Screening and purification of nanobodies from E. coli culture supernatants using the hemolysin secretion system
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.
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.
KeywordsE. coli/hemolysin Nanobodies Protein secretion Single-domain antibodies
bovine serum albumin
C-terminal domain of 218 amino acids of HlyA
complementary determining region
enterohemorrhagic E. coli
enzyme-linked immunosorbent assay
extracellular secreted protein A
heavy-chain only antibodies
immobilized metal affinity chromatography
extracellular 280 amino acid C-terminal region of Intimin
nanobody fused to C-HlyA
optical density at 490 nm
optical density at 600 nm
outer membrane protein
plaque forming units
polyvinylidene difluoride membrane
reverse transcription polymerase chain reaction
repeats in toxins
translocated intimin receptor middle domain
uropathogenic E. coli
variable heavy chain domain from heavy-chain only antibodies
Contrary to classical N-terminal signal peptides of proteins secreted to the periplasm by the Sec pathway , the signal for secretion of HlyA is located in the C-terminal end and is not removed during transport [20, 21]. The C-terminal location of HlyA signal also implies a post-translational mechanism of secretion. The last 50 amino acids of C-terminal end of HlyA appear to contain the signal recognized by HlyB/D complex [22, 23], but heterologous proteins have been secreted with this system fused to larger C-terminal fragments, comprising the last 60 amino acids [20, 24] and, more frequently, the last 218 residues (C-HlyA; 23 kDa), which includes three glycine- and aspartic-rich repeats, named as repeats in toxins (RTX) [7, 21]. Different heterologous proteins have been secreted in a functional form with C-HlyA, including the maltose binding protein , alkaline phosphatase [26, 27], β-lactamase , mammalian fatty acid binding protein , streptokinase , a single-chain Fv antibody , and a nanobody .
Nanobodies (Nbs) are recombinant single domain antibodies derived from the variable VHH domains from heavy chain-only antibodies (HCAbs) found in camelids (e.g., dromedaries, llamas, alpacas, etc.) [33, 34]. The VHHs have acquired important adaptations to be soluble and functional in the absence of the light chain, having longer and more diverse complementarity-determining regions (CDRs), strict monomeric behaviour, reversible folding properties, and higher resistance to proteolysis and thermal denaturation than VH domains from conventional antibodies . These intrinsic biophysical properties facilitate their expression in bacteria, yeast, and mammalian hosts [35, 36, 37, 38]. Their small size (ca. 14 kDa; 2–4 nm diameter) and long CDRs also allow binding of less accessible epitopes than those recognized by conventional antibodies, including active sites of enzymes [39, 40] and inner regions in the surface proteins of pathogens . VHHs are also highly similar to human VH3 sequences, which is important to have a low immunogenicity in therapeutic applications of Nbs [42, 43]. These characteristics have made Nbs very attractive molecules for multiple applications, including cell biology studies , protein crystallography , therapy and in vivo diagnosis of human diseases [46, 47, 48, 49]. Hence, methodologies for Nb selection, characterization and production are of great interest.
Secretion of Nbs to the extracellular medium of E. coli cultures could simplify the screening of clones binding an antigen of interest and their purification for in vitro characterization. Leakiness of the bacterial OM after overexpression in the periplasm allows, in some cases, the detection of Nbs in culture supernatants of E. coli [50, 51]. However, this is due to a non-specific release of periplasmic and cellular proteins due to bacterial lysis, and not to an actual secretion mechanism. Large accumulation of Nbs in the periplasm also leads to an impaired growth of the producing bacteria. Contrary to this, the Hly system specifically secretes the heterologous protein to the extracellular media, with little effect on the growth of the producing bacteria, given that the secreted protein is not accumulated in the periplasm and the integrity of the OM is not compromised . Considering that a Nb binding α-amylase had been secreted in an active form with Hly-secretion system of E. coli , we wondered whether the Hly-secretion system could secrete a large diversity of Nbs that can be found in an immune library. With this aim, we constructed and screened with the Hly-system an immune library of VHHs against three protein antigens from enterohemorrhagic E. coli (EHEC), namely the extracellular secreted protein A (EspA), the extracellular 280 amino acid C-terminal region of Intimin (Int280), and the translocated intimin receptor middle domain (TirM) [52, 53, 54]. Our data show that screening of E. coli culture supernatants after secretion of Nb-HlyA fusions allows the identification of a diverse set of Nbs binding these antigens. We also demonstrate that the secreted Nb-HlyA fusions present disulphide bonds, indicating a correct oxidation state of the cysteine pairs. Lastly, secreted Nb-HlyA fusions were directly purified in a single affinity chromatography step from the supernatants of small-scale E. coli cultures (200 ml), with yields ranging between ca. 200–800 μg of purified Nb-HlyA fusion (~ 0.3–1.3 mg/l per OD600).
Results and discussion
Screening of Nbs against EspA, Int280, and TirM antigens of EHEC using the Hly secretion system
Nanobodies identified against EHEC antigens by Hly screening
Disulphide bonds in the secreted Nb-HlyA fusions
In E. coli, the cytosol is a strongly reducing environment, and cytoplasmic proteins usually do not form stable disulphide bonds [56, 57]. In contrast, cysteines in extracellular and periplasmic proteins secreted by the Sec-pathway are frequently oxidized, forming disulphide bonds catalysed by specialized Dsb-enzymes located, or having their catalytic sites, in the periplasm of E. coli [58, 59]. Proteins secreted by the Hly-system do not have access to the periplasm but, despite this, disulphide bonds were formed in a secreted scFv-HlyA fusion independently of Dsb-enzymes . In contrast, when the same scFv was secreted fused to an autotransporter β-domain, which uses a periplasmic-dependent pathway, the presence of disulphide bonds required Dsb-enzymes (e.g. DsbA), which were not formed spontaneously in the extracellular medium .
Camelid VHHs frequently have, in addition to the conserved cysteine pair found in most immunoglobulin (Ig) domains, extra cysteine residues in the CDRs that also form disulphide bonds . Out of the Nb sequences selected in the Hly-screening (Fig. 3), three clones have the two cysteines of the canonical disulphide bond of Ig-domains (IC1, TD4, EC1), whereas the rest contain one extra pair of cysteines (IA1, IB10, TF2, TG10, TE4, TF1, TE1, EE6, EE7) or two (IB7). We chose two clones having either two (TD4) or four (TF1) cysteines and investigated the oxidation state of the Nb-HlyA fusions, secreted and in the producing bacteria, by incubation with the thiol alkylating reagent 4-acetamido-4′-maleimidylstilbene-2,2′-disulphonic acid (AMS). AMS has a molecular mass of ca. 500 Da and its covalent binding to free thiol groups increase the protein mass and induce a retardation of its mobility in non-reducing SDS-PAGE. In contrast, proteins having disulphide bonds do not react with AMS and show a faster electrophoretic mobility.
Purification of Nb-HlyA fusions from culture supernatants
Lastly, we confirmed the binding of the purified Nb-HlyA fusions for the corresponding antigen (i.e., Int280, TirM, EspA) by ELISA, using BSA as a negative control antigen. ELISA were developed with anti-E-tag mAb-POD and OD490 values to BSA (ca. ≤ 0.1) were subtracted from those obtained with the specific antigen at the different concentrations of the Nb-HlyA fusions (from 1 to 1000 nM; Fig. 6b). These data confirmed the activity of the purified Nb-HlyA fusions with curves indicating binding at low concentrations (1–10 nM). Hence, a single IMAC step allowed the purification of active Nb-HlyA fusions secreted in the culture supernatants of E. coli.
The Hly-system had been used previously for the secretion of a model Nb binding α-amylase , but its potential to secrete repertoires of Nbs had not been tested. This work has shown that VHH repertoires can be secreted as Nb-HlyA fusions with the Hly-system of E. coli. Culture supernatants can be used directly in ELISA to identify antigen-specific binders among the secreted Nb-HlyA fusions, as we did for an immune library of VHHs against Int280, TirM and EspA antigens of EHEC. In addition, we have shown that active Nb-HlyA fusions can be purified from culture supernatants using a single IMAC step, avoiding bacterial cell lysis. Secretion facilitates purification, reduces toxicity for the producing bacteria and minimizes the action of intracellular proteases [1, 2]. Finally, we have demonstrated that the cysteine pairs found in VHHs form disulphide bonds in the secreted Nb-HlyA fusions. In contrast, these cysteines are reduced in the cytoplasm of bacteria, indicating that oxidation of Nb-HlyA fusions occurs associated to protein secretion. Taken together, these data reveal the Hly-system as a suitable platform for the secretion of Nbs in screenings of VHH repertoires, which could be enriched by phage display or alternative yeast and E. coli display technologies . The amount of Nb produced in this work using small-scale shake-flask cultures is sufficient for in vitro characterization of the antigen-binding properties of the selected Nb clones. Higher yields of production may be attained in fed-batch cultures of E. coli (in bioreactors) in which very high cell densities can be achieved (OD600 > 50) and by optimization of the expression of Hly-export machinery and Nb-HlyA in the producing E. coli strain [1, 62, 63, 64].
Conditions for bacterial growth
Bacterial strains and plasmids used in this work
Source or reference
(F− λ−) mcrA Δmrr-hsdRMS-mcrBC φ80lacZΔM15, ΔlacX74, recA1, endA1, Δ(ara, leu)7697 galU galK rpsL(StrR), nupG tonA
Δlac-pro, ara, nalR, thi, F’(proAB lacIQ lacZΔM15)
Δ(lac-proAB) Δ(mcrB-hsdSM)5 (rK− mK−) thi-1 supE [F´ traD36 proAB lacIqZΔM15]
F−; ompT hsdSB(rB−, mB−) gal dcm lon λ(DE3[lacI lacUV5-T7 gene1 ind1 sam7 nin5])
ApR; phagemid vector for periplasmic production of Nb-pIII fusions with His and E-tag
ApR; pUC ori, lac promoter, N-terminal His tag, VHH, HA and E-tags, C-HlyA
CmR; expression of HlyB and HlyD
KmR; pBR ori, T7 promoter, N-terminal His-tagged fusions
pET28a derivative; expression of His-tagged TirM of EHEC
pET28a derivative; expression of His-tagged Int280 of EHEC
pET28a derivative; expression His-tagged EspA of EHEC
Plasmids, DNA constructs, and oligonucleotides
Oligonucleotides used in this study
Immunization and generation of the VHH library
Purified His-tagged Int280, TirM and EspA from EHEC were diluted in sterile water (2.5 ml) and mixed at 0.2 mg/ml to the same volume of adjuvant (Veterinary Vaccine Adjuvant, GERBU) reaching a total volume of 5 ml. This solution was injected subcutaneously to one male dromedary camel (Camelus dromedarius) corresponding to 0.5 mg of each antigen. After the initial immunization, four boosting immunizations were performed in the same manner once per week. Pre-immune serum was prepared from a small blood sample (5 ml) before the first injection. The immune serum was collected 7 days after the last immunization. Serial dilutions of pre-immune and immune sera were used in ELISA to confirm the immune response against the respective antigens with protein A-POD as secondary. Additional 50 ml of blood of the immunized animal were collected from the jugular vein in tubes containing EDTA as anticoagulant, using Venoject system. For lymphocyte isolation, 50 ml of RPMI-1640 medium (Sigma) were added to the 50 ml blood sample and the final mixture was divided in 4 aliquots of 25 ml. Each aliquot was added on top of a 25 ml of Ficoll-Paque Plus (StemCell Technologies) in sterile capped 50 ml Falcon tubes. After centrifugation (800g, 30 min, RT), lymphocytes were taken from the interphase, washed twice in RPMI-1640 by centrifugation (800g, 10 min), resuspended in 5 ml of RPMI-1640, and the number of cells determined in a Neubauer hematocytometer (Hausser Scientific). About 2 × 107 cells were lysed with 2 ml of Trizol (Invitrogen) for RNA extraction following manufacturer instructions. The poly-A+ mRNA was purified from total RNA using an oligo-dT resin (Oligotex mRNA Minikit, Qiagen) and directly employed as template for first-strand cDNA synthesis reactions with random hexamers and oligo-dT primers (iScript cDNA Synthesis, Bio-Rad). About 1 μl of each cDNA synthesis was used as template in 50 μl PCR reactions with oligonucleotides CALL001 and CALL002. The amplified fragments of ≈ 0.6 kb, corresponding to VHH-CH2 domains, and ≈ 0.9 kb, corresponding to conventional VH-CH1-CH2 domains, were separated in 1.2% (w/v) low melting agarose gel and the ~ 0.6 kb band was purified (Qiaex II Gel Extraction kit, Qiagen). This fragment was used as template in a second PCR reaction with oligonucleotides VHH-Sfi2 and VHH-Not2 (Table 3) to finally obtain the amplified fragments of ~ 0.4 kb, corresponding to the VHH domains. The amplified VHH fragments were cloned SfiI-NotI into the phagemid pCANTAB 5Ehis-backbone . Ligations were electroporated in E. coli TG1 cells (Stratagene) and a library size of approx. 2 × 107 clones were determined by plating dilutions on LB + Ap agar plates with 2% w/v glucose at 30 °C.
Cultures of E. coli BL21(DE3) carrying the corresponding pET28a-derivative were grown at 30 °C in 500 ml of LB + Km and induced at OD600 ~ 0.5 with 1 mM IPTG during 2 h. Cells were harvested by centrifugation (10 min,10,000g, 4 °C), resuspended in 20 ml of Solution A—NaPO4 pH 7, 300 mM NaCl, DNase (0.1 mg/ml; Roche) and protease inhibitor cocktail (Roche)—, and lysed by passing three times through a French-Press at 1200 psi. The resultant lysate was ultracentrifuged (60 min, 40,000g, 4 °C) to obtain a cleared lysate supernatant. For purification of the His-tagged Int280EHEC, EspAEHEC, TirMEHEC, the lysates were passed through 2 ml of pre-equilibrated Cobalt-containing resin (Clontech) in a chromatography column and were washed with HEPES buffer (20 mM HEPES pH 7.4, 200 mM NaCl). The bound His-tagged proteins were eluted adding the same buffer complemented with 100 mM imidazole and were collected in 0.5 ml aliquots. For the purification of the secreted His-tagged Nb-HlyA fusions, supernatants from induced cultures were loaded at ca. 1 ml/min onto chromatography columns with pre-equilibrated Cobalt-containing resin (Clontech). Columns were washed with PBS and eluted with the same buffer containing 150 mM imidazole. Fractions eluted from metal-affinity chromatography were concentrated to a final ~ 1 ml in PBS using a 3 kDa centrifugal filter unit (Amicon Ultra-15, Millipore). Protein concentration was estimated using the Bicinchoninic acid protein assay kit (Thermo Scientific).
Packaging of Phabs into M13 particles
For preparation of phage antibodies (Phabs) a mixture of E. coli TG1 cells representing the library or a subpopulation after panning, was incubated in 25 ml of LB-Ap supplemented with 2% (w/v) glucose at 30 °C until OD600 ~ 0.2. At this point, 1010 plaque forming units (PFU) of VCS-M13 helper phage (KmR; Stratagene) were added for 1 h incubation at 37 °C with gentle agitation. Then, E. coli cells were harvested by centrifugation (5 min, 4000g, RT) and resuspended in 250 ml of fresh LB + Ap + Km. After 16 h of incubation at 30 °C, the cultures were chilled on ice and centrifuged (10 min, 8000g, 4 °C). To recover the M13-particles from the supernatant, 50 ml of PEG-NaCl solution (20% w/v polyethylene glycol 8000; 2.5 M NaCl) were added and the resulting mixture was kept on ice for additional 45 min. Phage pellets obtained after centrifugation (20 min, 10,000g, 4 °C) were resuspended in 2 ml of TE (10 mM Tris–HCl, 1.0 mM EDTA, pH 8.0) and stored at − 80 °C.
Library enrichment by Phab panning
All steps were performed at room temperature (RT). Each antigen (10 µg/ml, in PBS) was adsorbed for 2 h to 4 wells (50 µl/well) of a microtiter immunoplate (Maxisorb, Nunc). These solutions were discarded, and the wells were blocked by adding 200 µl/well of PBS with 3% w/v skimmed milk and 1% w/v BSA (Sigma). After 2 h, the blocking solution was replaced by a total of 2 × 1011 PFU of Phabs in 50 µl of PBS with 3% w/v skimmed milk. Phabs were allowed to bind for 1 h, and the unbound Phabs were removed from the plates by 20 washes of 1 min employing 200 µl/well of PBS with Tween20 0.05% and another 20 washes with PBS. To collect the bound Phabs, wells were incubated during 5 min with 0.1 M glycine pH 2.5 (50 µl/well). The solution from the wells was pooled together and immediately equilibrated by addition of one volume (400 µl) of 1 M Tris–HCl, pH 7.5. Phabs in this solution were later used to infect TG1, which were plated on LB + Ap agar plates. After 24 h incubation at 30 °C, colonies grown on these plates were harvested as a pool and used for phagemid packaging.
Selection of individual specific binders
For rapid screening of individual Phab binders, a small-scale rescue of phagemids was performed in 150 µl cultures of TG1 grown in 96-well microtitre plates using 109 PFU of helper-M13 phages. After production of the phagemids the plate was centrifuged (10 min, 600g, RT) and the supernatants (containing the Phabs) were used in ELISA to determine their specific binding to the antigen. At least 2 × 106 independent colonies from each enriched Phab library were harvested from agar plates, and 50 units of OD600 were used for plasmid isolation (NucleoBond Xtra Midi, Macherey–Nagel). The VHH fragments were cloned SfiI-NotI into the pEHLYA5 backbone vector under the Plac promoter. The size of each library was 2–3 × 106 clones, as determined by plating dilutions on LB + Ap agar plates with 2% (w/v) glucose incubated at 30 °C. Isolated plasmids from the pools were transformed in strain HB2151 harbouring plasmid pVDL9.3 and individual colonies were isolated in 96-well microtitre plates with 200 µl of liquid LB-media with appropriated antibiotics and 1 mM IPTG for the secretion of the Nb-HlyA fusions.
Alkylation of thiol groups in Nb-HlyA fusions
Protein samples from extracellular media and whole-cell bacteria were treated with the alkylating reagent 4-acetamido-4′-maleimidylstilbene-2,2′-disulphonic acid (AMS; ThermoFisher Scientific) as reported previously . Briefly, for Nb-HlyA in supernatants, four aliquots of 800 µl from each induced culture supernatant were taken. Two of these aliquots were incubated with dithiothreitol (DTT, 25 mM) to obtain positive controls of reduced proteins. After incubation for 10 min at 37 °C, all four aliquots were precipitated with trichloroacetic acid (TCA, 10% w/v) for 1 h at 4 °C, and precipitated proteins were recovered by centrifugation (14,000g, 15 min). The protein pellets were washed twice with 1 ml of ice-cold acetone followed by centrifugation (14,000g, 15 min). Two of these protein pellets (AMS positive samples) were resuspended in 75 µl of a freshly prepared alkylation buffer (150 mM Tris HCl pH 7.5, 1% w/v SDS, 5% v/v glycerol, 15 mM AMS). The other two samples were resuspended in 75 µl of the same buffer without AMS. After 1 h incubation at 22 °C, 75 µl of non-reducing SDS-PAGE sample buffer was added (see below).
For intracelullar Nb-HlyA, bacteria from 1 ml of induced cultures were harvested, washed once, and resuspended in the same volume of LB. Samples were divided in four 200 µl aliquots and two of them were adjusted to 0.1 M DTT (DTT+ samples). Following 20 min incubation on ice, TCA was added (10% w/v). After 1 h incubation at 4 °C, the precipitated proteins collected by centrifugation (14,000g, 15 min, 4 °C). Pellets were washed once with 1 ml of ice-cold acetone, air-dried, and resuspended in 20 µl of alkylation buffer (AMS positive samples), or 20 µl of the same buffer without AMS. After incubation for 1 h at 22 °C, 10 µl of non-reducing SDS-PAGE sample buffer were added. All samples were boiled at 100 °C for 10 min before loading for SDS-PAGE (5–10 µl/well).
SDS-PAGE and Western blot
Sodium Dodecyl Sulfate–Polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot were performed following standard methods  using the Miniprotean III system (Bio-Rad). The sample buffer for reducing SDS-PAGE is 60 mM Tris–HCl pH 6.8, 1% w/v SDS, 5% v/v glycerol, 0.005% w/v bromophenol blue and 1% v/v 2-mercapto-ethanol (2-ME). In non-reducing SDS-PAGE, the sample buffer was devoid of 2-ME. Proteins separated by SDS-PAGE were either stained with Coomassie Blue R-250 (Bio-Rad) or subjected to Western blot. For the latter, the proteins were transferred to a polyvinylidene difluoride membrane (PVDF, Immobilon-P, Millipore) using semi-dry electrophoresis (Bio-Rad), as previously described . Nb-HlyA fusions were detected with primary mAb anti-E-tag (Phadia, 1:5000) and secondary polyclonal rabbit anti-mouse IgG antibodies fused to POD (1:5000, Sigma). Membranes were developed using a mixture of 100 mM Tris–HCl (pH 8.0) containing 1.25 mM luminol (Sigma), 0.22 mM cumaric acid (Sigma), and 0.0075% (v/v) H2O2 (Sigma). The membranes were then developed by exposure to X-ray films (AGFA).
Enzyme-linked immunosorbent assays (ELISAs)
ELISA conditions were based on those described previously . Briefly, 96-well immunoplates (Maxisorp, Nunc) were coated for 120 min at RT with purified antigens (as indicated) diluted in PBS at a concentration of 5 µg/ml. Bovine serum albumin (BSA, Roche) was used as a negative control for detection. Phabs, culture supernatants or purified Nb-HlyA fusions were added to the wells at the indicated dilutions. For detection of bound Phabs, an anti-M13-HRP mAb (GE Healthcare) was added (1:5000). For detection of Nb-HlyA fusions, anti-E-tag mAb (1:2000; Phadia) and anti-mouse IgG-POD (1:2000; Sigma), as secondary antibody, were added. The reaction was developed with o-phenylenediamine (OPD, Sigma) and H2O2 (Sigma) and the OD490 was determined using a microplate reader (iMark ELISA plate reader, Bio-Rad).
DRG and SF performed the experiments; CG performed camel immunization and collect camel blood samples; DR, SF, CG and LAF designed the experiments and analysed data; LAF conceived this study and secured funding. DRG and LAF wrote the manuscript and prepared the Figures and Tables. All authors read and approved the final manuscript.
We thank Prof. Víctor de Lorenzo (CNB-CSIC) and Prof. Gad Frankel (Imperial College London) for helpful discussions and support. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).
The authors declare that they have no competing interests.
Availability of data and materials
All data generated and/or analysed during this study are included in this published article.
Consent for publication
Ethics approval and consent to participate
The camel immunization protocol followed animal experimentation guidelines published by the regional government of the Canary Islands (Spain) and was approved by the Ethics Committee, Veterinary Medicine Service, Las Palmas de Gran Canaria University Foundation (Ref.: 0014/2011).
Work in the lab of LAF is partially supported by the Grants BIO2017-89081-R (Agencia Española de Investigación AEI/MICIU/FEDER, EU), ERC-2012-ADG_20120314 (European Research Council, EU) and LCCM_P59474 (Wellcome Trust, UK).
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