Screening of antibody (Ab) libraries by direct display on the surface of E. coli cells is hampered by the presence of the outer membrane (OM). In this work we demonstrate that the native β-domains of EhaA autotransporter and intimin, two proteins from enterohemorrhagic E. coli O157:H7 (EHEC) with opposite topologies in the OM, are effective systems for the display of immune libraries of single domain Abs (sdAbs) from camelids (nanobodies or VHH) on the surface of E. coli K-12 cells and for the selection of high affinity sdAbs using magnetic cell sorting (MACS). We analyzed the capacity of EhaA and intimin β-domains to display individual sdAbs and sdAb libraries obtained after immunization with the extracellular domain of the translocated intimin receptor from EHEC (TirMEHEC). We demonstrated that both systems displayed functional sdAbs on the surface of E. coli cells with little proteolysis and cellular toxicity, although E. coli cells displaying sdAbs with the β-domain of intimin showed higher antigen-binding capacity. Both E. coli display libraries were screened for TirMEHEC binding clones by MACS. High affinity binders were selected by both display systems, although more efficiently with the intimin β-domain. The specificity of the selected clones against TirMEHEC was demonstrated by flow cytometry of E. coli cells, along with ELISA and surface plasmon resonance with purified sdAbs. Finally, we employed the E. coli cell display systems to provide an estimation of the affinity of the selected sdAb by flow cytometry analysis under equilibrium conditions.

SummaryNanobodies (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 naïve, 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).