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

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.

Keywords: E. coli/hemolysin; Nanobodies; Protein secretion; Single-domain antibodies.

Fig. 1

The E. coli hemolysin system for secretion of nanobodies. a Schematic representation of the HlyB, HlyD and TolC components of the Hly secretion system that spans the inner membrane (IM), the periplasmic space with the peptidoglycan (PG) layer, and outer membrane (OM) of E. coli. TolC is a trimeric OM protein with a large periplasmic domain; HlyB is an ATPase and forms a dimer in the IM. HlyD is a trimeric adaptor IM protein that interacts with HlyB and with the periplasmic domain of TolC. For simplicity, a longitudinal section of the Hly-protein complex is represented showing only two subunits of HlyD and a continuous open channel for protein export. The HlyBD complex recognises the C-terminal domain of HlyA (C-HlyA) in the bacterial cytosol to export the fusion protein with the nanobody (Nb) VHH domain to the extracellular medium. b Plasmids pEHLYA5 and pVDL9.3 used in this work for the secretion of Nb-HlyA fusions on E. coli bacteria (TolC+). Plasmid pEHLYA5 is used to generate fusion of the VHH sequence with an N-terminal His-tag and C-HlyA secretion signal. The linker region between the VHH and C-HlyA sequences includes tags for immunodetection (HA-tag, E-tag) and a human rhinovirus 3C protease recognition site. Plasmid pVDL9.3 encodes HlyB and HlyD components. Expression of the VHH-HlyA, HlyB and HlyD is controlled under the Plac promoter in both plasmids

Fig. 2

Identification of Nb binders secreted with the Hly-system from a VHH immune library against EHEC antigens. a Coomassie staining of purified EHEC protein antigens EspA, Int280 and TirM used for camel immunization. b ELISA of camel serum after immunization to reveal antibody response against EspA, Int280, TirM and BSA (negative control). The camel antibody response against each of the proteins using the indicated serum dilutions was developed with protein-A peroxidase (POD). c The VHH sequences amplified from the immunized animal were used to generate a phage antibody (Phab) library. Phab binders were enriched by panning to obtain VHH repertoires against each antigen (Ag). The VHH repertoires were cloned into pEHLYA5 and the Nb-HlyA fusions were secreted in E. coli bacteria carrying pVDL9.3. The culture supernatants of individual clones from each VHH repertoire were tested by ELISA against their corresponding antigen (EspA, Int280, TirM) and BSA (negative control). Bound Nb-HlyA fusions were developed with anti-E-tag-mAb

Fig. 3

Amino acid sequence of VHHs clones identified after Hly-screening. Alignment of the amino acid sequences of the VHH domain in the clones selected by Hly-screening. The CDRs are labelled in colours: CDR1 (red), CDR2 (blue) and CDR3 (green). Cysteine residues are highlighted in yellow

Fig. 4

Secretion of Nb-HlyA fusions and antigen binding of representative clones. Representative clones of the identified VHH sequences against Int280, EspA and TirM were secreted as Nb-HlyA fusions. a Western blot developed with anti-E-tag-mAb of supernatants from induced cultures of the indicated clones showing a major protein band of ca. 42–45 kDa corresponding to Nb-HlyA fusions. The image shown is overexposed to visualize clearly protein bands of clones with lower expression levels. b ELISA of culture supernatants containing secreted Nb-HlyA fusions of the indicated clones against Int280 (top), EspA (middle) and TirM (bottom) developed with anti-E-tag mAb-POD. The represented OD490 values are the average from triplicate culture supernatants of each clone. Background signals to BSA are subtracted from the values obtained against the specific antigen (Int280, EspA, TirM)

Fig. 5

Disulphide bond formation in Nb-HlyA fusions. Thiol alkylation assay to determine the oxidation state of Cysteines (Cys) in secreted and cytoplasmic Nb-HlyA fusions from TD4 and TF1 clones, containing 2 and 4 Cys residues, respectively. Secreted proteins in culture supernatants and bacterial samples are treated (+) or not (−) with AMS (alkylating agent) and DTT (reducing agent), as indicated. Retarded mobility of alkylated polypeptides is indicative of reduced thiol groups. Protein bands of Nb-HlyA fusions developed after non-reducing SDS-PAGE and Western blot with anti-E-tag mAb

Fig. 6

Purification and antigen binding activities of secreted Nb-HlyA fusions. a Coomassie staining after SDS-PAGE of purified His-tagged Nb-HlyA fusions from induced culture supernatants of EC7, IB10 and TD4 clones produced in E. coli HB2151(pVDL9.3). b Antigen binding curves determined by ELISA of purified Nb-HlyA fusions from EC7, IB10 and TD4 clones at the indicated concentrations. Bound Nb-HlyA proteins were developed with anti-E-tag mAb. OD490 values indicated are obtained against the specific antigen (EspA for EC7, Int280 for IB10, TirM for TD4) after subtraction of OD490 values against BSA (control antigen). Data are the average of triplicate ELISA experiments