The E. coli strains used were DH5α [F⁻Φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (rk−,mk+) phoA supE44 thi-1 gyrA96 relA1 λ⁻] and BL21 (DE3) [F⁻ompT hsdS_B (rB−,mB−) gal dcm (DE3)] from Invitrogen (CA, USA). The plasmid used was pET28a(+) containing a 6-histidine tag (His-tag) at both the N- and C-terminal from Novagen (Darmstadt, Germany). T4 ligase and T4 buffer DNA ligase (2×) were purchased from Promega Corporation (WI, USA). The enzymes used (BamHI and HindIII) and the induction agent isopropyl β-d-1-thiogalactopyranoside (IPTG) were obtained from Thermo Scientific (MA, USA). The monoclonal anti-polyHistidine antibody produced in mouse, anti-mouse IgG (whole molecule) peroxidase antibody and 3′3′-diaminobenzidine (DAB) were purchased from Sigma-Aldrich (MO, USA). Luria–Bertani (LB) medium was from BD (NJ, USA), and kanamycin from Gibco (MA, USA).

The EspB synthetic gene was developed considering common regions of all known EspB variants to date, by alignment of α1, α2, α3, β, and γ EspB sequences (GenBank number: AAC38396.1, AEW69664.1, AEW69663.1, CAA74174.1, and CAA65654.1) using the Basic Local Alignment Search Tool (BLAST). The synthetic gene for the hybrid rEspB protein was assembled on the basis of the most prevalent amino acids among the variants for each position of the protein sequence (Figure 1). The restriction enzymes were selected with the support program BioEdit version 7.2.0, and the restriction sites for the enzymes BamHI and HindIII were inserted into the conserved sequence upstream and downstream, respectively (Figure S1 in Supplementary Material), while no stop codon was added in the sequence. The predicted recombinant protein had a molecular weight of 24.6 kDa. The EspB gene was manufactured by GenScript (NJ, USA) and cloned into pUC57 vector.

Chemically competent E. coli BL21 (DE3) were obtained using the Chung and Miller protocol, with modifications (26). The gene of interest was excised from pUC57 by restriction enzyme digestion and then cloned into the pET28a expression vector. The reaction mixture consisting of 2 µL of deionized water, 5 µL of the gene, 1 µL of the pET28a vector, 1 µL of T4 DNA ligase (3 IU), and 2 µL of T4 buffer DNA ligase (2×) was incubated at 24°C for 1 h, followed by a 4°C incubation for 18 h.

For E. coli BL21 (DE3) transformation, 1 µL of plasmid was incubated with 2 µL of 5× KCM buffer (0.5 M KCl, 0.15 M CaCl₂, and 0.25 M MgCl₂) and 7 µL of deionized water on ice for 5 min, followed by the addition of 10 µL of chemically competent cells; after 20 min, the solution was transferred to 24°C for 10 min. Subsequently, 200 µL of LB culture medium were added and the sample was incubated at 37°C for 1 h. The cells were then streaked on a LB agar plate containing 50 µg/mL of kanamycin and incubated at 37°C for 18 h.

BL21 His-EspB transformant was cultivated in 10 mL of LB medium containing 50 µg/mL kanamycin at 37°C for 18 h with stirring at 250 rpm. The culture was then added to 500 mL of LB medium supplemented with 0.2% glucose and 50 µg/mL of kanamycin, and further grown at 37°C for 2 h at 250 rpm. After reaching an optical density of 0.6–0.8 (OD₆₀₀), IPTG was added to a final concentration of 1 mM, and the culture was then incubated at 37°C for 4 h at 250 rpm. The cells were separated in a 5804 R centrifuge (Eppendorf, Hamburg, Germany) at 10,000 × g for 10 min, and the supernatant discarded. The pellet was resuspended in 60 mL of ligation buffer (20 mM Tris–HCl, pH 7.9, containing 0.5 M NaCl) with 1% 100× protease inhibitor cocktail and 50 µg/mL lysozyme, and allowed to stand in an ice bath for 30 min. The cells were lysed by three cycles of sonication for 10 min, with the amplitude set at 30% (Sonopuls Bandelin, Berlin, Germany). The lysate was centrifuged at 10,000 × g for 10 min and the resulting pellet was solubilized with 30 mL of buffer with 8 M urea, with stirring at 4°C for 18 h.

Purification was performed by metal affinity chromatography by gravity flow. Following urea treatment, 2 mL of Ni-NTA Agarose (Qiagen, NW, Germany) were added to the solubilized pellet and the suspension incubated at 4°C for 18 h with gentle shaking. The suspension was centrifuged at 30 × g for 1 min and pellet-containing agarose was gently transferred to a polypropylene column (Qiagen, NW, Germany). rEspB protein was eluted with buffer containing different concentrations of imidazole: 10, 20, 50, 100, 150, 200, 300, 400, and 500 mM. The eluted protein was refolded by long-term dialysis and subsequently concentrated by osmosis with PEG 4000. SDS-PAGE (12%) and immunoblotting were used to confirm the purification. The recombinant protein was quantified with a NanoDrop Lite Spectrophotometer (Thermo Scientific, MA, USA) and stored at 4°C. Identification of rEspB protein was performed by liquid chromatography coupled to mass spectrometry (LC–MS/MS). After SDS-PAGE analysis, protein bands were subjected to in gel trypsin digestion (27) and the resulting peptide mixture was analyzed by LC–MS/MS as described elsewhere (28). LTQ-Orbitrap Velos raw data were searched against a target database (UniProt restricted to E. coli; 22,940 sequences) using Mascot search engine (Matrix Science, UK).

Protein secondary structure was confirmed after refolding by circular dichroism (CD). The CD spectra were recorded between 190 and 260 nm using a quartz cuvette (0.1-mm path length) in a JASCO J-810 Spectropolarimeter (Jasco Corporation, Japan). After buffer-background subtraction (10 mM sodium phosphate buffer, pH 8.0), the CD data were converted to mean residue ellipticity [θ] units (degree × cm² dmol⁻¹). CD spectra were obtained at three different pH (7.0, 8.0, and 9.0) and at temperatures ranging from 5 to 95°C. The results were analyzed on the online server DICHROWEB (29–31), using the analysis program CDSSTR (32–34).

Antibody-binding epitopes were determined by designing a CelluSpots^® peptide array (INTAVIS Bioanalytical Instruments AG, NW, Germany), with 384 dots containing the full protein sequence divided in peptides with 11 amino acids/dots, having 8 overlapping amino acids. The sequences in the array were derived from the rEspB protein, which represents the α variants, as well as the full β and γ sequences (Table S1 in Supplementary Material) (5, 10, 11, 13). The assay was performed following the manufacturer’s recommendations: briefly, the slides were blocked by immersion in 1% BSA at 4°C for 18 h with shaking. Anti-EspB monoclonal antibody (mAb) (10 µg/mL) and polyclonal antibody (pAb) (30 µg/mL) were incubated at 24°C for 4 h with stirring. The slides were washed three times with 0.05% Tween in PBS (0.01 M, pH 7.2) for 5 min. The slides were then incubated with peroxidase-conjugated anti-mouse IgG antibody (1:5,000) at 24°C for 2 h with stirring. Detection was performed with DAB and hydrogen peroxide and the reaction stopped with distilled water.

Polyclonal serum was obtained from a New Zealand White female rabbit (60 days old) after immunizing intramuscularly, three times with 2-week intervals, using a dose of 100 µg of rEspB protein adsorbed to 2.5 mg alum (Al³⁺) as adjuvant. Serum was obtained 45 days after the first immunization. Immune serum reactivity was tested by indirect ELISA (35). Serum samples were obtained just before immunization from the auricular vein, which were used as the negative control in specific antibody evaluation. The anti-EspB mAb A5 was raised in the present study as in previous work by our group where mAb 4D9 was obtained (17).

The hybrid rEspB protein was obtained from the E. coli BL21 transformed with a plasmid harboring the hybrid espB gene. Restriction analysis confirmed that all clones had the same plasmid profile, and synthetic gene cloning was confirmed by sequencing. The protein was expressed in inclusion bodies; thus, urea treatment was necessary before the purification process. Since there was no stop codon in the cloned gene and since pET28a was the expression vector, the recombinant protein was expressed with two His-tag tails, one at each end of it. The protein was eluted using different imidazole concentrations, with effective elution occurring between 100 and 200 mM imidazole (Figure 2).

To confirm the identity of rEspB, protein bands indicated in Figure 2A were subjected to mass spectrometric analysis, which resulted in the identification of nine tryptic peptides (⁹⁵AGATAALIGGAISSVLGILGSFAAINSATK¹²⁴, ²¹⁶AGLLSR²²¹, ²²²FTAAAGR²²⁸, ²²⁹ISGSTPFIVVTSLAEGTK²⁴⁶, ²⁴⁷TLPTTISESVK²⁵⁷, ²⁵⁸SNHDINEQR²⁶⁶, ²⁸⁸AQDDISSR²⁹⁵, ²⁹⁸DMTTTAR³⁰⁴, and ³⁰⁵DLTDLINR³¹²), confirming the expression and isolation of protein EaeB (UniProt entry EAEB_ECO27).

The EspB CD spectra showed a negative ellipticity band at 222 nm, which corresponds to α-helix structure (36). This secondary structure was observed at all pH tested; however, ellipticity was closer to 0 at pH 7.0, indicating less α-helix content under this condition when compared to pH 8.0 and 9.0. Indeed, deconvolution analysis showed a higher level of unordered content at pH 7.0 and, on the other hand, an increase in α-helix content at pH 8.0 and 9.0 (Figure 3; Table S2 in Supplementary Material). Effects on protein secondary structures were observed at a higher temperature, indicating protein denaturation by heat (Figure 4; Table S3 in Supplementary Material).

Treatment with temperatures over 60°C resulted in altered secondary structure stability, showing this to be the necessary temperature to denature the rEspB protein. There was a slight reversible loss of secondary structure after heating to 95°C and gradual cooling to 5°C. Close to the storage temperature, at 5°C, EspB protein secondary structure exhibited approximately 18% α-helix, 20% β-sheet and 20% turns, and 42% irregular structures. As the temperature increased, the proportion of turns and irregular structures tended to be the same, while the α-helix content decreased and β-sheet content increased. Above 60°C, there were no more alterations in secondary structure, with α-helix around 5% and β-sheet 31%.

The anti-EspB pAb generated against the hybrid rEspB was able to recognize several dots on the peptide array, suggesting that those epitopes are involved in antibody binding. The pAb reacted with 11 points on α EspB, 4 on β EspB, and 5 on γ EspB (Table 1). Several spots were adjacent to each other, which resulted in three sequences for the α variant (¹⁹⁸ASQVAEEAADA²⁰⁸, ²³¹GSTPFIVVTSLAEG²⁴⁴, and ²⁶⁴EQRAKSVENLQASNLDTYKQDVRRAQDDISSR²⁹⁵) and the same two sequences for the β and γ variants (²³²STPFIVVTSLAEGT²⁴⁵ and ²⁷¹ENLQASNLDTYKQDVRRAQDDISS²⁹⁴). The common domain between all sequences could be defined as ²³²STPFIVVTSLAEG²⁴⁴ and ²⁷¹ENLQASNLDTYKQDVRRAQDDISS²⁹⁴.

The mAbs 4D9 and A5 were used for comparison with the pAb, since they were obtained against the α EspB variant. Individually, mAb 4D9 reacted with 2 points on α EspB, 3 on β EspB, and 2 on γ EspB, and mAb A5 reacted with 5 points on α EspB, 6 on β EspB, and 1 on γ EspB (Table 1). The common region between the mAbs was two dots on α EspB (¹²⁷DAAQELAEKAG¹³⁷ and ²²³ARDLTDLINRM²³³), one dot on β EspB (²⁹⁵AARDLTDLQNR³⁰⁵), and one dot on γ EspB (¹³⁹AAGAASEVANK¹⁴⁹) (Figure S2 in Supplementary Material).

Another BLAST alignment of the two major sequences recognized by the pAb was performed against non-redundant protein sequences (nr) within bacteria (taxid:2) to evaluate if the sequences actually correlated with EspB and had 100% identity to the enterobacterial EspB protein from E. coli (data not shown).