells in certain
types of cancers such as breast, colon, and lung.2 Surface
plasmon resonance (SPR) is employed to characterize interactions
between the membrane protein and immobilized anti-EGFR TK, an
antibody against the EGFR tyrosine kinase (TK) domain. Several
surface chemistries have been examined in an effort to find a
reproducible and functional biomimetic surface containing EGFR.
Conclusion
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Biotin Anti-EGFR
20 g/ml
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Time (min.)
Figure 7. Sensorgram showing the binding response between Erbitux and
EGFR expressing cells combined with PC lipid membrane. Erbitux is a
monoclonal antibody specific to an extracellular domain of EGFR.3 The lack
of a strong signal indicates that the EGFR is not properly oriented in the
membrane.
National Science Foundation
BRITE REU program
Cheng Lab Group