Parallelised Cloning, Mammalian Cell Expression, and Purification of Nanobodies Identified by Phage Display

Nanobodies are recombinant single-domain antibodies (VHHs) derived from the heavy chain-only subset of camelid immunoglobulins that can be reverse-engineered into bivalent antibodies by fusion to immunoglobulin Fc constant regions. Mammalian cells are the system of choice to produce VHH-Fcs to ensure authentic folding and post-translation glycosylation of the expressed VHH-Fcs. In a recent project to find neutralising VHH-Fc binders to the spike proteins of SARS-CoV-2 viruses, we identified a need for rapid expression and purification of multiple VHH-Fc fusions from nanobodies selected by phage display. Here, we present a protocol for the construction of expression vectors by parallel ligase-independent cloning, transient small-scale expression in mammalian cells (4 mL culture volume), screening antigen-binding activity, and midi-scale purification (30 mL culture volume) for downstream activity assays. The workflow is completely transferable between different vector formats, of which three are described herein: Fc fusion dimers, monomeric CD4 fusions, and His-tagged monomers. Key features • Miniaturised and parallelised methodology for the screening and production of large numbers of VHHs that bind antigens of interest. • Streamlined and unified, high-throughput cloning strategy for use in multiple modular vectors for monomeric and dimeric VHH production in mammalian culture.