Supporting Information For:
Site-Specific Biotinylation of an Ebola Detecting KZ52 Fab
Fragment at the Conserved Nucleotide Binding Site
Nur Mustafaoglu†,‡, Nathan J. Alves†,‡, and Basar Bilgicer*,†,§,┴
†
Department of Chemical and Biomolecular Engineering, University of Notre Dame
Advanced Diagnostics and Therapeutics, University of Notre Dame
┴
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN
46556, USA
‡
These authors contributed equally to this work.
§
KEYWORDS: Fab, UV-NBS Method, Nucleotide Binding Site (NBS), Biotinylation, Ebola, KZ52
* Basar Bilgicer
Assistant Professor
Department of Chemical and Biomolecular Engineering
Department of Chemistry and Biochemistry
University of Notre Dame
182 Fitzpatrick Hall
Notre Dame, IN 46556-5637
Voice: 574-631-1429
Fax: 574-631-8366
bbilgicer@nd.edu
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EXPERIMENTAL DETAILS:
Materials
Synthesis of IBA-EG11-Biotin
Preparation of KZ52 Fab Fragment
SUPPORTING INFORMATION FIGURES:
Fig. S1: Representation of Nucleotide Binding Site (NBS)
Fig. S2: Molecular structure and characterization of IBA-EG11-Biotin molecule
Fig. S3: Proposed mechanism for UV covalent bond formation of IBA to the antibody NBS.
Fig. S4: Schematic representation of ELISA assay for determining the effects of UV energy on antigen binding activity and KZ52 Fab fragment structure
Fig. S5: Effect of ligand concentration on the photocrosslinking efficiency by the UV-NBSBiotin
method
Fig. S6: Schematic representation of an ELISA assay for determination photocrosslinking efficiency of UV-NBSBiotin method.
Fig. S7: Biotinylation efficiency comparison of UV-NBSBiotin method and NHS-Biotin method
Fig. S8: Schematic representation of ELISA assay for UV-NBSBiotin method
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EXPERIMENTAL DETAILS
Materials
IBA, Biotin N-hydroxysuccinimide ester (Biotin-NHS), N,N-Diisoprophylethylamine (DIEA),
were purchased from Sigma-Aldrich (St. Louis, MO). NovaPEG Rink Amide resin was purchased
from Novabiochem (Billerica, MA). Mono-N-t-boc-amido-dPEG11-amide was purchased from Quanta Biodesign (Powell, OH). Rabbit anti-ZEBOV GP antibody was purchased from IBT Bioservices
(Gaithersburg, MD). KZ52 antibody and recombinant Ebola Zaire GPdTM protein were generously
provided by Luminex. Fab2 Fragment specific peroxidase-conjugated affiniPure goat anti-human IgG
and Fc Fragment specific peroxidase-conjugated affinipure goat anti-rabbit IgG were purchased from
Jackson ImmunoResearch (West Grove, PA). Heat shock isolated bovine serum albumin (BSA),
Amicon Ultra centrifugal filters (0.5 mL, 10K) and Coomassie R-250 were purchased from EMD
Millipore (Billerica, MA). Amplex Red Assay Kit was purchased from Invitrogen (Grand Island,
NY). Maleic Anhydride amine reactive 96-well plates and NeutrAvidin coated 96-well plates were
purchased from Thermo Scientific (Rockford, IL). Tris-Gly running buffer, transfer buffer, and tris
buffered saline (TBS) were purchased from Boston Bioproducts (Ashland, MA).
Synthesis of IBA-EG11-Biotin
To synthesize the IBA-EG11-amine, IBA was coupled to mono-N-t-boc-amido-dPEG11-amine in solution following HBTU activation in DMF and DIEA at room temperature (RT) for 3.5 h while stirring. DMF was evaporated by rotate evaporator, then t-boc protecting group was removed in a solution of 4% triisopropylsilane, 4% water, and 92% trifluoroacetic acid (TFA) for 45 min at RT. IBAEG11-amine was purified using RP-HPLC on a Zorbax C18 column (Semi-preparative 9.4 x 250 mm
5-Micron), and a Bruker MALDI-TOF MS was used for characterization of it. The calculated exact
mass for the IBA-EG11-amine (C36H63N3O12) was 729.44 Da; found 730.49 Da (product +H). IBAEG11-Biotin (Figure S2) is synthesized by conjugating Biotin to the purified IBA-EG11-amine. It was
purified and characterized using the above described instrumentation (calculated exact mass is
955.52 Da; found 956.765). The yield was 70%, and the product purity was confirmed using RPHPLC on the analytical Zorbax C18 column to be > 95%.
Preparation of KZ52 Fab Fragment
The antibodies were desalted using desalting columns in PBS buffer pH 7.4. 500 μL of antibody
(0.5 mg/mL) were cleaved using Papain enzyme resin for 5 hours at 37°C in digestion buffer including 35 mg cysteine in 10 mL of supplied digestion buffer pH 10. The cleaved Fab fragments were
purified using protein A column with PBS buffer. The purification of the Fab fragments was determined while running a 10% SDS-PAGE gel under reducing conditions. The concentration of Fab
fragments was calculated by Abs280 and an extinction coefficient of 140000 M-1cm-1.
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SUPPORTING INFORMATION FIGURES
Fig. S1: Representation of Nucleotide Binding Site (NBS) A) Crystal structure of Fab fragment of KZ52
(pdb: 3INU) and Rituximab (pdb: 2OSL) B) Alignment of Fab fragments of KZ52 and Rituximab, square
indicates the NBS C) Magnified representation of NBS; blue residues represent the KZ52 NBS, yellow
residues represent the Rituximab NBS. Red and purple backbone structures represent light chain, blue and
green backbone structures represent the heavy chain of the antibody.
Fig. S2: IBA-EG11-Biotin is synthesized by conjugating Biotin to the purified IBA-EG11-Amine (The
calculated exact mass for the IBA-EG11-Amine (C36H63N3O12) was 729.44 Da; found 730.49 Da). It is
also purified via RP-HPLC on the Zorbax C18 column and characterized with MALDI-TOF MS (calculated exact mass is 955.52 Da; found 956.765). The yield was 70%, and the product purity was confirmed
using RP-HPLC on the analytical Zorbax C18 column to be > 95%.
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Fig. S3: Proposed mechanism for UV covalent bond formation of IBA to the antibody NBS. The reaction
mechanism of UV-NBS method was investigated in detail in our previous study utilizing MS/MS technique 1. In summary, when NBS of an antibody is exposed to UV the final product is hydroxylation of the
phenyl ring resulting in the formation of a tyrosine like amino acid derivative. This hydroxylation can
occur at any site on the ring structure and is represented here as a hydroxylation at position G. UV exposure to NBS bound IBA causes the indole to become excited to the first triplet state (3Trp, 8 – 20 μs lifetime) resulting in radical-cation formation and deprotonation giving rise to the neutral indolyl radical.
From this state IBA most commonly undergoes photo-oxidation and through a complicated radical driven
reaction pathway and associated decomposition results in kynurenine formation. When this reaction is
carried out in the confined NBS the proposed result is covalent bond formation between the kynurenine
and tyrosine derivative at position 42 on the antibody light chain. The crosslink is pictured here as a covalent bond formed between positions D and H but this bond may exist at any of the other ring locations.
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Fig. S4: Schematic representation of the ELISA procedure for determining the effects of UV energy on
antigen binding activity and KZ52 Fab fragment structure stability determined by direct (A) and indirect
ELISA (B), respectively. 2 nM KZ52 Fab fragments after incubated with IBA-EG11-Biotin linker for 1 h,
exposed with increasing UV energies and then immobilized to high binding ELISA surface. The total
amount of bound Fab fragments was detected using HRP conjugated secondary antibody.
Fig. S5: Effect of ligand concentration on the photocrosslinking efficiency by the UV-NBSBiotin method.
The KZ52 Fab fragment (2 nM) or KZ52 antibody (2 nM) was incubated with increasing concentrations
of IBA-EG11-Biotin and then exposed to 1.5 J/cm2 UV. IBA-EG11-Biotin photocrosslinking efficiency at
the NBS was determined by directly adsorbing the biotinylated Fab fragments to a high binding ELISA
plate surface and using HRP conjugated Streptavidin as a reporter. All data represents means (± SD) of
triplicate experiments.
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Fig. S6: Schematic representation of an ELISA assay for determination photocrosslinking efficiency of
UV-NBSBiotin method. The photocrosslinking efficiency of IBA-EG11-Biotin to the KZ52 Fab fragment at
the NBS was determined by indirect (A) and direct (B) ELISA assay, where the total biotinylation levels
of KZ52 were detected HRP conjugated streptavidin after binding the surface immobilized ZEBOV
GPdTM.
Fig. S7: Determination of the amount of biotinylation using the UV-NBSBiotin method with 1 J/cm2 UV
exposure compared to biotinylation using the manufactures recommended protocol for antibody biotinylation with NHS-Biotin. Biotinylated Fab Fragment, 0 – 0.5 nM in 100 µL carbonate bicarbonate coating
buffer (pH 9.6), was directly adsorbed to high bind 96-well ELISA plates. Total biotinylation of the surface immobilized Fab Fragment was quantified using streptavidin-HRP. The slopes of the linear regression fits were used to determine the relative degree of biotinylation for each method with UV-NBS (S =
1620.2) having 1.25 fold more biotinylation compared to the NHS-Biotin (S = 1293.6). Data represents
means (± SD) of triplicate experiments.
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Fig. S8: Schematic representation of ELISA assay for UV-NBSBiotin method. Biotinylated Fab fragments
with UV-NBSBiotin method were incubated on streptavidin coated ELISA surface. Antigen binding activity of functionalized Fab fragments was determined via an indirect ELISA.
References
1. Alves, N. J.; Champion, M. M.; Stefanick, J. F.; Handlogten, M. W.; Moustakas, D. T.; Shi,
Y.; Shaw, B. F.; Navari, R. M.; Kiziltepe, T.; Bilgicer, B. Selective photocrosslinking of
functional ligands to antibodies via the conserved nucleotide binding site. Biomaterials
2013, 34, 5700-5710.
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