protein engineering to solve problems at nanoscale
DESCRIPTION
Background… To make a clinically useful MIG sensor: Streptavidin Concept of FET protein sensing Erroneous classical model (Schoning and Poghassian, 2002) Reality of bioHFET protein sensing (Gupta et al., 2008) To make a clinically useful MIG sensor: -Increase sensitivity 200X (Currently at 10-30nM) -Identify anti-MIG antibody fragmentsTRANSCRIPT
Protein Engineering to Solve Problems at Nanoscale
S.C. Lee
Department of Biomedical EngineeringDepartment of Chemical and Biomolecular Engineering
Ohio State University, Columbus OH
Dorothy M. Davis Heart and Lung Research CenterOhio State University Medical Center, Columbus OH
Background…
Concept of FET protein sensing Erroneous classical model (Schoning and Poghassian,2002)
Reality of bioHFET protein sensing (Gupta et al., 2008)
To make a clinically useful MIG sensor: -Increase sensitivity 200X (Currently at 10-30nM)-Identify anti-MIG antibody fragments
MIG
Streptavidin
HFET signal is inversely related to salt concentration…
(Schoning and Poghassian,2002)
(Shapiro et al., 2007, Gupta et al. 2008, Wen et al., submitted)
…so there is a sensitivity issue related to a nanoscale parameter, addressable with protein engineering…
Optimization opportunities…
Receptor -Size -Topology -Epitope specificity
Analyte -Size, conformation -Charge density -Charge distribution -Epitope distribution
FET -Composition -Dielectric -Gate bias -Surface chemistry
Polymer film -Thickness -Composition -Uniformity -Durability
Clinical relevance of sensing MIG…
• CXCL9 (or MIG) is a chemokine, involved in inflammatory processes – Made by monocyte/macrophages– Chemoattractant for T-lymphocytes
• Positively correlated with transplant rejection• Normal concentration: 40-100 pM• Disease concentration: as high as 34 nM• Highly positively charged protein
~Net 20 positive charges per molecule at pH 7.4
Antibody Fragments…
• scFv: one VariableHeavy (VH) and Variable Light (VL) chain
12 nm 3-4 nm
scFv
Phage Display for anti-MIG Phage Display for anti-MIG abab
yfpyfpyfgyfg
yfg-Your Favorite Geneyfg-Your Favorite Gene
yfp-Your Favorite Proteinyfp-Your Favorite Protein
•Phage display links variant gene to the protein it specifies
–Libraries screened by affinity–1011 variants/screen
Identify unique sequences from each
round of selection and express
Incubation with biotinylated-MIG—varying Concentrations
Elution of phage
Phage titer and amplification
Repeat 4-6 times
Use of solid support system to display MIG to phage
Phage Enrichment
5 x 104
5 x 106
1 x 107
1 x 105
Round of SelectionI II III IV
Round [MIG]
I 40 nM
II 1 nM
III 1 nM
IV 1 nM
Chemoselective scFv conjugation…
…orients scFvs…
Parent Mutant
P M M
Electrophile Nucleophile
N-end specific oxidation of ser-terminated proteins…
Unoxidized
Aldehyde-terminated(oxidized)
Aldehyde-terminated(oxidized)
Unoxidized
Intact Trypsin digested
SAMSAM
Electrophile Nucleophile
Eteshola et al., 2006, 2007, Shapiro et al., 2007
Lee et al., 2004
Differential epitope recognition…
MIG+
-
E2
E1
a
Anti-E2
MIG -+
E1
Anti-E1-
MIG+ E2
-
c
Anti-E2
b
SABio
HRP
MIG+
-Anti-E1
vs
..with chemoselectively orientedscFvs…
MIG structure and charge distribution….
3-4 nm
MIG
2.5-3 nm
White-NeutralBlue-PositiveRed-Negative
…makes differential epitope recognition a way to tune charge-surface distance…
Changing receptor topography: Scanning circular permutagenesis…
Eteshola et al., 2006, 2007
Improving the polymeric film…
Bhushan et al., 2009
Ideal APTES Real APTES
APDMES
Thinner, non-crosslinked film… -Better sensitivity -Saturates (biochemically) at lower analyte concentration -Smoother -More mechanically robust
Summary• The classical planar immunoFET analysis is inaccurate
– The flaws are conceptual: the model can’t be redeemed – Data from MIG detecting HFETs contradict the model– Distance between bound charges and sensing surface is
critical to magnitude of response• Protein engineering and bioconjugate chemistry can address
nanoscale issues – Modulating receptor topography (CP) can modulate the
critical distance– Epitope recognition specificity influences the critical
distance– The critical distance can be modulated by careful “SAM”
selection and construction
AcknowledgementsSurface Functionalization:Theo Nicholson IIISamit K. GuptaEdward EtesholaJohn P. ShapiroMark EliasMatt KeenerAlGaN Fabrication/Characterization:Wu LuLeonard BrillsonXuejin Wen
Surface Characterization:Bharat BhushanKwang Joo KwakDharma Tokachichu
Funds:National Science FoundationDepartment of Homeland Security