High-affinity antibodies against functional recombinant NhaA from Salmonella typhimurium generated with an avian immune phage-display library

Sodium/proton antiporters are essential for every cell as they regulate intracellular pH and cell volume. NhaA from Salmonella typhimurium (STNhaA), a homolog of the well characterized antiporter NhaA from Escherichia coli [1,2], was produced by heterologous overexpression in E. coli. The recombinant His-tagged protein was purified by immobilized metal-affinity chromatography. The antiporter is functional as shown by growth complementation assays with sodium/proton antiporter deficient E.coli strains, which cannot grow at high-salt concentrations. Chicken were immunized with pure, detergent-solubilized STNhaA. An immune-chicken scFv antibody library was constructed and sub-nanomolar affinity binders against STNhaA were isolated which recognize conformation specific and linear epitopes. The latter was mapped to a surface exposed loop connecting transmembrane segments VIII and XI of STNhaA. The binders form stable complexes with the native transporter in solution, ideal for functional and structural studies. Avian immune phage display libraries provide an efficient way to generate high affinity antibodies.