Nanobodies are the small (15 kDa) and stable single-domain fragments harboring the full antigen-binding capacity of camelid heavy chain–only antibodies. Nanobodies are exquisite chaperones for crystallizing membrane proteins, multiprotein assemblies, transient conformational states and intrinsically disordered proteins. Nanobodies can also be used for other applications in structural biology. Domain-specific Nanobodies have been used in single-particle electron microscopy (EM) to track these domains in particle projections. Because Nanobodies can be functionally expressed as intrabodies in eukaryotic cells, these single-domain antibodies can also be used to track their targets inside a living cell.
Nanobody discovery pipeline: The production of Nanobodies consists of the following steps. First, llamas are immunized with the appropriate conformers of the proteins (protein complex). In parallel, assays are developed to screen and characterize the nanobodies with the desired properties. After immunizations, a blood sample is collected, the in vivo matured nanobody repertoire is amplified by RT-PCR and cloned in phage display or yeast display libraries. Target specific nanobodies are then selected by panning on displayed antigens. Finally, nanobodies with the appropriate properties for applications in structural biology and cellular imaging are selected and characterized. A typical Nanobody discovery project/visit comprises of a protein complex or maximum 3 different proteins.
Project-Provided Antigen Requirements: Only small amounts (<2mg) of the folded and quality controlled protein are required to produce Nanobodies that recognize conformational epitopes on the native proteins. In case no purified antigen is available, cells expressing purified protein or genetic vaccination may be used to induce an in vivo matured humoral response. By learning more about the nature of each project, the team will advise in designing better antigens and work out optimal immunization schemes, panning strategies and screening methods.
Active partnership: We store immune libraries that can recurrently be rescreened. Phage display and yeast display libraries that have been generated within the framework of one integrated Instruct program for one partner that focusses on one particular technology can later be screened within few days for other partners within the same program according to newly specified properties. We do acknowledge that binders that have been selected for affinity purification purposes may be suboptimal for X-ray crystallography, X-ray tomography or X-ray imaging.