Escherichia coli surface display for the selection of nanobodies.

Summary Nanobodies (Nbs) are the smallest functional antibody fragments known in nature and have multiple applications in biomedicine or environmental monitoring. Nbs are derived from the variable segment of camelid heavy chain-only antibodies, known as VHH. For selection, libraries of VHH gene segments from naive, immunized animals or of synthetic origin have been traditionally cloned in E. coli phage display or yeast display systems, and clones binding the target antigen recovered, usually from plastic surfaces with the immobilized antigen (phage display) or using fluorescence-activated cell sorting (FACS; yeast display). This review briefly describes these conventional approaches and focuses on the distinct properties of an E. coli display system developed in our laboratory, which combines the benefits of both phage display and yeast display systems. We demonstrate that E. coli display using an N-terminal domain of intimin is an effective platform for the surface display of VHH libraries enabling selection of high-affinity Nbs by magnetic cell sorting and direct selection on live mammalian cells displaying the target antigen on their surface. Flow cytometry analysis of E. coli bacteria displaying the Nbs on their surface allows monitoring of the selection process, facilitates screening, characterization of antigen-binding clones, specificity, ligand competition and estimation of the equilibrium dissociation constant (KD).