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Review
. 2015 Jun 8;209(5):633-44.
doi: 10.1083/jcb.201409074.

Nanobodies and recombinant binders in cell biology

Jonas Helma  1 , M Cristina Cardoso  2 , Serge Muyldermans  3 , Heinrich Leonhardt  4
Affiliations
Review

Nanobodies and recombinant binders in cell biology

Jonas Helma et al. J Cell Biol. .

Abstract

Antibodies are key reagents to investigate cellular processes. The development of recombinant antibodies and binders derived from natural protein scaffolds has expanded traditional applications, such as immunofluorescence, binding arrays, and immunoprecipitation. In addition, their small size and high stability in ectopic environments have enabled their use in all areas of cell research, including structural biology, advanced microscopy, and intracellular expression. Understanding these novel reagents as genetic modules that can be integrated into cellular pathways opens up a broad experimental spectrum to monitor and manipulate cellular processes.

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Figures

Figure 1.
Figure 1.
Comparison of immunoglobulin derivatives and nonimmunoglobulin binder formats. (A) Immunoglobulins and their derivatives. (left) A conventional IgG molecule is comprised of two heavy and two light chains. Heavy chains comprise three constant domains (CH1–3, dark blue) and one variable domain (VH, dark green); light chains comprise one constant (CL, light blue) and one variable domain (VL, light green). The naturally functional antigen binding unit is formed by noncovalent association of the VH and the VL domain. This association is mediated by hydrophobic framework regions, indicated in pink. IgG can be derivatized to Fab, scFv, and single domain VH or VL binders. (right) Heavy chain antibodies (hcAb) are found in Camelidae, lack the light chain and the CH1 domain, and comprise a single, antigen binding domain, the VHH domain. (B) Nonimmunoglobulin binders are based on natural and designed protein scaffolds. Shown are fibronectin-derived Adnectins/monobodies that are characterized by an Ig-like β-sandwich structure, anticalins that are based on the lipocalin fold, affibodies that derive from protein A and comprise three α helices, and DARPins, which are designer proteins composed of ankyrin repeats. The randomized residues that mediate the respective ligand binding are marked in red. Protein Data Bank accession numbers are given in parentheses. N, N terminus; C, C terminus.
Figure 2.
Figure 2.
Applications of recombinant binders in biochemistry and structural biology. (A) Use of recombinant binders for purification and detection in vitro. Fields of applications involve classic capture assays such as ELISA, high-throughput screening (HTS) techniques and immunoprecipitation (IP) as well as proteomic and array technologies. (B) Recombinant binders for assisted crystallography. Shown are three different structures and binder formats (Nanobody, Affibody, and DARPin) that have been used for the elucidation of challenging structures. Protein Data Bank accession numbers are given in parentheses.
Figure 3.
Figure 3.
Recombinant binders as intracellular biosensors, effectors, and tools for nanoscopy. (A) Categories of affinity-based live-cell biosensors and effectors. (top left) Fluorescently tagged tracers are used to monitor antigen-specific localization patterns. (top right) Conformation-specific and PTM-specific binders are recruited to respective sites of action. (bottom left) Binders that block biologically active target sites or modulate target function upon binding. (bottom right) An F3H assay to investigate PPIs in living cells. A GFP-specific nanobody is anchored to defined subcellular structures such as the artificially introduced LacO array visible as a spot in the nucleus or the endogenous nuclear lamina or centrosomes. In all of these cases, RFP-preys colocalize with GFP-bait fusions in the presence of an interaction. (B) Recombinant binder tools for nanoscopy. Superresolution techniques such as 3D-structured illumination microscopy require repetitive imaging of a single cell, which leads to severe bleaching of FPs. Nanobodies can thus be used to stabilize or enhance FPs and enable high quality image acquisition conditions. Importantly, their small size reduces the linkage error, as the distance between the fluorescent label and the actual specimen is minimized. Bar, 10 µm.
Figure 4.
Figure 4.
Recombinant binders as modular entities in conceptual, cell-based assay design. (A) A targeted protein knockout via an engineered proteasomal degradation device. The natural WD40 domain that mediates the interaction with a substrate to be degraded via the Skp1-Cul1-F-box-protein (SCF) ubiquitin (Ub) ligase complex is substituted with a recombinant binder, allowing targeted ablation of a protein of interest (POI; Caussinus et al., 2012). (B) A scaffold-induced system to manipulate gene activity. Two different binders, recognizing distinct epitopes of a scaffold protein of interest are fused to a DNA-binding protein (DBP) and a transcriptional activator (TA) enabling protein of interest–dependent gene activation (Tang et al., 2013).
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