Please use this identifier to cite or link to this item: http://hdl.handle.net/10553/69843
Title: Screening and purification of nanobodies from E. coli culture supernatants using the hemolysin secretion system
Authors: Ruano-Gallego, David
Fraile, Sofía
Gutiérrez Cabrera, Carlos Javier 
Fernández, Luis Ángel
UNESCO Clasification: 310907 Patología
310801 Bacterias
Keywords: E. Coli/Hemolysin
Nanobodies
Protein Secretion
Single-Domain Antibodies
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)
Appears in Collections:Artículos
Thumbnail
PDF
Adobe PDF (3,22 MB)
Show full item record

SCOPUSTM   
Citations

12
checked on May 9, 2021

Page view(s)

82
checked on May 10, 2021

Download(s)

54
checked on May 10, 2021

Google ScholarTM

Check

Altmetric


Share



Export metadata



Items in accedaCRIS are protected by copyright, with all rights reserved, unless otherwise indicated.