Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation.

Tino Pleiner,Sergei Trakhanov,Holger Stark,Chung-Tien Lee,Jan Erik Schliep,Dirk Görlich,Henning Urlaub,Hema Chug,Marc Böhning,Mark Bates

doi:10.7554/elife.11349

Abstract

Nanobodies are single-domain antibodies of camelid origin. We generated nanobodies against the vertebrate nuclear pore complex (NPC) and used them in STORM imaging to locate individual NPC proteins with <2 nm epitope-label displacement. For this, we introduced cysteines at specific positions in the nanobody sequence and labeled the resulting proteins with fluorophore-maleimides. As nanobodies are normally stabilized by disulfide-bonded cysteines, this appears counterintuitive. Yet, our analysis showed that this caused no folding problems. Compared to traditional NHS ester-labeling of lysines, the cysteine-maleimide strategy resulted in far less background in fluorescence imaging, it better preserved epitope recognition and it is site-specific. We also devised a rapid epitope-mapping strategy, which relies on crosslinking mass spectrometry and the introduced ectopic cysteines. Finally, we used different anti-nucleoporin nanobodies to purify the major NPC building blocks – each in a single step, with native elution and, as demonstrated, in excellent quality for structural analysis by electron microscopy. The presented strategies are applicable to any nanobody and nanobody-target.

Highlights

Nanobodies represent antigen-binding domains of ’heavy-chain-only’ camelid antibodies and are typically selected by phage display from an immune library (Hamers-Casterman et al, 1993; Arbabi Ghahroudi et al, 1997; Muyldermans, 2013)
Nucleoporins are organized in multiple subcomplexes around a central eightfold rotational symmetry axis
We developed a well-characterized toolset of high-affinity nanobodies against the vertebrate nuclear pore complex (NPC) and established novel strategies to use these nanobodies to natively purify large NPC subcomplexes and to reliably label them with fluorophores for precise super-resolution localization

Summary

Introduction

Nanobodies represent antigen-binding domains of ’heavy-chain-only’ camelid antibodies and are typically selected by phage display from an immune library (Hamers-Casterman et al, 1993; Arbabi Ghahroudi et al, 1997; Muyldermans, 2013). Their small size (~13 kDa), monoclonal nature and high specificity are ideal for applications like affinity purification or protein detection and localization (Helma et al, 2015). It often results in low final yield (Baneyx and Mujacic, 2004), probably due to saturation of the secretion machinery and aggregation of precursor proteins in the cytoplasm.

Methods

Results

Conclusion