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Production and Application of Nanobodies for Membrane Protein Structural Biology

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

Abstract

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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