1 Neuropharm and Genetics Lab, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, Københavns Universitet2 VIB Structural Biology Brussels, Vrije Universiteit Brussels3 Pharma Research and Early Development (pRED), Small Molecule Research, Discovery Technologies, F. Hoffmann-La Roche, Basel, Switzerland.4 Department of Biochemistry, Biomolecular Structure Center, School of Medicine, University of Washington, Seattle, Washington, USA.5 Department of Molecular and Cellular Physiology, Stanford University School of Medicine6 Neuropharm and Genetics Lab, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, Københavns Universitet
There is growing interest in using antibodies as auxiliary tools to crystallize proteins. Here we describe a general protocol for the generation of Nanobodies to be used as crystallization chaperones for the structural investigation of diverse conformational states of flexible (membrane) proteins and complexes thereof. Our technology has a competitive advantage over other recombinant crystallization chaperones in that we fully exploit the natural humoral response against native antigens. Accordingly, we provide detailed protocols for the immunization with native proteins and for the selection by phage display of in vivo-matured Nanobodies that bind conformational epitopes of functional proteins. Three representative examples illustrate that the outlined procedures are robust, making it possible to solve by Nanobody-assisted X-ray crystallography in a time span of 6-12 months.
Nature Protocols (print), 2014, Vol 9, Issue 3, p. 674-93