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Engineering nanobodies as precision tools for cell biology

Antibodies are one of the most powerful tools our adaptive immune system produces to protect us against pathogens. Conventional antibodies are made up of 2x light and 2xheavy chains to form a 150 kDa tetrameric molecule that is capable of binding antigens via two paratopes formed from the variable heavy and light chain domains. These large molecules cannot be produced in bacteria or in the cytosol of mammalian cells, as they don't fold under reducing conditions that do not support disulfide bond formation. 

Conventional antibodies.png

In contrast to humans, camels, alpacas and llamas possess atypical antibodies that lack light chains. These "heavy-chain antibodies" arose from point mutations that cause skipping of the light-chain binding constant domain CH1 of the heavy chain. In the absence of the light chain, the variable heavy chain domain (VHH) became fully functional in antigen binding by acquiring additional mutations at the former interface with the light chain variable domain that increase solubility. Additionally, the CDR loop 3 is frequently enlarged to provide additional antigen contacts. The VHH domain can easily be cloned and expressed recombinantly in bacteria and mammalian cells, often fully functional and in high yields. 

During Tino's PhD at the Max Planck Institute for Biophysical Chemistry, he helped established one of the few academic facilities worldwide for the generation of alpaca-derived nanobodies. As part of his own work and in collaboration, he generated nanobodies against a wide variety of targets. In the Görlich lab, Tino used nanobodies as tools to study nucleocytoplasmic transport: first, as crystallization chaperones for an essential heterotrimeric complex that lines the nuclear pore complex (Chug et al., 2015, Science); and then, as intracellular inhibitors of a nuclear transport receptor (NTR), which allowed the lab to demonstrate that the NTR could unexpectedly carry-out bidirectional protein transport (Aksu* and Pleiner* et al., 2018, JCB).


Further, Tino established a close collaboration with the group of Stefan Hell and used nanobodies as ultra-small immunolabels for super-resolution microscopy (Pleiner et al., 2015, eLife; Göttfert et al., 2017, PNAS; Pleiner et al., 2018, JCB). For this, he developed a site-specific fluorescent labeling strategy that maximizes nanobody brightness, while maintaining specificity and stability (Pleiner et al., 2015, eLife). This was essential to generate bright anti-mouse and rabbit IgG ‘secondary nanobodies’ that could rival the signal amplification effect of conventional polyclonal antibodies (Pleiner et al., 2018, JCB). The resulting reagents have been distributed by Addgene to hundreds of labs worldwide. This study was also selected for a special collection of the Journal of Cell Biology (JCB65: Methods) and awarded with the ‘animal welfare prize’ of the German government.

Short film about Tino's PhD work (in German) produced by a professional film team on behalf of the Max Planck Society to promote better understanding for the use of animals in research. Copyright: MPG/ WagnerVision. This work was awarded with the "37th Animal welfare research prize" by the Federal Ministry of Food and Agriculture, Germany. Click the link below to watch video:

Relevant publications:

Aksu, M.*; Pleiner, T.*; Karaca, S.; Kappert, C.; Dehne, H.J.; Seibel, K.; Urlaub, H.; Bohnsack, M.; Görlich, D. (2018) Xpo7 is a broad-spectrum exportin and a nuclear import receptor. J. Cell Biol. 217, 1143-1154.

Pleiner, T.†; Bates, M.†; Görlich, D.† (2018) A toolbox of anti-mouse and anti-rabbit IgG secondary nanobodies. J Cell Biol. 217(3):1143-1154.
Press release, F1000 recommendation; Spotlight by Ewers (2018); Addgene Blue Flame Award; JCB65 anniversary methods collection; Animal welfare prize by German government.

Göttfert, F.; Pleiner, T.; Heine, J.; Westphal, V.; Görlich, D.; Sahl, S.; Hell, S. (2017) Strong signal increase in STED fluorescence microscopy by imaging regions of subdiffraction extent. PNAS. 114(9):2125-2130.

Pleiner, T.; Bates, M.; Trakhanov, S.; Lee, C.T.; Schliep, J. E.; Chug, H.; Böhning, M.; Stark, H.; Urlaub, H.; Görlich, D. (2015) Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation. eLife. 4; e11349.
Highlighted by Nature Methods 13, 116 (2016).

Chug, H.; Trakhanov, S.; Hülsmann, B.; Pleiner, T.; Görlich, D. (2015) Crystal structure of the metazoan Nup62-Nup58-Nup54 nucleoporin complex. Science. 350(6256): 106-10.

Perspective by Ullmann and Powers, Science (2015).

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