Surface display of a single-domain antibody library on Gram-positive bacteria

Abstract

Combinatorial protein engineering for selection of proteins with novel functions, such as enzymes and affinity reagents, is an important tool in biotechnology, drug discovery, and other biochemical fields. Bacterial display is an emerging technology for isolation of new affinity proteins from such combinatorial libraries. Cells have certain properties that are attractive for directed evolution purposes, in particular the option to use quantitative flow-cytometric cell sorting for selection of binders. Here, an immune library of around 10(7) camelid single-domain antibody fragments (Nanobodies) was displayed on both the Gram-positive bacterium Staphylococcus carnosus and on phage. As demonstrated for the first time, the antibody repertoire was found to be well expressed on the bacterial surface and flow-cytometric sorting yielded a number of Nanobodies with subnanomolar affinity for the target protein, green fluorescent protein (GFP). Interestingly, the staphylococcal output repertoire and the binders from the phage display selection contained two slightly different sets of clones, containing both unique as well as several similar variants. All of the Nanobodies from the staphylococcal selection were also shown to enhance the fluorescence of GFP upon binding, potentially due to the fluorescence-based sorting principle. Our study highlights the impact of the chosen display technology on the variety of selected binders and thus the value of having alternative methods available, and demonstrates in addition that the staphylococcal system is suitable for generation of high-affinity antibody fragments.

A truncated and dimeric format of an Affibody library on bacteria enables FACS‐mediated isolation of amyloid‐beta aggregation inhibitors with subnanomolar affinity

“…It has previously been demonstrated that staphylococcal cell surface display in combination with fluorescenceactivated cell sorting (FACS) is well suited for combinatorial affinity maturation efforts, since it allows for high precision affinity discrimination between binders [35][36][37][38]. Recently, the method was used for affinity maturation of a HER3-specific Affibody, and generated binders with picomolar affinities [39].…”

“…It has been shown that there is a correlation between the affinity of peripherally-administered Ab-specific agents and the efflux of Ab from the brain to the serum [29]. As levels of Ab peptides in the blood are low (subnanomolar) [30][31][32][33][34], it is probably critical to use capturing-agents of high affinity for such applications.It has previously been demonstrated that staphylococcal cell surface display in combination with fluorescenceactivated cell sorting (FACS) is well suited for combinatorial affinity maturation efforts, since it allows for high precision affinity discrimination between binders [35][36][37][38]. Recently, the method was used for affinity maturation of a HER3-specific Affibody, and generated binders with picomolar affinities [39].…”

Intracellular Expression of Camelid Single-Domain Antibodies Specific for Influenza Virus Nucleoprotein Uncovers Distinct Features of Its Nuclear Localization

“…After two consecutive rounds of phage display and panning, we obtained four unique VHHs specific for influenza virus. Three additional VHHs specific for influenza virus were then obtained by selection using staphylococcal surface expression (38). All seven VHHs were equipped with a C-terminal sortase motif (41,42) followed by a 6ϫHis tag, expressed in bacteria, and purified on a Ni-NTA affinity matrix followed by size exclusion chromatography.…”

“…The second method utilized recombinant influenza virus protein with a C-terminal sortase motif expressed in 293T cells, biotinylated, and immobilized on streptavidin beads. A third method utilized a Staphylococcus aureus display platform, based on a strategy developed for Staphylococcus carnosus (38), which will be described in detail elsewhere.…”

Camelid single-domain antibody fragments: Uses and prospects to investigate protein misfolding and aggregation, and to treat diseases associated with these phenomena

“…In all cases, antigen-specific nanobodies are retrieved from these libraries by phage display or other selection protocols such as bacterial display [57], yeast display [58] or ribosome display [59]. Due to its robustness, the phage display method is the most often used one.…”