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Production and Application of Nanobodies for Membrane Protein Structural Biology

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Expression, Purification, and Structural Biology of Membrane Proteins

Part of the book series: Methods in Molecular Biology ((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.

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Author information

Authors and Affiliations

  1. Laboratory of Biomolecular Research, Division of Biology and Chemistry, Paul Scherrer Institute, Villigen PSI, Switzerland

    Janine Denise Brunner & Stephan Schenck

Authors
  1. Janine Denise Brunner
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  2. Stephan Schenck
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Corresponding author

Correspondence to Janine Denise Brunner .

Editor information

Editors and Affiliations

  1. Biozentrum, University of Basel, Basel, Basel Stadt, Switzerland

    Camilo Perez

  2. Biozentrum, University of Basel, Basel, Basel Stadt, Switzerland

    Timm Maier

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Brunner, J.D., Schenck, S. (2020). Production and Application of Nanobodies for Membrane Protein Structural Biology. In: Perez, C., Maier, T. (eds) Expression, Purification, and Structural Biology of Membrane Proteins. Methods in Molecular Biology, vol 2127. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0373-4_12

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  • DOI: https://doi.org/10.1007/978-1-0716-0373-4_12

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0372-7

  • Online ISBN: 978-1-0716-0373-4

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