Bioconjugate Chem. 2008, 19, 1753–1756
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A Designed Protein for the Specific and Covalent Heteroconjugation of Biomolecules Christopher Chidley, Katarzyna Mosiewicz, and Kai Johnsson* Ecole Polytechnique Fe´de´rale de Lausanne (EPFL), Institute of Chemical Sciences and Engineering, CH-1015 Lausanne, Switzerland. Received July 1, 2008; Revised Manuscript Received August 12, 2008
Bioconjugations often rely on adaptor molecules to cross-link different biomolecules. In this work, we introduce the molecular adaptor covalin, which is a protein chimera of two self-labeling proteins with nonoverlapping substrate specificity. Covalin permits a selective and covalent heteroconjugation of biomolecules displaying appropriate functional groups. Examples for the use of covalin include the specific heteroconjugation of a reporter enzyme to an antibody and of molecular probes to the surface of living cells. The efficiency and specificity of covalin-based bioconjugations together with the availability of a large variety of substrates create immediate and ubiquitous applications for covalin in bioconjugate chemistry.
The specific heteroconjugation of two different biomolecules generally relies on cross-linkers with two independent reactive groups or binding sites. Examples of cross-linkers include bifunctional synthetic molecules, oligonucleotides, and proteins (1-3). Streptavidin is the most widely used protein in bioconjugations, as it can stably conjugate biotinylated molecules with high efficiency (3, 4). However, for the specific heteroconjugation of two different molecules the streptavidin-biotin technology has the limitation that streptavidin possesses four identical binding sites and that its simultaneous incubation with two different biotinylated molecules therefore leads to a heterogeneous mixture of conjugates. Mutants of streptavidin with a reduced number of binding sites have been described (5), but these mutants also do not allow a specific heteroconjugation of two different biotinylated molecules. Here, we introduce a designed protein for the specific and covalent heteroconjugation of two different (bio)molecules displaying appropriate functional groups. Using an antibody or cell surface proteins as examples, we demonstrate how this protein, named covalin, can be used to specifically conjugate proteins to other proteins or to synthetic probes. The simplicity, efficiency, and general applicability of the method should allow it to become an important tool for heteroconjugations in bioconjugate chemistry. To achieve covalent and specific bioconjugations, covalin was designed as a fusion protein of two monomeric, self-labeling proteins with nonoverlapping substrate specificity (Figure 1). The first protein is a mutant of human O6-alkylguanine-DNA alkyltransferase (known as SNAP-tag; 182 residues) that reacts with benzylguanine (BG) derivatives (6). The second protein is a mutant of a bacterial dehalogenase (known as HaloTag; 293 residues) that reacts with primary chloroalkanes (7). The rate constants of the reactions of both proteins with their substrates are high (104 s-1 M-1 for SNAP-tag and 106 s-1 M-1 for HaloTag). This makes them well-suited for bioconjugations, especially when compared to typical bioorthogonal reactions such as copper-free click chemistry or the Staudinger ligation, which are at least 104-fold slower (8, 9). Numerous substrates are available for SNAP-tag and HaloTag (Figure 1b) and both tags have proven their versatility in vitro and in * Corresponding author. E-mail:
[email protected]. Phone: +41-21-6939356. Fax: +41-21-6939365.
vivo (6, 7). Covalin possesses an N-terminal His-tag for purification and a human rhinovirus 3C protease (PreScission protease) cleavage site between SNAP-tag and HaloTag for an optional separation of covalently conjugated biomolecules. Its size (55 kDa) is comparable to that of streptavidin (54 kDa). Although covalin is currently not commercially available, it can be readily produced through overexpression in E. coli (Supporting Information). We first characterized the reactivity of covalin toward simple SNAP-tag and HaloTag substrates. Incubation of covalin with BG-547 and Halo-DAF, substrates for the labeling of SNAPtag and HaloTag with the fluorescent dyes DY-547 and fluorescein, respectively, resulted in the labeling of covalin with both fluorophores (Figure 1c). Digestion of the labeled protein with PreScission protease yielded DY-547-labeled SNAP-tag and fluorescein-labeled HaloTag (Figure 1c), showing that covalin has two independent self-labeling sites. Incubation of covalin with either BG-DAF or Halo-DAF, substrates for the labeling of SNAP-tag and HaloTag with fluorescein, yielded fluorescein-labeled covalins with almost identical fluorescence intensities ((5%; Figure 1c, lane 1 and 2), indicating that in covalin SNAP-tag and HaloTag are active to the same extent. We then used covalin in a series of proof-of-principle experiments for the conjugation of an antibody to a synthetic fluorophore, a reporter enzyme and magnetic beads (Figure 2a). In a first step, this requires a chemical labeling of the antibody with one of the two covalin substrates. Subsequently, the antibody can be functionalized through simultaneous incubation with covalin and the (bio)molecule of interest displaying the other covalin substrate. The first step of the procedure is similar to a chemical biotinylation of an antibody for subsequent bioconjugations with streptavidin. However, a subsequent simultaneous incubation of a biotinylated antibody with streptavidin and other biotinylated objects would inevitably lead to a complex mixture of different products. The commonly used monoclonal antibody 12CA5 that recognizes the HA epitope tag was chosen as the antibody for the bioconjugation experiments. 12CA5 was labeled with primary chloroalkane through incubation of the antibody with a commercially available N-hydroxysuccinimide (NHS) ester (Figure 1b), a labeling strategy that should result in antibodies displaying varying amounts of the HaloTag substrate (1). Derivatized 12CA5 was stored for weeks at 4 °C without
10.1021/bc800268j CCC: $40.75 2008 American Chemical Society Published on Web 08/28/2008
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Figure 1. Covalin-dependent bioconjugations. (a) General scheme for covalin-dependent bioconjugations. An object represents synthetic probes, biomolecules, beads, or cells. (b) Covalin substrates used in this study. (c) SDS-PAGE and laser-based in-gel fluorescence scanning of covalin incubated with: BG-DAF (lane 1); Halo-DAF (lane 2); BG-547 (lane 3); Halo-DAF and BG-547 (lane 4); Halo-DAF, BG-547, and PreScission protease (lane 5). DY-547 and fluorescein fluorescence are shown in red and green, respectively.
Figure 2. Covalin-dependent derivatization of an anti-HA antibody. (a) General scheme for the covalin-dependent conjugation of an antibody to a fluorophore (b), to a reporter protein (c), and to magnetic beads (d). (b) Derivatized anti-HA antibody 12CA5 (3 µM) was incubated with covalin (10 µM) and BG-547 (15 µM). Aliquots of the reaction mixture drawn at indicated time points were analyzed by SDS-PAGE and laser-based in-gel fluorescence scanning. (c) Detection of different dilutions of ACP-HA by Western blotting using 12CA5-covalin-HRP (0.17 µg/mL in 12CA5) or a commercially available 12CA5HRP conjugate (0.15 µg/mL). (d) Analysis of the pulldown experiment using 12CA5 immobilized on magnetic beads via covalin and incubated with an equimolar mixture of Cy3-labeled ACP-HA and ACP-CaM-PCP. Samples of the pulldown were analyzed as in (b). Lane 1, sample before pulldown; lane 2, flowthrough of pulldown; lanes 3 and 4, wash fractions; lane 5, elution of captured proteins from beads.
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Bioconjugate Chem., Vol. 19, No. 9, 2008 1755
Figure 3. Use of covalin for the bioconjugation of molecular probes to cell surfaces. (a) General scheme for the bioconjugation of molecular probes via covalin to cell surfaces. In the first step, cell surfaces could either be derivatized with the SNAP-tag substrate or with the HaloTag substrate, but for reasons of clarity only one of the two orientations is shown. (b-e) Micrographs of derivatized and nonderivatized CHO cells incubated with covalin and a fluorescent covalin substrate. (b) CHO cells derivatized with BG and first incubated with covalin (10 µM) and then with Halo-DAF (2 µM). (c) CHO cells not derivatized with BG and first incubated with covalin (10 µM) and then with Halo-DAF (2 µM). (d) CHO cells derivatized with primary chloroalkane and first incubated with covalin (10 µM) and then with BG-547 (2 µM). (e) CHO cells not derivatized with primary chloroalkane and first incubated with covalin (10 µM) and then with BG-547 (2 µM). Scale bar, 10 µm.
decrease in reactivity toward covalin. To label 12CA5 with a fluorophore, the derivatized antibody (3 µM) was incubated with covalin (10 µM) and BG-547 (15 µM). Aliquots of the reaction mixture drawn at different time points were then analyzed by SDS-PAGE and in-gel fluorescence scanning (Figure 2b). Under these conditions, the fluorescence labeling of 12CA5 with covalin-DY-547 is near completion after one hour and heavy and light chains conjugated to one or multiple covalin-DY-547 could be detected. On average, approximately 2.5 covalin-DY547 were bound per anti-HA antibody (Supporting Information; Figure S1a). This set of experiments serves as a first example to illustrate how covalin can be used for the straightforward conjugation of molecular probes to a derivatized protein. It should be noted that the covalent cross-linking of fluorescent probes to derivatized biomolecules such as 12CA5 allows the quantification of the initial derivatization step before use of the same derivatized biomolecule in further applications (vide infra). One of the main applications that we foresee for the use of covalin is in the selective conjugation of two different proteins to each other, as it resolves the problem to derivatize one of the two proteins with a reactive group that specifically reacts only with the other protein. To demonstrate the potential of covalin for such applications, we attempted to conjugate 12CA5 via covalin to horseradish peroxidase (HRP) and to use the resulting conjugate in Western blotting. HRP is commonly used as a reporter enzyme in immunodetection experiments and HRPantibody conjugates are therefore widely used. Toward this end, HRP was incubated with a BG-NHS ester. Derivatized HRP (3 µM) was incubated with covalin (3 µM) and Halo-DAF (4 µM) to verify that BG-labeled HRP is a substrate of covalin and to determine the degree of labeling of HRP with BG. In these
experiments, 40% of HRP was derivatized with one covalin (Supporting Information; Figure S1b,c). The derivatization of HRP with NHS esters is known to be inefficient due to the low number of amino groups available (1), and no attempts were made to improve the labeling of HRP with BG. To conjugate HRP to the anti-HA antibody, derivatized 12CA5 (3 µM) was incubated with covalin (15 µM) and derivatized HRP (30 µM total HRP) for 5 h and then stored at 4 °C for later use. To evaluate the activity of the self-assembled 12CA5-covalin-HRP, we compared it to a commercially available 12CA5-HRP conjugate (Roche Molecular Biochemicals) optimized for applications in Western blotting. Using recombinant acyl carrier protein with a C-terminal HA tag (ACP-HA) as an arbitrary test antigen, 12CA5-covalin-HRP and the commercially available 12CA5-HRP permitted the detection of similar amounts of ACP-HA in Western blotting (Figure 2c). This demonstrates that the conjugation of HRP to 12CA5 via covalin does neither impede the antibody-antigen interaction nor the HRP activity. While the specific heteroconjugation of antibodies to HRP can be achieved via a number of different procedures using synthetic bifunctional cross-linkers (1), these procedures are not generally applicable to heteroconjugations of proteins, as they chemically exploit the fact that antibodies and HRP are glycosylated and/ or that both proteins in their native form do not possess any free cysteines. This is in contrast to covalin-mediated heteroconjugations of proteins as the SNAP-tag and HaloTag substrates are chemically inert toward the functional groups found in proteins and other biomolecules. Covalin should furthermore allow a selective immobilization of biomolecules on surfaces such as polymer beads as both SNAP-tag (10) and HaloTag (7) have already been successfully
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used for this purpose. We therefore attempted the immobilization of the anti-HA antibody 12CA5 on magnetic beads for pulldown experiments. Primary chloroalkane-derivatized 12CA5 (6 µM) was incubated with covalin (9 µM) and magnetic beads displaying BG. For a mock pulldown experiment, derivatized beads were incubated with an equimolar mixture of ACP-HA and of a similar protein without HA-tag (a fusion protein of ACP with calmodulin and peptidyl carrier protein (ACP-CaMPCP)) which was diluted into a crude cell lysate. For detection, ACP-HA and ACP-CaM-PCP were both labeled via ACP with Cy3 (11) before dilution into the cell lysate. After several washing steps, protein bound to the beads was eluted with SDS sample buffer and samples of different steps of the pulldown were analyzed (Figure 2d). The enrichment of ACP-HA over ACP-CaM-PCP in the pulldown was 80-fold and no enrichment was observed when either derivatized 12CA5 was replaced by original 12CA5 or when covalin was omitted (Supporting Information; Figure S1d). The different experiments with antibody 12CA5 show that once an antibody is derivatized with a covalin substrate it can be either conjugated to various probes or immobilized, providing the approach with a high degree of flexibility. A further application for covalin is the conjugation of (bio)molecules to the surfaces of cells or viruses previously derivatized with appropriate substrates (Figure 3a). For a proofof-principle experiment, we used covalin to conjugate fluorophores to the surface of CHO cells. CHO cells were first derivatized either with primary chloroalkane or with BG by a brief incubation of the cells with the corresponding NHS ester. Both NHS esters were utilized in order to test covalin in both orientations. The derivatized CHO cells were subsequently incubated first with covalin (10 µM) and then with either BG547 or Halo-DAF (each 2 µM) for visualization. Labeling of derivatized cells with either DY-547 or fluorescein was detectable by fluorescence microscopy, whereas no labeling could be detected when nonderivatized CHO cells were incubated with covalin and either BG-547 or Halo-DAF (Figure 3b-e). The synthetic fluorophores used here could be easily exchanged for more complex (bio)molecules, thereby allowing a stepwise assembly of synthetic structures on cells and viruses. In conclusion, covalin is a versatile and easy to use fusion protein for the specific conjugation of different molecules or objects displaying appropriate functional groups. In contrast to the popular adaptor protein streptavidin, it has two binding sites with nonoverlapping substrate specificity, ensuring the formation of complexes with defined composition and stoichiometry. Just as for streptavidin, covalin substrates are available for a large variety of applications, allowing the immediate and ubiquitous use of covalin in bioconjugate chemistry. Furthermore, the orthogonal substrate specificities of streptavidin and covalin should permit their simultaneous use in bioconjugations. Finally, the existence of other self-labeling proteins with nonoverlapping substrate specificities should allow the generation of a family of orthogonal covalins as well as covalins with different valencies (12, 13).
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ACKNOWLEDGMENT This work was supported by the Swiss National Science Foundation. We thank Marlon Hinner for valuable comments. Supporting Information Available: Detailed procedures for all experiments are available. This material is available free of charge via the Internet at http://pubs.acs.org.
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