Escherichia coli surface display for the selection of nanobodies.

Valencio Salema,Luis Ángel Fernández

doi:10.1111/1751-7915.12819

Valencio Salema, Luis Ángel Fernández

Open Access

https://doi.org/10.1111/1751-7915.12819

Copy DOI

Abstract

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).

Highlights

Nanobodies (Nbs) are the smallest, intact antigen-binding fragments derived from a functional immunoglobulin (Ig)
Nbs are recombinant single domain antibody (Ab) fragments with a molecular weight of ~14 kDa and ~2– 4 nm in size. They comprise the variable domain of a heavy chain-only antibody (HCAb; Fig. 1A), which was discovered in the serum of camelids in the early 1990s (Hamers-Casterman et al, 1993), and are likely an outcome of adaptive changes occurring in conventional Abs within the Camelidae lineage, playing a role in the immune response of these animals (Nguyen et al, 2002; Flajnik et al, 2011; Muyldermans and Smider, 2016)
We have demonstrated that flow cytometry analysis of E. coli bacteria displaying N-terminal fragment of EHEC intimin (Neae)-VHH fusions gives a good estimation of the apparent KD of Nbs, with values similar to those obtained by surface plasmon resonance (SPR) with purified Nbs (Salema et al, 2013, 2016a,b)

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. Libraries of VHH gene segments from na€ıve, 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). 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.

Introduction

Conclusions and future directions