For L. lactis, GM17 broth (M17 broth (Oxoid) supplemented with 0.5% glucose) was used as a standard culture medium. Luria-Bertani (LB) broth (10 g peptone from casein, 5 g yeast extract and 5 g sodium chloride per litre) was used for Escherichia coli cultures. Solid media were prepared by adding technical agar (Merck) to the corresponding broth with a final concentration of 1.5% w/v. L. lactis cultures were incubated statically at 30°C while E. coli was incubated at 37°C with shaking when applicable. When required, ampicillin (Amp) was used at a concentration of 100 μg/ml for E. coli while chloramphenicol (Cm) was used at 10 μg/ml for both E. coli and L. lactis.

Bacterial strains and vectors used in the present study are described in Table 1 while PCR primers are summarized in Table 2. High fidelity KOD hot start DNA polymerase (Novagen) was used in all PCR reactions throughout the whole procedures following manufacturer's instructions. Restriction enzymes and T4 DNA ligase were purchased from Roche Diagnostics (Mannheim, Germany). The gene encoding listeriolysin O, hly, of L. monocytogenes EGD-e was PCR-amplified without the signal peptide coding sequence from the chromosomal DNA [GenBank: AL591824] using primers 1 and 2. The resulting PCR product was sequentially digested by BamHI and PstI respectively. Ligation to a similarly digested pQE30 vector (Qiagen) was successfully performed using T4 DNA ligase. The ligation reaction mixture was transformed into chemically-competent E. coli BL21 (Novagen) by heat shock following manufacturer's instructions and plated onto LB agar containing 100 μg/ml ampicillin. Positive colonies were identified by PCR and plasmid was extracted from BL21 using Qiagen Miniprep Kit (Qiagen). The resulting plasmid (pQE30/hly) had an N-terminus six-histidine tagged hly gene and the correct nucleotide sequence was confirmed by DNA sequencing (Lark Technologies Inc., UK). pQE30/hly was utilised as a template for further cloning steps outlined below.

The lactococcal plasmid pNZ8048 13 was manipulated to allow insertion of the His-tagged hly gene. Plasmid pNZ8048 contains the PnisA promoter (nisin inducible) with a downstream start codon (ATG) inside an NcoI restriction site (CC

ATG

Filter-sterilized culture supernatant of the nisin-secreting strain L. lactis NZ9700 was used as a source of nisin 18. Sterile culture supernatant of L. lactis NZ9700 was stored in small aliquots at -20°C and one aliquot was thawed and used as required. The use of the same filter-sterilized batch of supernatant throughout the study ensured consistency of the nisin content between induction experiments. Overnight culture of L. lactis NZ9000 (pNZPnisA:CYTO-LLO) was subcultured into fresh GM17 broth (Cm 10 μg/ml) and incubated statically at 30°C. Nisin was added when the optical density at 600 nm (OD600) reached 0.5. Induction was conducted statically at 30°C after which cells were pelleted (3200 × g for 10 min) and washed once with buffer (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, pH 8). Pellets were frozen at -80°C prior to sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) or protein purification.

Frozen induced pellets from 500 ml cultures were thawed on ice and resuspended in 10–15 ml ice-cold lysis buffer (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, pH 8) containing 30 mg/ml lysozyme (Sigma) and kept for 30 min on ice. This was followed by sonication at maximum amplitude (Soniprep 150, MSE UK Ltd.) using eight ten-second pulses with intervening ten-second pauses on ice. For small volume cultures (10–50 ml), pellets were resuspended in 500 μl lysis buffer in eppendorf tubes and acid-washed glass beads (Sigma) were added followed by beating the tubes in a bead beater (Mini beadbeater-8, Biospec products) for three one-minute beats with one-minute pauses in between on ice. Clear supernatant was obtained after centrifugation at 10000 × g for 30 min at 4°C.

For SDS-PAGE, 12% SDS-polyacrylamide separating gel and 4% stacking gel were used and gels were stained with coomassie blue stain followed by destaining and gel imaging. For Western blot, pre-stained protein marker was used (Amersham Biosciences UK, Ltd) and gels were blotted against a nitrocellulose membrane (Hybond-ECL™, Amersham Biosciences UK, Ltd.) using a semi-dry Western transfer apparatus. Membranes were blocked overnight at 4°C in 5% skimmed milk in TBS buffer (0.8% NaCl, 20 mM Tris-HCl, pH 7.6). Primary rabbit anti-LLO antibody (Diatheva, Italy) and secondary anti-rabbit antibody (Amersham ECL Western Blotting System) were used at 1/1000 and 1/1500 dilutions in 5% and 10% skimmed milk in TBS buffer respectively. Western blot detection was done using Amersham ECL Western Blotting System (Amersham Biosciences UK, Ltd.) using the protocol recommended by the manufacturer.

Clear supernatant obtained after cell lysis (performed as described in the previous section) was passed through a disposable chromatography column (Biorad) containing 2 ml Ni-NTA affinity gel (Qiagen). Column was washed twice with wash buffer (50 mM NaH₂PO₄, 300 mM NaCl, 20 mM imidazole, pH 8) then eluted in fractions of 0.5 ml with elution buffer (50 mM NaH₂PO₄, 300 mM NaCl, 250 mM imidazole, pH 8). Elutions were checked by SDS-PAGE and combined together and dialysed overnight at 4°C against 2 l of dialysis buffer (500 mM NaCl, 10 mM Na₂HPO₄, 0.5 mM EDTA, 0.02% NaN₃, pH 7.0). Listeriolysin O concentration was measured as total protein concentration by Micro Lowry total protein kit (Sigma) and also by absorbance at 280 nm using the molar extinction coefficient for His-tagged LLO as 71,950 cm^-1 (calculated based on the actual sequence of the His-tagged LLO) 19. The purified protein was stored at 4°C.

Haemolytic activity of LLO was measured according to the method described by Khoda et al 20 with slight modification. Briefly, defibrinated sheep blood was centrifuged at 800 × g for 10 min at 4°C, then washed twice with phosphate buffered saline (PBS) (Gibco) pH 5.5. The pelleted red blood cells (RBCs) were diluted with PBS (pH 5.5) to obtain 0.5% RBCs volume/volume (v/v). Aliquots of 100 μl of the RBCs suspension were distributed in 1.5 ml tubes and twofold serial dilutions of LLO (in PBS pH 5.5) was added to each tube to a final volume of 1 ml. LLO final concentrations covered the range from 8 μg ml^-1 to 29 pg ml^-1. A positive control (distilled water, 100% haemolysis) and a negative control (PBS pH 5.5) were also included. Tubes were incubated statically at 37°C for 45 min after which they were centrifuged at 1700 × g for 5 min and supernatants were collected. Absorbance was measured colorimetrically at 415 nm and haemolytic units were calculated. One haemolytic unit (HU) was defined as the amount of protein required to cause 50% haemoglobin release from sheep RBCs as compared to the 100% haemoglobin release of the positive control (distilled water) 20.

Native LLO secreted by Listeria monocytogenes EGD-e is composed of a secretory signal peptide (25 amino acids) and the actual active portion of LLO (504 amino acids). In the cloning described in this paper we omitted the native secretory signal sequence (coding for the first 25 amino acids) of LLO and added six histidine amino acids (a His-tag) at the N-terminus (Figure 2). DNA sequencing of the constructed pNZPnisA:CYTO-LLO confirmed the addition of the six-His-tag upstream of the hly gene and the integrity of the remainder of the hly gene sequence. Overall we added a total of 10 amino acids (including the 6 histidine residues) at the N-terminus of LLO, in place of the signal sequence, to facilitate tagging and purification of the protein (Figure 2). These few amino acids did not affect protein activity as shown below.

We performed a series of induction experiments on L. lactis NZ9000 (pNZPnisA:CYTO-LLO) to determine the optimum conditions for protein expression. Preliminary induction time-course experiments showed the optimum duration for nisin-induction to be 3–4 h (data not shown). Consequently, induction was performed at mid-log phase (OD600 = 0.5) for three hours. Initially, different nisin concentrations were examined: 0.02%, 0.1%, and 0.2% (v/v), to determine the optimum concentration for induction and SDS-PAGE was performed to assess the amount of LLO produced (Figure 3A). Subsequently, for protein over-production experiments, 0.2% v/v nisin concentration was chosen for induction to maximize the amount of LLO produced. Western blot was also performed and confirmed the production of pure non-degraded LLO (Figure 3B). It is noteworthy that we detected minimal basal expression of LLO in the uninduced L. lactis NZ9000 (pNZPnisA:CYTO-LLO) with no apparent toxicity on bacterial growth (data not shown). The inducer of basal PnisA activity in our experiments in the absence of nisin is not clear although other inducers such as lactose and galactose have been previously reported to induce this promoter at low levels 21. In our experiments the basal LLO expression was negligible compared to the amount of LLO produced upon nisin induction.

To obtain rapid and reproducible purification of LLO, we used the Ni-NTA technology to capture and purify the six-His-tagged protein by affinity chromatography under native purification conditions. Figure 4 shows a representative purification procedure of the His-tagged LLO (about 57 kDa) checked by SDS-PAGE. The average yield of pure LLO (measured as total protein) after 3 h-induction at 30°C using a concentration of 0.2% v/v of nisin was 3 mg per litre culture as measured by both Lowry and A₂₈₀ absorbance methods. When the induction time was increased to 4 h at 30°C, the yield ranged from 4.43–5.9 mg per litre culture. However, to increase the total yield of LLO, we modified the LLO extraction method by applying double sonication to the 4 h-induced pellets of L. lactis NZ9000 (pNZPnisA:CYTO-LLO). In brief, after the first sonication was completed (as described in the Methods section), the supernatant was collected for Ni-NTA purification and the remaining pellets were frozen again at -20°C for 24 hours then thawed, resuspended in lysis buffer and sonicated again. Supernatant was collected and then Ni-NTA-gel-purified. This second sonication of the same pellets simply extracted LLO which had not been released from Lactococcus cells upon the first treatment. Eluted proteins from the two sonication treatments were tested by SDS-PAGE for purity (Figure 5) then combined and dialysed as mentioned earlier. This double sonication approach increased the total combined yield of LLO to the range of 9.3–12.9 mg per litre culture (average 11.6 ± 1.56 mg per litre culture). However, when the resulting LLO was examined by Western blot, minor lower molecular weight bands appeared below the major LLO band. This indicated slight degradation of LLO upon the double sonication procedure (Figure 6).

To check the functionality of LLO, the haemolytic titre test was carried out and specific LLO haemolytic activity ranged from 5 × 10⁵ to 5 × 10⁷ HU per mg total protein. This haemolytic activity was still detectable after at least 8 months of storage at 4°C.