Production and Application of Nanobodies for Membrane Protein Structural Biology.

TitleProduction and Application of Nanobodies for Membrane Protein Structural Biology.
Publication TypeJournal Article
Year of Publication2020
AuthorsBrunner, J. D., and S. Schenck
JournalMethods Mol Biol
Volume2127
Pagination167-184
Date Published2020
ISSN1940-6029
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.

DOI10.1007/978-1-0716-0373-4_12
Alternate JournalMethods Mol. Biol.
PubMed ID32112322
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