Peptides in headlock – a novel high-affinity and versatile peptide-binding nanobody for proteomics and microscopy

Abstract

Nanobodies are highly valuable tools for numerous bioanalytical and biotechnical applications. Here, we report the characterization of a nanobody that binds a short peptide epitope with extraordinary affinity. Structural analysis reveals an unusual binding mode where the extended peptide becomes part of a β-sheet structure in the nanobody. This interaction relies on sequence-independent backbone interactions augmented by a small number of specificity-determining side chain contacts. Once bound, the peptide is fastened by two nanobody side chains that clamp it in a headlock fashion. Exploiting this unusual binding mode, we generated a novel nanobody-derived capture and detection system. Matrix-coupled nanobody enables the fast and efficient isolation of epitope-tagged proteins from prokaryotic and eukaryotic expression systems. Additionally, the fluorescently labeled nanobody visualizes subcellular structures in different cellular compartments. The high-affinity-binding and modifiable peptide tag of this system renders it a versatile and robust tool to combine biochemical analysis with microscopic studies.

Modular assembly of the nucleolar pre-60S ribosomal subunit

“…The beads were washed four times in ice-cold buffer A before the bound proteins were eluted using TEV-protease cleavage (1 h, 4 °C). The eluate was then incubated with NHS–sepharose beads (Sigma) coupled with anti-beta-catenin nanobody 19 in buffer B (50 mM Tris-HCl pH 7.6 (20 °C), 150 mM NaCl, 1 mM EDTA, 1 mM DTT) for 1 h at 4 °C with agitation. For electron microscopy sample preparation, the anti-beta catenin beads were washed once with buffer B. Cleavage by 3C protease for 1 h at 4 °C released the Nsa1–Nop2 containing nucleolar pre-60S particles.…”

Minimalist Antibodies and Mimetics: An Update and Recent Applications

“…Historically, full‐length antibodies have been used as bioanalytical tools—western blot being an obvious example. However, relative challenges in the expression of these molecular Winnebagos opens the door for smaller and simpler proteins, and nanobodies have recently been used in this context …”

“…Whereas nanobodies have largely been used to recognize large conformational regions on proteins, they can also be subjected to directed evolution to recognize small unfolded peptide epitopes. Recently, Rothbauer, Braun, and co‐workers generated a nanobody called BC2‐nb that recognizes a short linear epitope corresponding to residues 16–27 of β‐catenin (BC2T) . The structure of this complex was solved by X‐ray crystallography, revealing complete encapsulation of the epitope by an extended CDR3 loop (Figure D ) .…”

Reflections on the use of protein binders to study protein function in developmental biology

“…Therefore, the use of recently reported intrabodies against small tags, such as SunTag (19aa) Degraded in absence of the antigen (GFP) (Tang et al, 2016) Lysine-less VHH4 (GFP, YFP, Venus) VHH4 in which lysines have been substituted for Arginines (Daniel et al, 2018) mCherry VHH (Fridy et al, 2014) MoonTag (gp41, 15aa linear epitope) (Boersma et al, 2019) BC2 VHH (BC2 tag, 12aa) Not tested as intrabody. (Braun et al, 2016) NbALFA (ALFAtag, 15aa) (Götzke et al, 2019) Darpin 3G86.32 Anti-GFP DARPin (GFP) (Brauchle et al, 2014) 2 m22 Anti-mCherry DARPin (mCherry) (Brauchle et al, 2014) E11 and G01 Anti-TFP DARPins (TFP) (Vigano et al, 2018) scFv HA frankenbody (HA, 9aa linear epitope) (Zhao et al, 2018) Suntag (GCN4 v4, 19aa linear epitope) Aggregation at high levels unless fused to sfGFP-GB1 scaffold (Tanenbaum, Gilbert, Qi, Weissman, & Vale, 2014) BGP7 ScFv (BGP7, 7aa linear epitope) (Lim, Ichinose, Shinoda, & Ueda, 2007;Wongso, Dong, Ueda, & Kitaguchi, 2017) (15aa), promises to be a great addition to the protein binder toolbox (Boersma et al, 2019;Gotzke et al, 2019;Tanenbaum et al, 2014;Zhao et al, 2018). In most cases, the short peptide tags recognized by these intrabodies has been used in multimerized form such as to recognize single protein particles; whether these short tags also work well when integrated as a single tag remains to be tested.…”