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Site-Specific, Covalent Labeling of Recombinant Antibody Fragments via Fusion to an Engineered Version of 6-O-Alkylguanine DNA Alkyltransferase Florian Kampmeier,†,# Markus Ribbert,†,# Thomas Nachreiner,‡ Sofia Dembski,§ Florent Beaufils,| Andreas Brecht,| and Stefan Barth*,†,⊥ Fraunhofer Institute for Molecular Biology and Applied Ecology, Forckenbeckstrasse 6, 52074 Aachen, Germany, Institute for Neuropathology, University Hospital, RWTH Aachen University, Pauwelsstrasse 30, 52074 Aachen, Germany, Fraunhofer-Insitut fu¨r Silicatforschung ISC, 97082 Wuerzburg, Germany, Covalys Biosciences AG, Benkenstrasse 254, CH4108 Witterswil, Switzerland, and Department of Experimental Medicine and Immunotherapy, Helmholtz Institute for Biomedical Engineering, RWTH Aachen University, Pauwelsstrasse 20, 52074 Aachen, Germany. Received January 21, 2009; Revised Manuscript Received March 13, 2009
Recombinant antibodies are promising tools for a wide range of bioanalytical and medical applications. However, the chemical modification of such molecules can be challenging, which limits their broader utilization. Here we describe a universal method for the site-specific labeling of antibody fragments and protein ligands by genetically fusing them to an engineered version of the human DNA-repair enzyme O(6)-alkyllguanine DNA alkyltransferase (AGT), known as SNAP-Tag (1-3). Substrates containing O(6)-benzylguanine are covalently bound to the fusion proteins via a stable thioether bond in a rapid and highly specific self-labeling reaction. The coupling is sitedirected, allowing the design and synthesis of antibody conjugates with predefined stoichiometry. We cloned a series of ligand SNAP-Tag fusion proteins and expressed them in HEK 293T cells. The antibody/ligand-fusions were characterized by labeling with different fluorophores, labeling with biotin, or by coupling them to fluorescent nanobeads, followed by analysis by flow cytometry and confocal microscopy. All ligands retained their original antigen-binding properties when fused to the SNAP-Tag. The combination of recombinant antibodies or protein ligands with the SNAP-Tag facilitates simple and efficient covalent modification with a broad range of substrates, thus providing a useful and advantageous alternative to existing coupling strategies.
INTRODUCTION Recombinant antibodies and protein ligands have many applications in nanobiotechnology, analytical assays, diagnosis, and therapy, but there is an increasing demand for efficient strategies to further functionalize these proteins. Available methods often rely on the random modification of sulfhydryl, amine, or carboxyl groups and thus have unpredictable side effects on the stability, solubility, and affinity of the modified proteins. Directed coupling via carbohydrate residues works well for many full-size antibodies, but this approach is not easily transferred to other recombinant proteins, particularly antibody fragments that lack glycan chains. Genetic engineering can be used to introduce or remove additional reactive groups, such as cysteine residues, but this requires laborious screening procedures to identify suitable positions on the antibody for modification, and complex chemistry may be necessary to achieve site-specific coupling (4-7). An alternative method for the site-specific labeling of proteins is the introduction of short peptide tags that function as label acceptor sites. Here the label is attached either through an intrinsic activity of the tag or the activity of an enzyme supplied in trans. Site-specific biotinylation, as mediated by the biotin holoenzyme synthetase BirA, * Corresponding author. Phone: +49-241-6085.11060; fax: +49241-6085.10000; e-mail:
[email protected]. † Fraunhofer Institute for Molecular Biology and Applied Ecology. ‡ Institute for Neuropathology, RWTH Aachen University. § Fraunhofer-Insitut fu¨r Silicatforschung ISC. | Covalys Biosciences AG. ⊥ Helmholtz Institute for Biomedical Engineering, RWTH Aachen University. # These authors contributed equally to this work.
allows tagged proteins to be combined with a broad range of avidin/streptavidin reagents but does not allow direct labeling (8-10). Proteins fused to the LUMIO-tag (a hexapeptide containing a tetracysteine motif) can be stained directly with dyes containing two arsenic atoms, but the modification is noncovalent and thus far limited to a small number of fluorescent dyes (11, 12). In addition the significant toxicity of arsenic prevents the application of this technology in vivo. When using recombinant antibodies for analytical, diagnostic, or therapeutic applications, it is useful to have a broad range of modification options available, facilitating the adaptation of particular antibodies to suit diverse purposes. The SNAP-Tag technology allows us to equip a given fusion partner with many different dyes, with biotin, or with other effector molecules. It also facilitates coupling to nanoparticles and directed immobilization on chemically modified surfaces. The Tag is derived from the human DNA-repair enzyme O(6)-alkylguanine DNA alkyltransferase (AGT) (1). It rapidly, specifically, and covalently binds substrates that contain O(6)-benzylguanine. The SNAP-Tag reacts with para-substituted O(6)-benzylguanine (BG) derivatives by transferring the substituted benzyl group to its active site through a nucleophilic substitution reaction while releasing free guanine. The technology has been applied in a variety of experimental systems, ranging from in-cell labeling of tagged proteins to the immobilization of proteins on chip surfaces (3, 13). We cloned and expressed a series of single-chain antibodies (scFv) and natural protein ligands as SNAP-Tag fusions, to develop a platform for the directed modification of these molecules (Figure 1). The fusion proteins were expressed efficiently in 293T and CHO cells and retained the antigenbinding properties of their parent ligands. To demonstrate the versatility of the method, we present fluorescence-activated cell
10.1021/bc9000257 CCC: $40.75 2009 American Chemical Society Published on Web 04/23/2009
scFv-SNAP-Tag Fusions for Site-Specific Labeling
Figure 1. Site-directed modification of recombinant antibody fragments mediated by the SNAP-Tag. Para-substituted O(6)-benzylguanine (BG) derivatives are bound by transfer of the substituted benzyl group to the active site of the SNAP-Tag through a nucleophilic substitution reaction while releasing free guanine. Small compounds like fluorophores as well as nanometer- to micrometer-sized particles can be bound without affecting the antibody or protein ligand activity.
sorting (FACS) and confocal microscopy data for an anti-CD30 scFv and its natural ligand CD30L labeled with different dyes, labeled with biotin and coupled to fluorescent silica beads.
EXPERIMENTAL PROCEDURES Bacterial Strains, Oligonucleotides, and Cloning. All plasmids were prepared using E. coli strain XL1-blue (supE44 hsdR17 recA1 endA1 gyrA46 thi relA1 lacF0[proAB+ lacIq lacZDM15 Tn10(tetr)]). Molecular cloning steps were performed by standard methods. Synthetic oligonucleotides were supplied by MWG (Martinsried, Germany). The source plasmid containing the SNAP-Tag sequence was provided by Covalys (Witterswil, Switzerland). Single Chain Antibodies and Ligands. The ligands used to generate the SNAP-Tag fusions have been described (14-18). The CD30 receptor is highly overexpressed on Hodgkin Reed-Sternberg cells. The human CD30L as well as scFvKi4, a high affinity antibody fragment generated from a monoclonal anti-CD30 antibody, has been shown to target L540 Hodgkin lymphoma cells efficiently (19, 20). Construction of Mammalian Expression Vectors. We used a modified version of the pSecTac vector (21) and introduced the SNAP-Tag sequence to generate both N-terminal and C-terminal fusions. An internal SfiI restriction site was removed by using splice overlap PCR to introduce two silent mutations, allowing scFv fragments and other fusion partners to be introduced by standardized SfiI/NotI restriction. Culture and Transfection of HEK-293T Cells. HEK-293T cells were transfected with RotiFect (Carl Roth GmbH; Karlsruhe, Germany) using 1 µg of plasmid DNA and 3 µL of RotiFect, according to the manufacturer’s instructions. The expression and functionality of each fusion protein was tested 3 days after transfection by FACS analysis of the culture supernatant, using an antipenta-HisAlexa488 antibody (Qiagen). SNAP-Tag activity was tested by staining 10 µL of supernatant with a BG derivative modified with carboxyfluorescein (Vista-Green, Covalys) followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and visualization of the labeled protein with a UV-transilluminator. Further subcultivation of transfected cells and expression of the recombinant SNAP-Tag
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fusion proteins were carried out as previously described (21). Established transfected cultures were grown in RPMI 1640 medium supplemented with 10% (v/v) heat inactivated fetal calf serum (FCS), 50 µg/mL penicillin, 100 µg/mL streptomycin, 2 mM L-glutamine, and 100 µg/mL Zeocin (all components were obtained from Invitrogen, Carlsbad, CA). For the production of larger amounts of protein, triple flasks were inoculated and 500-1000 mL of supernatant was collected for purification. Protein Purification. His-tagged proteins were purified from cell-free supernatants by Ni-NTA metal affinity chromatography as described (21). Larger volumes were purified on an ¨ kta FLPC system with a 5 mL Ni-NTA Superflow cartridge A (Qiagen) after equilibration with 4× buffer (200 mM NaH2PO4, 1.2 M NaCl, 40 mM imidazole, pH 8). After elution, proteins were dialyzed against phosphate-buffered saline (PBS) containing 1 mM dithioerythritol. After concentration to 0.5-1 mg/ mL (if necessary) with a 5 kDa MWCO VivaSpin Column (Sigma-Aldrich), ectoine was added as cryopreservative (22) to a final concentration of 50 mM and aliquots were stored at -20 °C until further use. Protein Labeling with BG Derivatives of Organic Fluorophores or Biotin. Purified SNAP-Tag fusion proteins were conjugated with the BG-modified dyes (Covalys AG, Witterswil) by incubation in the dark with 1.5-3-fold molar excess of dye for 2 h at room temperature or overnight at 4 °C. Residual dye was removed by gel filtration chromatography using PD10 columns (GE) or dialysis. Alternatively, the conjugation was performed with the protein still bound to the Ni-NTA resin with one bed volume of a 2 µM dye solution in 1× Ni-NTA wash buffer (300 mM NaCl, 50 mM sodium phosphate, pH 7.5). The labeled protein was eluted with 500 mM imidazole after washing away residual dye. Coupling efficacy was determined photometrically using the extinction coefficients of the corresponding dyes and the theoretical extinction coefficient of the fusion protein. Stained proteins were visualized after separation by SDS-PAGE with either a UV transilluminator (BioRad Gel Doc XR gel documentation) or with a CRi Maestro imaging system using the blue, red, and near-infrared filter sets. Biotinylation was performed by incubating the fusion proteins with a 2-5 fold molar excess of BG-Biotin using the same procedure described above. Coupling SNAP-Tag Fusion Proteins to Luminescent Silica Beads. We used spherical silica nanoparticles labeled with carboxyrhodamine to demonstrate coupling of the antibodies. The particles were prepared using a modified Sto¨ber synthesis method (23) and had an average diameter of 90 nm. Amine functions were introduced at the bead surface using bifunctional aminosilane (24). The average number of amino functionalities on the bead surface was determined with the fluorescamine test and adjusted to ∼320 functions per bead as described (25). To facilitate benzylguanine coupling, the beads were dispersed in dimethylformamide (DMF) and incubated with a 60-fold molar excess of BG-NHS (Covalys) over amino groups for 3 h at room temperature. The beads were washed three times with PBS (pH 7.4) and dispersed in the same buffer. For coupling, beads where dispersed in RPMI containing 10% FCS and 0.1% Tween 20 (R10-T) and sonicated to disrupt aggregates. Approximately 0.2 nmol of BG-beads was incubated with 50 µg of Ki4-SNAP (1 nmol) in 100 µL of R10-T for 2 h at room temperature. Coupled beads were washed four times with R10-T and dispersed in 200 µL of RPMI-10% FCS, and 60 µL of the bead preparation was used for analysis by flow cytometry and confocal microscopy. To block SNAP-Tag mediated coupling, Ki4-SNAP was incubated beforehand with a 3-fold molar excess of bromothenylpteridine (BTP, Covalys) for 1 h at room temperature.
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Figure 2. Expression cassettes for C-terminal (A) and N-terminal (B) SNAP-Tag fusions. The coding sequences for the protein ligands are introduced using SfiI/NotI either upstream or downstream of the SNAP26m gene, modified to delete an internal SfiI site. The fusion proteins are secreted into the culture supernatant of transfected cells and can be purified via the C-terminal His-tag.
Flow Cytometry. The labeled fusion-proteins were analyzed by flow cytometry with a FACSCalibur (Becton & Dickinson) and CellQuest software. The CD30+ cell line L540 was used as the target for scFv Ki4-SNAP and SNAP-CD30L, whereas the CD30- histiocytic lymphoma cell line U937 was used as control. Approximately 4 × 105 cells were incubated in 300 µL of PBS containing 1 ng to 1 µg of labeled protein for 20 min on ice. For detection of biotinylated proteins, cells were washed and incubated with 1.5 µg/mL StreptavidinDyLight488 for 20 min on ice. The cells were then washed twice with 1.8 mL of PBS in a conventional cell washer. Confocal Microscopy. All images were taken with a LEICA confocal microscope (TCS SP5). Cells were prepared as described above for flow cytometry. The cells were resuspended in 30 µL of PBS, and 10 µL of the cell suspension was incubated for 5 min with 2 µL of a 1:100 diluted Draq5 solution (Biostatus, Shepshed, UK) to counterstain the nucleus.
Figure 3. Purified SNAP-Tag fusion proteins labeled with different fluorophores. (1) scFv425-SNAP-782; (2) scFvKi4-SNAP-VistaGreen; (3) SNAP-EGF-Alexa Fluor647; (4) SNAP-CD30L-505 (glycosylation leads to band broadening and an increase in size); (5) scFvH22-SNAPTMR-star. Approximately 1 µg of labeled protein was separated by SDS-PAGE and visualized with a CRi Maestro Imaging System (lower part) and then stained with coomassie brilliant blue (upper part). Different dye spectra were unmixed using Maestro software.
RESULTS Expression of SNAP-Tag Fusion Proteins in HEK 293 Cells. We cloned and expressed several N- and C-terminal SNAP-fusion proteins by transfecting 293T cells and selecting transformants on the basis of Zeocin resistance and reporter GFP fluorescence. To facilitate cloning, the internal SfiI restriction site in the vector was removed by splice overlap PCR, and the modified SNAP-Tag sequence was inserted into different pMS vectors for the generation of C- and N-terminal fusions (Figure 2). A selection of the functionally expressed constructs is shown in Table 1. Ki4-SNAP and SNAP-EGF expression was also assessed in CHO-K1 cells. Active protein could be purified from the culture supernatant, but more intensive selection and screening was required and expression levels as achieved with the 293T cells were not reached. The SNAP-Tag was active irrespective of the position in the fusion protein. The single chain antibodies were usually used in the C-terminal position, in accordance with other fusion proteins based on these ligands. In contrast, the ligands CD30L and EGF were functional only with the SNAP-Tag at the N-terminal position. High expression levels were observed for most of the fusion proteins, e.g., up to
Figure 4. FACS analysis with Ki4-SNAP and SNAP-CD30L. (A) Increasing amounts of Ki4-SNAP labeled with BG-Alexa Fluor647 on L540 (CD30+) and U937 (CD30-) cells. (B) SNAP-CD30L labeled with BG-biotin and detected with StreptavidinDyLight488.
17.5 mg/L culture supernatant for scFv 425-SNAP, and 5-10 mg/L for the others (Table 1). Comparisons of the fusion proteins and their parental counterparts indicated that the SNAPTag did not have a negative impact on expression levels. Stability was not tested in detail for every fusion protein, but scFv Ki4-SNAP showed no significant loss of binding activity
Table 1. Selection of Successfully Expressed Ligand-SNAP-Tag Fusion Proteins fusion protein scFv Ki4-SNAP (49 kDa) SNAP-CD30L (41 kDa + glycosylation) scFv 425-SNAP (48 kDa) SNAP-EGF (28 kDa) SNAP-Myelin Oligodendrocyte Glycoprotein (MOG) (38 kDa) Annexin V-SNAP (59 kDa) scFv H22-SNAP (48 kDa) scFv RFT5-SNAP (48 kDa)
target
tag position
expression rate in 293T cells
binding confirmed on
CD30 CD30 EGFR EGFR anti MOG Ig
N-term. C-term. N-term. C-term. N-term.
>10 mg/L ∼5 mg/L >15 mg/L >10 mg/L >10 mg/L
L540 L540 A431/L3.6pl A431/L3.6pl 8.18-C5 murine hybridoma cells
Phosphatidyl-serine CD64 CD25
N-term. or C-term. N-term. N-term.
nd ∼ 8 mg/L nd
apoptotic L3.6pl cells U937 L540
scFv-SNAP-Tag Fusions for Site-Specific Labeling
Figure 5. Confocal microscopy of Ki4-SNAP and SNAP-CD30L on CD30+ L540 cells. (A) Ki4-SNAP labeled with BG-505. 1) 505 fluorescence signal, 2 ) nucleus stained with Draq5, 3 ) transmission light picture, 4 ) overlay of 1-3. (B) SNAP-CD30L labeled with BGAlexa Fluor 647. One ) Alexa Fluor 647 fluorescence signal, 2 ) overlay of fluorescence and transmission light picture. The high CD30 expression on the L540 cell surface results in a strong membrane staining mediated by the Ki4 single chain antibody, as well as the natural human CD30L, fusion protein.
or SNAP-Tag activity when concentrated at ∼1 mg/mL and stored at 4 °C for 4 weeks. All the constructs we tested were still active after at least 4 months when stored at -20 °C. Labeling of SNAP-Tag Fusion Proteins with Organic Fluorophores. Figure 3 shows a selection of five fusion proteins labeled with different fluorophores and stained with Coomassie Brilliant Blue after separation by SDS-PAGE. Coupling efficiencies were independent of the SNAP-Tag fusion partner. We achieved rates of 90-100% efficiency after overnight incubation using different constructs and different dyes (BGAlexa Fluor647, BG-TMR-star, BG-505 and BG-Vista-Green, BG-782), and in most cases 60-70% efficiency after 1 h at
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room temperature. A 1.5-fold molar excess of dye was sufficient to achieve these labeling efficiencies. Confocal Microscopy and FACS Analysis. To evaluate the activities of the different fusion proteins, we carried out FACS analyses with proteins purified from the supernatant of transfected 293T cells and stained with various BG-modified fluorescent dyes. All the labeled proteins we tested gave a strong fluorescence signal on the corresponding target cell line but not on control cells. To exemplify these results, FACS and confocal microscopy data are shown for the anti-CD30 scFv Ki4-SNAP and the corresponding human ligand SNAP-CD30L. For Ki4SNAP labeled with BG-Alexa Fluor647, less than 15 ng/mL was sufficient to detect binding to target cells by flow cytometry (Figure 4A). Confocal microscopy of Ki4-SNAP labeled with BG-505 confirmed homogeneous membrane staining on L540 cells (Figure 5A). A similar staining pattern was observed with the natural ligand of CD30, CD30L, labeled with Alexa Fluor647 (Figure 5B). We also used BG-biotin to label the fusion proteins, and FACS analysis of cells treated with biotinylated CD30L followed by StreptavidinDyLight 488 is shown in Figure 4B. SNAP-Tag Coupling to Silica Beads. To test the versatility of the coupling procedure, we used carboxyrhodamine-labeled silica beads and modified them with an N-hydroxysuccinimideester derivativeof BG (BG-GLA-NHS) via surface amino groups. The anti-CD30 scFv Ki4 was coupled to these beads, and the labeling and binding properties were analyzed by FACS (data not shown) and confocal microscopy. The bead conjugates showed specific labeling of CD30+ L540 cells (Figure 6 A) while only minimal background was observed when the SNAPTag was blocked with bromothenylpteridine (Figure 6 B). No binding of the beads was observed on CD30- U937 cells (not shown). The multivalency of the conjugated beads led to the
Figure 6. Confocal microscopy with Ki4-SNAP coupled to benzylguanine-modified fluorescent beads on L540 cells. (A) Ki4-SNAP-coupled beads. (B) Beads incubated with Ki4-SNAP with the SNAP-Tag previously blocked with bromothenylpteridine. In each panel, 1 ) signal from carboxyrhodamine silica beads, 2 ) nucleus stained with Draq5, 3 ) transmission light picture, and 4 ) overlay of 1-3. The Ki4-SNAP coupled beads bind to and lead to aggregation of the L540 target cells while only minimal attachment of bead aggregates is observed with the blocked Ki4-SNAP-Tag.
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formation of cell aggregates, linking several cells via their surface receptors.
DISCUSSION To demonstrate the feasibility of directed coupling of protein ligands and antibodies using the SNAP-Tag method, we cloned and expressed a series of SNAP-Tag fusion proteins containing either a single chain antibody fragment or a corresponding natural ligand directed against well-characterized tumor markers. All constructs listed in Table 1 retained both the binding activity of the parental molecule and SNAP-Tag activity. The fact that such activity could be demonstrated for a range of different ligand formats, the extracellular domains of membrane glycoproteins (CD30L, MOG), a secreted peptide ligand (EGF), and engineered antibody fragments, provides ample evidence for the broad applicability of this method. SNAP-Tag fusions have previously been expressed in E. coli, yeast, and mammalian cells (13, 26) but never with the intention of producing large amounts of a reagent for further applications. Expression in 293T cells provides a convenient system that allows rapid screening of the fusion proteins, since 293T cells are easy to transfect and expression of the target protein can be assessed directly by correlation with eGFP expression levels (21). The average yield of purified fusion protein was 10 mg/L supernatant (Table 1), and this was achieved without further enrichment for highperformance clones, indicating that much higher yields should be possible. Several different approaches have been used to engineer antibody fragments for site-specific coupling/labeling, including the introduction of additional cysteine residues at the C-terminus (27) or at the end of each linker connecting the VH and VL chains in a scFv diabody (28). The site-specific immobilization of a scFv fusion protein by transglutaminase has also been reported (29) as well as the directed biotinylation of different antibody fragments by coexpression with the bacterial biotin ligase BirA in HEK293 cells (30). Despite these successes, it would be advantageous to increase the versatility of site-specific labeling systems to allow diverse labels to be used with the same parental protein. Labeling with the SNAP-tag is advantageous because large quantities of very pure ligands can be produced and made directly available for coupling, and the activity of the ligand is not affected by the fusion or by coupling to a substrate. Furthermore, conjugation is covalent, stable, is site-specific, and has a predefined stoichiometry. The approach is not limited to a single class of recombinant proteins but is applicable to a wide range of protein ligands. The targeted delivery of nanoparticles for imaging and drug delivery has become a very active area of biomedical research. Quantum dots, iron oxide or gold nanoparticles, liposomes and polymers have all been equipped with antibodies for targeted delivery in vivo (31-33). The coupling strategy described here enables the efficient modification of such particles with recombinant proteins, as demonstrated for BG-functionalized silica nanobeads. By genetically fusing antibody fragments and other targeting ligands to the SNAP-Tag, we have developed and evaluated a new platform for the directed modification of diverse protein molecules. A series of different ligands has been cloned, expressed, and successfully labeled with several fluorophores, biotin, and silica nanoparticles, showing the versatility of the approach. Experimental data is shown for a single chain antibody fragment directed against CD30 and the natural human ligand CD30L. This new platform could have a significant impact on the labeling of recombinant proteins used as reagents in
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nanobiotechnology, molecular biology, and cell biology, as well as proteins used in analytical assays, disease diagnosis, and therapy.
ACKNOWLEDGMENT We would like to thank Dr. Richard Twyman for the critical reading of the manuscript.
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