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Background  The hemolysin (Hly) secretion system of  E. coli  allows the one-step translocation of hemolysin A (HlyA) from the bacterial cytoplasm to the extracellular medium, without a periplasmic intermediate. In this work, we investigate whether the Hly secretion system of  E. coli  is competent to secrete a repertoire of functional single-domain VHH antibodies (nanobodies, Nbs), facilitating direct screening of VHH libraries and the purification of selected Nb from the extracellular medium.    Results  We employed a phagemid library of VHHs obtained by immunization of a dromedary with three protein antigens from enterohemorrhagic  E. coli  (EHEC), namely, the extracellular secreted protein A (EspA), the extracellular C-terminal region of Intimin (Int280), and the translocated intimin receptor middle domain (TirM). VHH clones binding each antigen were enriched and amplified by biopanning, and subsequently fused to the C-terminal secretion signal of HlyA to be expressed and secreted in a  E. coli  strain carrying the Hly export machinery (HlyB, HlyD and TolC). Individual  E. coli  clones were grown and induced in 96-well microtiter plates, and the supernatants of the producing cultures directly used in ELISA for detection of Nbs binding EspA, Int280 and TirM. A set of Nb sequences specifically binding each of these antigens were identified, indicating that the Hly system is able to secrete a diversity of functional Nbs. We performed thiol alkylation assays demonstrating that Nbs are correctly oxidized upon secretion, forming disulphide bonds between cysteine pairs despite the absence of a periplasmic intermediate. In addition, we show that the secreted Nb-HlyA fusions can be directly purified from the supernatant of  E. coli  cultures, avoiding cell lysis and in a single affinity chromatography step.    Conclusions  Our data demonstrate the Hly secretion system of  E. coli  can be used as an expression platform for screening and purification of Nb binders from VHH repertories.

Screening and purification of nanobodies from E. coli culture supernatants using the hemolysin secretion system