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1
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
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Results
Introduction
Conclusion
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.
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).

• 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.
• Detected a slightly higher signal between cells
  over-expressing 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.

Figure 3. Sensorgram showing the response between
unbiotinylated BSA and NeutrAvidin
Future Work
Figure 4. Sensorgram showing the response between
Amine-PEG3-Biotinylated BSA and NeutrAvidin

• Determine ideal membrane interface design for
  effective and functional EGFR immobilization for
  protein binding.
• Create an interface based on the calcinated chip
  (glassified layer on gold) for direct
  immobilization of the EGFR in a membrane. 4
• Apply best interface design to a microarray
  format for high-throughput screening with SPR
  imaging.

Methods
Surface plasmon resonance is a label-free
biosensing technique that is capable of real-time
monitoring of biological interactions.
P-polarized 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 (lt 200 nm).

As molecules bind to the surface, the signal
increases proportionally to the amount of
accumulated mass.
References
Figure 5. Sensorgram showing enhanced signal to
mAb for EGFR, Erbitux, in the presence of
overexpressed EGFR cells compared to control
cells.

• Hopkins, A. L. Groom, C. R. Nat.Rev. Drug
  Discovery 2002, 1, 727730.
• Hubbard, S. R. Cancer Cell 2005, 7, 287-288.
• Li, Shiqing et al. Cancer Cell 2005, 7, 301-311.
  
• Linman, M. J. Culver S.P. Cheng Q. Langmuir
  2009, 25, 3075-3082.

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.
Acknowledgements
Figure 2. Cartoon representation of the desired
molecular layers on gold.
National Science Foundation BRITE REU
program Cheng Lab Group
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.
Figure 6. Sensorgram showing the binding
responses of control cells and cells
overexpressing EGFR to anti-EGFR TK immobilized
on a functionalized self-assembled monolayer
(SAM). The signal for the EGFR-expressing cells
is slightly higher than the control cells.
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.