Title | Production and Application of Nanobodies for Membrane Protein Structural Biology. |
Publication Type | Journal Article |
Year of Publication | 2020 |
Authors | Brunner, J. D., and S. Schenck |
Journal | Methods Mol Biol |
Volume | 2127 |
Pagination | 167-184 |
Date Published | 2020 |
ISSN | 1940-6029 |
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. |
DOI | 10.1007/978-1-0716-0373-4_12 |
Alternate Journal | Methods Mol. Biol. |
PubMed ID | 32112322 |
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