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 biocon- jugations, as it can stably conjugate biotinylated molecules with high efficiency (3, 4). However, for the specific heteroconju- gation 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 heterocon- jugation 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 O 6 -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 (10 4 s -1 M -1 for SNAP-tag and 10 6 s -1 M -1 for HaloTag). This makes them well-suited for bioconju- gations, especially when compared to typical bioorthogonal reactions such as copper-free click chemistry or the Staudinger ligation, which are at least 10 4 -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 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 (Sup- porting 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 SNAP- tag 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 strepta- vidin 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 * Corresponding author. E-mail: kai.johnsson@epfl.ch. Phone: +41-21-6939356. Fax: +41-21-6939365. Bioconjugate Chem. 2008, 19, 1753–1756 1753 10.1021/bc800268j CCC: $40.75  2008 American Chemical Society Published on Web 08/28/2008