Designing an SPR biointerface for transmembrane proteins

1
Designing an SPR biointerface for transmembrane
proteins

• Heather Ferguson
• Matthew Linman, Dr. Quan Jason Cheng
• BRITE Research Presentation
• August 20, 2009

2
Outline

• Background information
• SPR and EGFR
• Experimental approach
• Results
• Conclusions
• Future work

3
Background - SPR

• Metals with free electrons
• Gold, silver
• P-polarized light at resonance angle excites
  electrons
• Angle at which photons couple with plasmons
• Plasmons are collective vibrations of electron
  gas

4
Experimental Setup for SPR
Characteristic Sensorgram (2) Association (3)
Equilibrium (4) Dissociation
Cooper, M.A. Nat. Rev. Drug Discovery 2002, 1, 517
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Membrane Proteins

• Target of 60 of drugs
• Epidermal Growth Factor (EGFR)
• Overexpressed in epithelial cancers
• Lung, ovary, breast, colon
• 3 domains
• Intracellular tyrosine kinase
• lipophilic transmembrane
• Extracellular ligand binding
• Antibodies
• Polyclonal anti-EGFR TK
• mAb Erbitux (cetuximab)
• Provided by Eureka Therapeutics Inc.

Huang S., Invest New Drugs 1999, 259-269
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Methods Protein Concentration Assay

• Purify cells
• Extract proteins
• Bio-Rad Protein Assay kit
• Colorimetric, similar to ELISA
• Folin reagent alkaline copper tartrate
• UV/vis spectroscopy to measure absorbance
• Use standards to make calibration curve (1.6
  0.2 mg/ml)
• Determined EGFR 4.06 mg/ml

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Methods - SAM
1-Ethyl-3-3-dimethylaminopropylcarbodiimide
(EDC) N-hydroxysulfosuccinimide (Sulfo-NHS)
SH(CH2)10 COOH

• Stable bond between sulfur and gold
• Short hydrocarbon chain
• Change functional groups

Johanna Stettner, Institute of Solid State
Physics, Graz University of Technology
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Covalent Coupling mAb to SAM

• mAb binding significantly stronger to EGFR cells
  than the control

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SAM-Based Design on Polyclonal Ab

• EGFR cells 84.3 mdeg
• Control 62.9 mdeg
• Control signal still large

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EGFR in Tethered Bilayer Membrane

• Small signal increase after injection of
  Anti-EGFR (2 µg/ml)
• Co-injected PC vesicles along with cells
• EGFR cells 413 mdeg
• Control cells 352 mdeg
• Tethered membrane provides space
• Biologically relevant

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Interface Design II Biotinylation

• Biotin-Avidin bonds
• Very strong (Ka 1015 M-1 )
• orientation specific
• Biotinylate TK antibody
• Sulfo-NHS-LC-Biotin kit

Biotin
NHS
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Effect of Biotinylation
Biotin BSA signal 3X greater than control
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Complete Surface System

• Biotin BSA, NeutrAvidin, biotin anti-EGFR, PC
  vesicles cells (EGFR and control)
• Ideally, the signal should be greater from the
  EGFR cells, and Erbitux should have a greater
  signal

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Conclusions

• Biotinylation procedure is effective.
• Erbitux shows preferential binding to cells
  overexpressing EGFR compared to control cells.
• Current method of combining EGFR and PC vesicles
  can be improved.
• Lack of signal between EGFR cells in lipid
  membrane and Erbitux may indicate improper
  orientation within the membrane
• Both SAMs and biotinylated surfaces show promise

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Next Steps

• Determine ideal membrane interface design for
  effective and functional EGFR immobilization for
  protein binding.
• Try different lipid mixtures to more closely
  mimic natural membrane
• Create an interface based on the calcinated chip
  (glassified layer on gold) for direct
  immobilization of the EGFR in a membrane.
• Use mAb Erbitux once ideal interface design is
  determined
• Apply best interface design to a microarray
  format for high-throughput screening with SPR
  imaging.

16
Acknowledgements

• Matt Linman and Dr. Cheng
• National Science Foundation
• Jun Wang and BRITE REU program

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References

• Hopkins, A. L. Groom, C. R. Nat.Rev. Drug
  Discovery 2002, 1, 727730.
• Hubbard, S. R. Cancer Cell 2005, 7, 287-288.
• Kim, Edward S., et al. Epidermal growth factor
  receptor biology. Current Opinion in Oncology
  2001, 13, 506-513.
• Li, Shiqing et al. Cancer Cell 2005, 7, 301-311.
  
• Liedberg, B., I. Lundstrom, and E. Stenberg.
  1993. Principles of biosensing with an extended
  coupling matrix and surface plasmon resonance.
  Sensors and Actuators B 11 63-72.
• Linman, M. J. Culver S.P. Cheng Q. Langmuir
  2009, 25, 3075-3082.
• Macher, Bruce A., Yen, Ten-Yang. Proteins at
  membrane surfaces a review of approaches. Mol.
  Biosyst. 2007, 3, 705-713.