Production and Application of Nanobodies for Membrane Protein Structural Biology

  • Janine Denise BrunnerEmail author
  • Stephan Schenck
Part of the Methods in Molecular Biology book series (MIMB, volume 2127)


Nanobodies, small recombinant binders derived from camelid single chain antibodies, have become widely used tools in a diversity of disciplines related to membrane proteins. They are applied as chaperones in crystallization and blockers or modifiers of protein activity among numerous other applications. Their simple architecture as a single polypeptide chain, in contrast to classical antibodies, enables straightforward cloning, library generation, and recombinant expression. The small diameter and the pointed wedge-like shape of the antigen-binding site underlies binding to hollows and crevices of membrane proteins and renders nanobodies often conformation specific making them a preferred type of chaperone. Here we describe a simple protocol for the recombinant production of nanobodies in E. coli and their purification. We expand the current repertoire of usage further by describing a procedure for enlarging nanobodies on their C-terminal end to generate “macrobodies,” without interfering with their original characteristics. These enlarged nanobodies extend the application as a chaperone in crystallography and can serve to increase the mass for small targets in single particle electron cryo-microscopy, a field where nanobodies had so far only limited effect because of their small size.

Key words

Nanobody Macrobody Nanobody expression Complex formation Nanobody enlargement Nanobody generation MBP fusion protein 


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Laboratory of Biomolecular Research, Division of Biology and ChemistryPaul Scherrer InstituteVilligen PSISwitzerland

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