MEASURING SINGLE-DOMAIN ANTIBODY
INTERACTIONS WITH EPITOPES IN JET FUEL
USING MICROSCALE THERMOPHORESIS
Richard R Chapleau,1,2,* Jeanette S Frey,1,2 David S Riddle,1,2 Oscar N Ruiz,3 and
Camilla A Mauzy1
1
Molecular Bioeffects Branch, Bioeffects Division, Human Effectiveness Directorate, 711 Human
Performance Wing, Air Force Research Laboratory (711 HPW/RHDJ), Wright Patterson AFB, OH;
2
Henry M. Jackson Foundation for the Advancement of Military Medicine, Wright Patterson AFB, OH;
3
Fuels and Energy Branch, Turbine Engine Division, Aerospace Systems Directorate, Air Force Research
Laboratory, Wright Patterson AFB, OH.
SUPPLEMENTAL MATERIAL
SUPPLEMENTARY METHODS
Expression and Purification of Recombinant LcrV.
Genomic DNA from Y. pestis KIM5 (a gift of Dr. Matthew Nilles, University of North
Dakota, Grand Forks) was used as a template for PCR amplification of lcrV. Primers specific for
the gene were designed to include NdeI and XhoI restriction sites for directional cloning into the
pET-24 expression plasmid. Sequence verification was performed by the Ohio State University
Plant-Microbe Genomics Facility following plasmid ligation. The plasmid was transformed into
E. coli BL21(DE3) and selection was performed using kanamycin. Using an overnight starter
inoculation, 10L Luria-Bertani media was used in a BioFlo 310 Fermenter (New Brunswick) for
expression induced by the addition of isopropyl-β-D-thiogalactoside for 4 hours.
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Bacteria were harvested by centrifugation, resuspended in a small volume of 20 mM Tris
pH 8, 150 mM NaCl, 10% glycerol prior to mechanical cell lysis. Cellular debris were removed
by centrifugation at 10,000 x g for 20 minutes. LcrV containing supernatant was filtered through
a 0.45 µm filter; buffer exchange occurred by FPLC using a HiPrep Desalt column. Crystalline
ammonium sulfate was added to a final concentration of 1.5 M for precipitation at room
temperature for 1 hour followed by another filtration step of LcrV containing supernatant. 5 mg
total protein were then applied to a Source 15PHE hydrophobic interaction column and elution
was performed against an ammonium sulfate free buffered solution (50 mM sodium phosphate,
pH 7) over 10 column volumes. Purified LcrV was buffer exchanged again prior to loading on a
MonoQ anion exchange column with final elution into 25 mM Tris pH 8, 2 M NaCl, 0.05%
Tween-20, 1 mM EDTA.
Expression and Purification of Single-Domain Antibodies
The genes for L1 and L3 were amplified from a recombinant pHEN4 plasmid (a gift of
Dr. Serge Muyldermans, Vrije Universiteit Brussel, Belgium) using common primers
(VHH_FR1: DDDDD; VHH_FR4: AAAA). The amplified products were digested with PstI
and BstEII for cloning into pHEN6c for expression in E. coli BL21 cells. Bacterial expression
was performed as for LcrV.
Purification was performed from the periplasmic extract by hypotonic shock. Cells were
harvested by centrifugation, washed, and resuspended in chilled TES buffer (0.2 M Tris pH 8,
0.5 mM EDTA, 0.5 M sucrose). Lysis occurred on ice by centrifugation and resuspension in 30
mL chilled TES diluted 1:5 with chilled water. Cell debris was removed by centrifugation and
the supernatant was filtered through an 0.45 µm filter. Buffer exchange into PBS (20 mM
2
sodium phosphate pH 7.4, 0.5 M NaCl) with 20 mM imidazole was accomplished by FPLC
using the HiPrep Desalt column prior to purification over a HisTrap FF affinity column. Elution
was performed using PBS with 500 mM imidazole and a final buffer exchange into PBS with no
imidazole resulted in pure sdAbs.
Determining Dissociation Constants in Buffered Conditions by Surface Plasmon Resonance
Pure LcrV was buffer exchanged out of Tris and into HEPES buffered saline with 0.5%
v/v P20 surfactant (HBS-P+, GE Healthcare) for immobilization on a Biacore CM5 SPR chip
(GE Healthcare). Flow channel 1 (FC1) was used for a control surface and the protein was
immobilized on flow channel 2 (FC2). Using a Biacore T200, the channels were first activated
by addition of a 1:1 mixture of 400 mM N-hydroxysuccinimide and 100 mM N-(3dimethylaminopropyl)-N’-ethylcarbodiimide for 8 minutes at 5 µL/min. LcrV was flowed
through FC2 in 30 second pulses at 5 µL/min until 400 resonance units (RU) had accumulated.
Free succinimide ester groups were quenched on both FC1 and FC2 using 1 M ethanolamine for
10 minutes at 10 µL/min. A final wash with HBS-P+ resulted in a prepared chip surface. Each
sdAb was diluted to 500 nM and diluted 1:2 serially to 0.195 nM. All kinetic curves were
trimmed and aligned to injection time before determining the dissociation constant using the
equilibrium method as described in the text.
Characterizing Binding by Microscale Thermophoresis in Buffer and Jet Fuel
Pure LcrV was fluorescently labeled with amine reactive DyLight 650 (Pierce) according
to the manufacturer’s instructions. 100 µL of 1 mg/mL LcrV was incubated with 8 µL of borate
buffer (provided) and added directly to a single vial of DyLight 650 reagent. Protein and label
were incubated at room temperature protected from light for 1 hour, then purified twice by
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centrifugation at 1000 rpm using 100 µL purification medium (provided). LcrV sdAbs were
diluted from PBS to 2 µM in MST buffer (NanoTemper Technologies). 20 µL of 2 µM sdAb
was transferred to well A1 of a 384 well microplate. 10 µL of MST buffer or a 50% mixture of
jet fuel sump water (obtained from the US Air Force Research Laboratory Fuels and Energy
Branch) and MST buffer was placed into the remaining 15 wells of column 1. A 1:1 serial
dilution of 10 µL was removed from the top well and the column was filled out. The final 10 µL
were discarded. A stock labeled LcrV solution was made at 10.76 nM and 10 µL were added to
each well in the microplate. The proteins were incubated for 5 minutes before being loaded
simultaneously into capillaries preloaded in a Monolith Capillary Tray. The capillaries were
scanned to confirm uniformity of fluorescent signal (±10% RFU) and to identify optimal location
of the capillary. MST experiments were performed using an LED power of 50% with a 5 second
equilibration step, an infrared laser pulse at 60% for 30 s, and another 5 second recovery with the
laser off. Each capillary was measured once and each dilution series was performed in triplicate.
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