Production and Crystallization of Nanobodies in Complex with the Receptor Binding Domain of the SARS-CoV-2 Spike Protein.
Bio Protoc
; 12(9): e4406, 2022 May 05.
Article
em En
| MEDLINE
| ID: mdl-35800465
ABSTRACT
The receptor binding domain (RBD) of the spike protein of SARS-CoV-2 binds angiotensin converting enzyme-2 (ACE-2) on the surface of epithelial cells, leading to fusion, and entry of the virus into the cell. This interaction can be blocked by the binding of llama-derived nanobodies (VHHs) to the RBD, leading to virus neutralisation. Structural analysis of VHH-RBD complexes by X-ray crystallography enables VHH epitopes to be precisely mapped, and the effect of variant mutations to be interpreted and predicted. Key to this is a protocol for the reproducible production and crystallization of the VHH-RBD complexes. Based on our experience, we describe a workflow for expressing and purifying the proteins, and the screening conditions for generating diffraction quality crystals of VHH-RBD complexes. Production and crystallization of protein complexes takes approximately twelve days, from construction of vectors to harvesting and freezing crystals for data collection.
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Bases de dados:
MEDLINE
Idioma:
En
Revista:
Bio Protoc
Ano de publicação:
2022
Tipo de documento:
Article
País de afiliação:
Reino Unido