Nanobodies CRCM platform

The specificity of this platform is to yield single domain antibodies (nanobodies). These small and compact fragments (13 kDa), which correspond to variable domains of a subtype of llama antibodies, are characterized by outstanding properties in terms of production, stability, and their propensity to bind buried epitopes inaccessible to conventional antibodies. Most nanobodies can be expressed as soluble and active fragments without disulfide bond formation, in a reducing environment such as the cell cytoplasm or nucleus. This particularity allows their use as intracellular tool to precisely knock out a specific interaction of a target protein without interfering with other interactions of this target. The mission of the nanobody platform is to help research teams with the isolation of specific nanobodies (single domain antibodies which can bind to buried epitopes inaccessible to conventional antibodies). The selection and screening steps, based on phage display, can be performed on recombinant proteins, transfected cells, intact cells or lysate of cell lines. 
Nanobodies are produced and purified from E. coli, and can be fused to various tags (c-Myc, 6-His), modified with an extra C-terminal free cystein allowing a site-directed conjugation to nanoparticles, or biotinylated in vivo. Genes coding for nanobodies can also be fused to the gene of a reporter molecule such as GFP or mRFP1. The resulting constructs can be directly used as probe in living cells or for protein localization.

After exchanging and discussing the project, its acceptance will depend on its feasibility, its adequacy with the skills and availability of the platform.

The platform allows performing llama immunization with purified proteins, membrane extract or intact cell, followed by the creation of a E. coli nanobody library after cloning of nanobody genes in a phagemid. This library will be used to generate libraries of phage displayed nanobodies that can be enriched by panning on immobized antigens or intact cells, following depletion steps on irrelevant surfaces. Positive binders are sequenced and representative clones are produced in E. coli and purified in milligram scale for affinity/specificity determination by flow cytometry, ELISA, and biolayer interferometry.