Production and Crystallization of Nanobodies in Complex with the Receptor Binding Domain of the SARS-CoV-2 Spike Protein
Audrey Le Bas,
Halina Mikolajek,
Jiandong Huo,
Chelsea Norman,
Joshua Dormon,
James Naismith,
Raymond Owens
Affiliations
Audrey Le Bas
Structural Biology, The Rosalind Franklin Institute, Harwell Science Campus, UK
Halina Mikolajek
Diamond Light Source Ltd, Harwell Science Campus, UK
Jiandong Huo
Structural Biology, The Rosalind Franklin Institute, Harwell Science Campus, UKDivision of Structural Biology, The Wellcome Centre for Human Genetics, University of Oxford, UK
Chelsea Norman
Structural Biology, The Rosalind Franklin Institute, Harwell Science Campus, UK
Joshua Dormon
Structural Biology, The Rosalind Franklin Institute, Harwell Science Campus, Uk
James Naismith
Structural Biology, The Rosalind Franklin Institute, Harwell Science Campus, UKDivision of Structural Biology, The Wellcome Centre for Human Genetics, University of Oxford, UK
Raymond Owens
Structural Biology, The Rosalind Franklin Institute, Harwell Science Campus, UKDivision of Structural Biology, The Wellcome Centre for Human Genetics, University of Oxford, UK
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.
