- Methodology article
- Open Access
Efficient, chemoselective synthesis of immunomicelles using single-domain antibodies with a C-terminal thioester
© Reulen et al; licensee BioMed Central Ltd. 2009
- Received: 15 April 2009
- Accepted: 20 July 2009
- Published: 20 July 2009
Classical bioconjugation strategies for generating antibody-functionalized nanoparticles are non-specific and typically result in heterogeneous compounds that can be compromised in activity. Expression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester, providing a unique handle for site-specific conjugation using native chemical ligation (NCL). However, current methods to generate antibody fragments with C-terminal thioesters require cumbersome refolding procedures, effectively preventing application of NCL for antibody-mediated targeting and molecular imaging.
Targeting to the periplasm of E. coli allowed efficient production of correctly-folded single-domain antibody (sdAb)-intein fusions proteins. On column purification and 2-mercapthoethanesulfonic acid (MESNA)-induced cleavage yielded single-domain antibodies with a reactive C-terminal MESNA thioester in good yields. These thioester-functionalized single-domain antibodies allowed synthesis of immunomicelles via native chemical ligation in a single step.
A novel procedure was developed to obtain soluble, well-folded single-domain antibodies with reactive C-terminal thioesters in good yields. These proteins are promising building blocks for the chemoselective functionalization via NCL of a broad range of nanoparticle scaffolds, including micelles, liposomes and dendrimers.
- Antibody Fragment
- Periplasmic Fraction
- Native Chemical Ligation
The ability to raise antibodies with high affinity and specificity to almost any biomolecular target has made antibodies essential components in many biomedical fields, both in diagnostics and in the active targeting of drugs and contrast agents for molecular imaging . For many of these applications there has been a drive to move towards smaller antibody formats, both to allow efficient recombinant production in E. coli and to potentially avoid unwanted immunogenic problems . The ability to express these smaller antibody fragments in E. coli has also allowed the application of phage display approaches to allow in vitro screening of large libraries of antibody fragments. Nowadays, a wide range of smaller antibody formats are available including monovalent antibody fragments (Fab), single-chain antibody fragments (scFv), and single-domain antibodies (sdAb) . The latter, which are sometimes also referred to as nanobodies, are derived from heavy-chain-only antibodies that have been found in camels, dromedaries, llamas and sharks [3, 4]. Single-domain antibodies are the smallest antibody fragments available to date and have unique features including high solubility and thermal stability .
Current methods for bioconjugation of antibody fragments are non-specific and usually rely on amine and cysteine functionalities present on the protein surface . This lack of control over the conjugation reaction gives rise to heterogeneous protein-nanoparticles. Moreover, the smaller size of single-domain antibodies compared to full size antibodies significantly increases the risk of affecting key residues near the antigen binding site when using non-specific conjugation strategies. In recent years several bioorthogonal ligation reactions that were originally developed in peptide chemistry have been applied for chemoselective protein functionalization of nanoparticles and chip surfaces [6–14]. Two examples of antibody conjugation using oxime chemistry were recently reported that take advantage of novel methods to selectively oxidize the N-terminus of antibodies or introduce genetically-encoded aldehyde tags at any position in the antibody sequence [15, 16]. While promising, the applicability of oxime chemistry is still hampered by the incomplete introduction of ketone functionalities and the inability to use N-terminal acetylated proteins [17, 18]. We and others have therefore explored the use of native chemical ligation (NCL) as an alternative chemoselective conjugation reaction, demonstrating its potential for the ligation of proteins to chip surfaces, dendrimers, supported lipid bilayers, micelles and liposomes [6, 11, 12, 19–23]. Native chemical ligation is a chemoselective reaction under aqueous conditions between a C-terminal thioester and an N-terminal cysteine yielding a native peptide bond . Site-specific coupling via NCL was made possible by the development of expression systems with self-cleavable intein domains to generate recombinant proteins with C-terminal thioesters .
Intein fusion proteins are normally expressed in the cytoplasm of E. coli, a reducing environment that prevents the proper formation of disulfide bonds that are essential for antibody stability. In vitro refolding of scFv-intein fusion proteins followed by on-column NCL has been reported , but the requirement to perform NCL on the column limits the applicability of this method. We recently reported a refolding procedure based on the redox couple sodium 2-mercaptoethanesulfonate (MESNA)/sodium 2,2-dithio-bis(ethanesulfonate) (diMESNA) to generate disulfide-containing proteins with a C-terminal MESNA thioester . However, also for this method the requirement to do in-vitro refolding presents an important practical limitation. Here, we present an efficient strategy to obtain well-folded single-domain antibodies with a reactive C-terminal thioester by targeting the intein fusion protein to the periplasm of E. coli. Targeting antibody fragments to the oxidizing environment of the bacterial periplasm is known to increase the amount of active antibody fragments by allowing proper disulfide bond formation. Following this new procedure single-domain antibodies with C-terminal thioesters are obtained that can be directly coupled to cysteine-functionalized micelles to generate immunomicelles via native chemical ligation.
Production of single-domain antibodies with a C-terminal thioester
Figure 2 shows the purification of the MESNA thioester of sdAb-aGST (sdAb-aGST-MESNA) starting form the periplasmic fraction containing the sdAb-aGST-intein-CBD fusion protein. Upon loading the periplasmic fraction on a chitin column the sdAb-aGST-intein-CBD fusion protein binds to the chitin resin. The single-domain antibody with a C-terminal MESNA thioester was eluted from the chitin column after overnight incubation with the thiol MESNA. Yields for thioester terminated single-domain antibodies varied between 2 – 5 mg/L, which compares favorably with the 0.1–6 mg/L yields typically reported for single-domain antibodies via periplasmic expression in E. coli . Since SDS-PAGE analysis showed the presence of two bands of slightly different molecular weight around 18 kDa, we performed LC-MS analysis to characterize the obtained proteins (Figure 2B). The main peak at 5.5 min contained sdAb-aGST-MESNA with the pelB leader attached (pelB). This peak also contained sdAb-aGST-MESNA with the first amino acid of the pelB leader removed (pelBΔ1). The minor peak at 5 min contained two sdAb-aGST-MESNA protein species with the first 14 and 15 amino acids of the pelB leader removed. The double band visible on the SDS-PAGE gel thus corresponds to multiple species of sdAb-aGST, all of which contain a C-terminal MESNA thioester. Addition of protease inhibitors during the purification procedure could not prevent this heterogeneous N-terminal processing. Similar incomplete pelB removal was observed before in E. coli BL21(DE3) strains and has been suggested to be due to the inability of its amino peptidase to efficiently cleave the pelB leader peptide . The minor band observed on the SDS-PAGE gel around 70 kDa was identified using LC-MS as DnaK, a molecular chaperone protein that prevents aggregation of misfolded proteins in E. coli.
Preparation of immunomicelles with single-domain antibodies using NCL
An efficient strategy was developed that generates active, folded single-domain antibodies with a reactive C-terminal thioester for direct application in nanoparticle functionalization via native chemical ligation. Two applications were reported here to illustrate their potential as generic building blocks for the generation of targeted nanoparticles, the generation of biotinylated single-domain antibodies and the efficient preparation of immunomicelles. Finally, the approach of targeting intein fusion proteins to the bacterial periplasm may provide a generic, attractive alternative to existing methods to obtain disulfide-containing proteins with a C-terminal thioester.
Unless stated otherwise, all reagents and chemicals were obtained from commercial sources and used without further purification. Cysteine-functionalized 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(poly(ethylene glycol))2000] (Cys-PEG-DSPE) was prepared according to a literature procedure . The expression vectors pTXB1 and pTYB1, restriction enzymes and chitin beads were purchased from New England Biolabs (Beverly, MA). Competent cells were purchased from Novagen (Darmstadt, Germany). Monoclonal mouse anti-vsv glycoprotein antibody (V5507) was purchased from Sigma (St. Louis, MO). HRP-conjugated rabbit anti-mouse immunoglobulin polyclonal antibody (P0260) was purchased from DakoCytomation (Heverlee, The Netherlands). UV-Vis spectra were recorded on a Shimadzu Multispec 1501 spectrophotometer. Primers used for all the cloning procedures were supplied by MWG (Ebersberg, Germany).
The GST-specific single-domain antibody was obtained from a naïve llama-derived nanobody library described previously . Selection of phages for binding to bacterial expressed recombinant GST (pGEX-4T-2 vector) was performed essentially as described in [35, 36]. The gene encoding for sdAb-aGST was amplified by PCR from vector pHENIX sdAb-α GST C11 with the primer pair pUC119pelB_f (5'-GGTGGTCATATGAAATACCTATTGCCTACGGCAGC-3') and pUC119vsv_r (5'-GGTGGTTGCTCTTCCGCATGCGGCCCCCTTTCCAAG-3'). The PCR products and the pTXB1 and pTYB1 vectors were double-digested with the restriction endonucleases Nde I and Sap I followed by ligation of the amplified DNA fragments in the open plasmids yielding the expression plasmids pTXB1-sdAb-aGST and pTYB1-sdAb-aGST. DNA sequencing (BaseClear, Leiden, The Netherlands) using T7 promoter and intein-specific reversed primers (New England Biolabs) confirmed the correct in-frame fusion of the proteins with the intein sequence.
Protein Expression and Purification
The expression plasmids were transformed into E. coli BL21 (DE3) cells. Expression experiments were performed in 0.5 L LB cultures supplemented with 100 μg/mL ampicillin by inoculating with a 5 mL overnight culture. The cultures were grown at 37°C and 250 rpm until the OD600 reached a value between 0.5–0.6. Protein expression was induced with 0.2 mM IPTG and cultures were incubated for 3 hours at 30°C. Cells were harvested by centrifugation for 10 min at 8,000 g at 4°C, after which the medium was discarded and the cell pellets were resuspended in 85 mL TES (30 mM Tris-HCl pH 8.0, 1 mM EDTA, 20% (w/v) sucrose) and incubated at room temperature for 10 minutes with continuous shaking. After centrifugation for 10 min at 8,000 g and 4°C, the pellet was resuspended in 85 mL of ice-cold 5 mM MgSO4 and incubated on ice for 10 minutes with occasional shaking. After centrifugation for 10 min at 8,000 g at 4°C, 2 mL 0.5 M Tris-HCl pH 7.5 was added to the supernatant. ATP (3 mM) was added to the supernatant and incubated for 1–3 hours at 4°C to suppress binding of DnaK to the sdAb-aGST intein fusion protein. The periplasmic protein fraction was directly applied to a chitin column that was equilibrated with 10 column volumes of column buffer (20 mM sodium phosphate, 0.1 mM EDTA, 0.5 M NaCl, 3 mM ATP, pH 8). The column was washed with 10 volumes of column buffer after which the columns were quickly flushed with 3 column volumes of cleavage buffer (20 mM sodium phosphate, 0.1 mM EDTA, 0.5 M NaCl, 100 mM MESNA, pH 6) and incubated overnight at 4°C. Proteins were eluted in cleavage buffer without MESNA and pooled. The protein concentration was determined by UV-Vis using ε280 nm = 24,535 M-1cm-1 or using a Quant-it protein assay (Invitrogen) according to the manufacturers instructions.
Samples for LC-MS analysis were concentrated with Biomax centrifugation filters (MWCO 5 kDa) for small volumes and Amicon centrifugational filters (MWCO 10 kDa) for large volumes. Samples were buffer exchanged to a mixture of 9:1 H2O/acetonitrile or to 50 mM ammonium acetate pH 6.8. Reversed phase HPLC was performed on a Vydac protein column with a mobile phase of H2O/acetonitrile with 0.1% TFA. ESI-MS spectra were measured on a Thermo Finnigan LCQ Deca XP MAX in positive mode. MagTran software was used for deconvolution.
All washing steps were performed with 200 μl MPBST (5% milk powder, 0.05% Tween20 in PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.4)) unless stated otherwise. 96 wells Corning EIA/RIA Microplates were coated overnight at 4°C with 0.5 μg/well (50 μl) GST or BSA in 0.1 M NaHCO3 pH 8.6. After overnight incubation the plates were washed 3 times with PBS/0.05% Tween20. Plates were blocked with 50 μl MPBST for 1 hour at room temperature. After washing the plates 3 times, 50 μl samples of sdAb-aGST-MESNA were loaded and incubated for 1 hour at room temperature. Plates were washed 6 times and subsequently incubated with 50 μl mouse Anti-VSV monoclonal antibody (5000× diluted) for 1 hour. Plates were washed 6 times and subsequently incubated with 50 μl alkaline HRP-conjugated rabbit anti-mouse immunoglobulin polyclonal antibody (2500× diluted), for 1 hour. Plates were washed 6 times and subsequently incubated with 50 μl/well detection solution (1:1 peroxide buffer (UP)/Tetramethylbenzidine (TMB), BioMérieux, Boxtel, The Netherlands). Reaction was stopped after 5 to 10 min with 50 μl 2 M sulfuric acid. The absorbance was measured at 450 nm in duplicate on a Thermo multiskan ascent platereader.
Solid phase binding assay with immunomicelles
All washing steps were performed with 200 μl HBS. 96 wells Corning EIA/RIA Microplates were coated overnight at 4°C with 0.5 μg/well (50 μl) GST or milk powder in HBS (10 mM HEPES, 135 mM NaCl, pH 7.4). After overnight incubation the plates were washed 3 times. Plates were blocked with 50 μl MHBS (5% milk powder in HBS) for 3 hours at room temperature. After washing the plates 3 times, 50 μl samples of sdAb-aGST micelles were loaded and incubated for 4 hours at room temperature. Plates were washed 8 times after which the fluorescence of the rhodamine-containing micelles was measured in duplicate on a Thermo Fluoroskan Ascent FL plate reader (excitation at 578 nm, emission at 620 nm).
Surface Plasmon Resonance
Sensorgrams were obtained on a Biacore T100 (GE Healthcare) using a CM5 chip functionalized with GST using standard EDC/NHS protocols. All binding experiments were performed at 25°C using PBS with 0.05% Tween-20 (PBST) as running buffer at a flow rate of 30 μL/min. Samples containing 0–500 nM of sdAb-aGST-MESNA in PBST were injected on a CM5 chip functionalized with GST (160 RU immobilized) for 400 sec, followed by a 10 min dissociation phase. Regeneration of the chip was performed with a 30 s injection of 10 mM NaOH. Aspecific binding and buffer effects were taken into account by subtracting the simultaneous response from a reference surface functionalized with ethanolamine.
Ligation of cysteine-functionalized biotin to sdAb-aGST
Cysteine-functionalized biotin was prepared according to a literature procedure and checked with NMR and LC-MS analysis . A reaction mixture was prepared containing 7 μM sdAb-α GST-MESNA in 20 mM sodium phosphate, 0.1 mM EDTA, 0.5 M NaCl, pH 6 to which 1 mM cysteine-functionalized biotin, 50 mM MPAA and 10 mM tris(2-carboxyethyl) phosphine hydrochloride (TCEP) were added. The final pH of the reaction mixture was 5.5. The reaction was incubated overnight at 20°C and analyzed using LC-MS analysis.
Ligation of sdAb-αGST to cysteine-functionalized micelles
A mixture of 97.5 mol percent Cys-PEG-DSPE and 2.5 mol percent rhodamine-DPPE in chloroform was placed in a vial. After chloroform evaporation the obtained lipid film was rehydrated in 0.1 M sodium phosphate pH 8.5 ([lipid] = 640 μM) with 50 mM MPAA and 10 mM TCEP, vortexed for 2 min followed by 5 min sonication. SdAb-aGST-MESNA was added to a final concentration of 50 μM (final pH was 6.7). After overnight incubation at room temperature the protein micelles were analyzed using SDS-PAGE to check for complete conversion to the lipidated protein.
The authors thank Danielle Hof for guidance during the initial expression experiments of the single-domain antibodies, SyMOChem (Eindhoven) for synthesizing the cysteine-functionalized phospholipid, and Bert Meijer for general support. This work was supported by an NWO VIDI grant 700.56.428 to M.M. and the BSIK program entitled Molecular Imaging of Ischemic Heart Disease (project number BSIK03033).
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