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


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2019-12

AUTHORS

David Ruano-Gallego, Sofía Fraile, Carlos Gutierrez, Luis Ángel Fernández

ABSTRACT

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. More... »

PAGES

47

References to SciGraph publications

Identifiers

URI

http://scigraph.springernature.com/pub.10.1186/s12934-019-1094-0

DOI

http://dx.doi.org/10.1186/s12934-019-1094-0

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1112682875

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/30857538


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