Steps Towards Generation of an Anti-GFP Nanobody Escherichia coli Surface Display System Using the BrkA Autotransporter System

Abstract

Nanobodies, the smallest antigen-binding fragments, are promising for targeted therapies due to their small size, ease of expression, and low immunogenicity. Escherichia coli surface display offers a rapid and controllable alternative to phage and yeast systems for directed evolution to generate nanobodies. Here, we present a method to use the Bordetella pertussis autotransporter, Bordetella serum-resistance killing protein A (BrkA), known for displaying exogenous proteins, as a scaffold for nanobody display. An anti-green fluorescent protein (GFP) nanobody was used as a model nanobody in the design. The proposed BrkA anti-GFP nanobody construct was predicted to fold correctly and exhibit GFP binding ability based on AlphaFold modeling. Two new plasmids with varying flexible linker lengths were generated. The constructs were identified to contain frameshift mutations that introduced premature stop codons, inhibiting the ability to properly express an anti-GFP nanobody within this E. coli system. Steps to generate two new functional anti-GFP nanobody constructs within the BrkA passenger domain are outlined in this manuscript.