Designing an SPR biointerface for transmembrane proteins
Heather FergusonMatthew Linman, Dr. Quan “Jason” Cheng
BRITE Research Presentation
August 20, 2009
Outline
Background informationSPR and EGFR
Experimental approach
Results
Conclusions
Future work
Background - SPRMetals 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
Cooper, M.A. Nat. Rev. Drug Discovery 2002, 1, 517
Characteristic Sensorgram
(2) Association
(3) Equilibrium
(4) Dissociation
Experimental Setup for SPR
Membrane ProteinsTarget of 60% of drugs Epidermal Growth Factor (EGFR)
Overexpressed in epithelial cancers
Lung, ovary, breast, colon
3 domainsIntracellular tyrosine kinaselipophilic transmembraneExtracellular ligand binding
AntibodiesPolyclonal: anti-EGFR TKmAb: Erbitux® (cetuximab)
Provided by: Eureka Therapeutics Inc.
Huang S., Invest New Drugs 1999, 259-269
Methods – Protein Concentration Assay
Purify cellsExtract 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
Methods - SAM
Johanna Stettner, Institute of Solid State Physics, Graz University of Technology
SH(CH2)10 COOH
1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC)
N-hydroxysulfosuccinimide (Sulfo-NHS)
•Stable bond between sulfur and gold
•Short hydrocarbon chain
•Change functional groups
Covalent Coupling mAb to SAM
mAb binding significantly stronger to EGFR cells than the control
62.0
62.2
62.4
62.6
62.8
63.0
63.2
63.4
63.6 Red = Control CHO CellsBlack = EGFR overexpressed in cells
Signal = 360 mdeg
Signal = 190 mdeg
5.5 x 106 cells (CHO or EGFR overexpressed CHO)
0.25 mg/mL Erbitux
EDC/NHS
Min
. An
gle
(d
eg.)
Time (min.)
SAM-Based Design on Polyclonal Ab
EGFR cells = 84.3 mdegControl = 62.9 mdegControl signal still large
0 25 50 75 100 125 15062.0
62.2
62.4
62.6
62.8
63.0
63.2
63.4
63.6
63.8
Rinse
Control cells and EGFR cells
Control cells and EGFR cells
Anti-EGFR TK (20 g/ml)
Rinse
EDC/NHS4:1
Min
. An
gle
(d
eg.)
Time (min.)
EGFR-expressing Control
EGFR in Tethered Bilayer Membrane
0 25 50 75 100 125 150 175
62.0
62.2
62.4
62.6
62.8
63.0
63.2
63.4
63.6
63.8
Rinsed
PEG-amineblocking agent
Anti-EGFR TK
Rinsed
EDC/NHS 4:1
Rinsed
PC vesicles and cellsMin
An
gle
(d
eg.)
Time (min.)
EGFR cells Control
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
Interface Design II: Biotinylation
Biotin-Avidin bondsVery strong (Ka = 1015 M-1 )
orientation specific
Biotinylate TK antibodySulfo-NHS-LC-Biotin kit
BiotinNHS
Effect of Biotinylation
0 20 40 60 80 10063.20
63.25
63.30
63.35
63.40
63.45
Rinse
NeutrAvidin (0.5 mg/ml)
Rinse
Biotin-BSA
Min
. An
gle
(d
eg.)
Time (min.)
0 20 40 60 80 100
63.20
63.25
63.30
63.35
Rinse
NeutrAvidin (0.5 mg/ml)Rinse
BSA (0.5 mg/ml)Min
. An
gle
(d
eg.)
Time (min.)
Biotin BSA signal 3X greater than control
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
0 25 50 75 100 125 150 175 200
62.4
62.6
62.8
63.0
63.2
63.4
63.6 Control Response
Rinse
PC vesicles and control cells
switched buffers from PBS to HEPES
Rinse
biotinylated Anti-EGFR20 g/ml
NeutrAvidin0.25 mg/ml
Biotin BSA0.25 mg/ml
Min
. An
gle
(d
eg.)
Time (min.)
-25 0 25 50 75 100 125 150 175 200 225 250 275 300
62.2
62.4
62.6
62.8
63.0
63.2
63.4EGFR
switched buffersPBS to HEPES
Rinse
Erbitux0.25 mg/ml
Rinse
PC vesicles + EGFR overexpressing cells
biotin Anti-EGFR20 g/ml
NeutrAvidin0.25 mg/ml
biotin-BSA0.25 mg/ml
Min
. An
gle
(d
eg.)
Time (min.)
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
Next StepsDetermine 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.
Acknowledgements
Matt Linman and Dr. Cheng
National Science Foundation
Jun Wang and BRITE REU program
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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.