|Title:||Screening and purification of nanobodies from E. coli culture supernatants using the hemolysin secretion system||Authors:||Ruano-Gallego, David
Gutiérrez Cabrera, Carlos Javier
Fernández, Luis Ángel
|UNESCO Clasification:||310907 Patología
|Issue Date:||2019||Journal:||Microbial Cell Factories||Abstract:||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.[Figure not available: see fulltext.]||URI:||http://hdl.handle.net/10553/69843||DOI:||10.1186/s12934-019-1094-0||Source:||Microbial Cell Factories, v. 18 (1)|
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