Please use this identifier to cite or link to this item: http://hdl.handle.net/10553/69843
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dc.contributor.authorRuano-Gallego, Daviden_US
dc.contributor.authorFraile, Sofíaen_US
dc.contributor.authorGutiérrez Cabrera, Carlos Javieren_US
dc.contributor.authorFernández, Luis Ángelen_US
dc.date.accessioned2020-02-05T12:50:40Z-
dc.date.available2020-02-05T12:50:40Z-
dc.date.issued2019en_US
dc.identifier.otherScopus-
dc.identifier.urihttp://hdl.handle.net/10553/69843-
dc.description.abstractThe 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.]en_US
dc.languageengen_US
dc.relation.ispartofMicrobial Cell Factoriesen_US
dc.sourceMicrobial Cell Factories, v. 18 (1)en_US
dc.subject310907 Patologíaen_US
dc.subject310801 Bacteriasen_US
dc.subject.otherE. Coli/Hemolysinen_US
dc.subject.otherNanobodiesen_US
dc.subject.otherProtein Secretionen_US
dc.subject.otherSingle-Domain Antibodiesen_US
dc.titleScreening and purification of nanobodies from E. coli culture supernatants using the hemolysin secretion systemen_US
dc.typeinfo:eu-repo/semantics/articleen_US
dc.typeArticleen_US
dc.identifier.doi10.1186/s12934-019-1094-0en_US
dc.identifier.scopus85062858895-
dc.contributor.authorscopusid55786459300-
dc.contributor.authorscopusid6508063810-
dc.contributor.authorscopusid7202545218-
dc.contributor.authorscopusid16238777700-
dc.identifier.issue1-
dc.relation.volume18-
dc.investigacionCiencias de la Saluden_US
dc.type2Artículoen_US
dc.utils.revisionen_US
dc.identifier.ulpgces
dc.description.sjr1,356
dc.description.jcr4,187
dc.description.sjrqQ1
dc.description.jcrqQ1
dc.description.scieSCIE
item.grantfulltextopen-
item.fulltextCon texto completo-
crisitem.author.deptGIR IUIBS: Medicina Veterinaria e Investigación Terapéutica-
crisitem.author.deptIU de Investigaciones Biomédicas y Sanitarias-
crisitem.author.orcid0000-0003-0764-7408-
crisitem.author.parentorgIU de Investigaciones Biomédicas y Sanitarias-
crisitem.author.fullNameGutiérrez Cabrera,Carlos Javier-
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