The expression plasmid pET24-BthTX-1, which contains codons for translation of a protein with a Met residue, followed by the sequence of BthTx-1, was transformed by electroporation in competent E. coli BL21 cells. For BthTx-1 expression, a culture was carried out in a 1000 ml shake flask containing 250 ml of rich culture medium (2x-HKSII) supplemented with kanamycin (50 μg/ml). The flasks were inoculated with a pre-cultivated medium (20 ml) containing E. coli harboring the expression plasmid and incubated (37°C) in a rotary shaker until optical density (OD_600 nm) of the culture reached the value of 3.0. Protein expression was then induced with addition of isopropyl β-d-thiogalactopyranoside IPTG (0.5 mM, final concentration) and the cultures were grown for 16 additional hours and harvested by centrifugation. The pellets were resuspended in Tris–HCl (50 mM, 50 ml, pH 7.5), containing EDTA (2 mM), Triton X-100 (0.1%, v/v) and 50 μg/ml lysozyme and incubated at 25°C for 15 min. The suspension was sonicated and centrifuged (8,000×g, 4°C, 10 min). The supernatant was discarded; the pellet was resuspended in 50 ml Tris–HCl (50 mM, pH 7.5), EDTA (2 mM), and Triton X-100 (0.1%, v/v). The centrifugation and resuspension steps were repeated five times; the resulting pellet was dissolved in Tris–HCl (50 mM, pH 7.5), resuspended in the same buffer and stored (−20°C) for further assays.

Suspensions of IBs were diluted in refolding buffer (50 mM Tris–HCl, pH 7.5, 1 mM EDTA) containing different proportions of GSH/GSSG and concentrations of the redox pair, GdnHCl or different pHs, as indicated in Table 1 and Fig. 1. Suspension samples (1.0 ml) were placed into plastic bags, which were sealed and placed into a larger plastic bag, which was also sealed under vacuum. This bag was then placed into a pressure vessel (High Pressure Equipment, Model R4-6-40) filled with a mixture of water and oil as pressure-transmitting fluid and, high pressure (2 kbar) was applied (16 h) using an air compressor fitted to the system (High Pressure Equipment, Model PS-40). After decompression, the samples were centrifuged (12,000×g, 15 min) to remove the insoluble aggregates. The supernatants were dialyzed, centrifuged again to remove insoluble aggregates formed during dialysis and stored (−20°C) for later analysis. All assays were performed at least twice.Table 1

Analysis of the soluble BthTx-1 obtained by pressurization of IBs suspensions

Insoluble (IBs) and soluble BthTx-1 were diluted in Tris–HCl (50 mM, pH 7.5) buffer containing urea (8 M) at appropriate ratios, and submitted in duplicate to total protein determination (Bradford assay). An absorbance curve (A_nm) for pure bovine serum albumin (BSA; 15–500 μg/ml; Bradford assay) samples was used as a standard. The same samples were also submitted to both SDS-PAGE and staining with Coomassie blue. Digital densitometry of BthTx-1 bands was determined using the Image J software.

Scanning electron microscopy (SEM) was performed after drying water-dialyzed insoluble aggregates (IB or the non-refolded aggregates) onto clean polished Philips stubs and sputter-coated in a sputter coater (Balzer, model SCD-040, 38 mA, 120 s). The samples were then examined and photographed in a Field Emission Scanning Electron Microscope (JEOL, JSM 670-1F). Size analysis of IBs was performed using the Image Tools software.

C2C12 (ATCC CRL-1772), a murine skeletal myoblast cell line which can fuse and differentiate into myotubes, was grown in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma D-5796) supplemented with 10% fetal calf serum, 2 mM glutamine, 1 mM pyruvic acid, penicillin (100 U/ml), streptomycin (0.1 mg/ml), and amphotericin B (0.25 μg/ml), in a humified atmosphere with 5% CO₂, at 37°C. C2C12 cells were seeded in 96-well microplates, at an approximate initial density of 1–4 × 10³ cells per well. After reaching near confluence, usually in 3–5 days, medium was replaced by differentiation medium, which consisted of DMEM supplemented with 1% FCS. After 4–6 additional days of culture, cells were incubated with BthTx-1 purified from crude venom, used as a control or recombinant BthTx-1 refolded at high pressure, diluted in a total of 250 μl culture medium/well. After 4 h incubation, aliquots of the supernatant were collected and LDH activity was determined using a commercial kit (LDH Liquiform-Labtest) and expressed as percentage of LDH release [24]. Reference control for 100% LDH release consisted of supernatant of cells incubated with medium from cells incubated with 0.1% (v/v) of Triton X-100. The assays were carried out in triplicates.

The extraordinarily high number of free cysteine thiol groups present in the native BthTx-1 molecule was regarded as an indication that refolding of this protein could be complicated by the presence of non-native intra- and inter-protein covalent disulfide bonds in IBs. Previous works reported that HHP in combination with oxido-shuffling agents, such as glutathione, can effectively refold covalently cross-linked aggregates of recombinant proteins produced as IBs in E. coli [16, 20]. In order to evaluate the potential benefit of this approach, suspensions of IBs were incubated (2 kbar) in refolding buffer (50 mM Tris–HCl, pH 7.5; 2 M GdnHCl) containing varying ratios of reduced (GSH) and oxidized (GSSG) glutathione molecules at a 10 mM final concentration. Presence of BthTx-1 in the supernatants after compression followed by dialysis and centrifugation, performed to withdraw the denaturing reagent and insoluble aggregates eventually formed during dialysis, was analyzed by SDS-PAGE (Table 1) and quantification of the bands of protein with a molecular mass of about 14 kDa, corresponding to BthTx-1. The sample of HHP-treated BthTx-1 exhibiting the most intense band, was obtained with a 2:3 ratio of the GSH:GSSG redox pair. For a fixed 2 GSH:3 GSSG proportion, 3 mM proved to be the total molar concentration of the redox mixture in which the highest level of soluble BthTx-1 was obtained for pressure-induced refolding. Low (non-detectable) yields of soluble BthTx-1 was observed for supernatant of suspensions of IBs incubated at HHP in the absence of the redox pair, indicating that these reagents are essential for BthTx-1 refolding.

Incubation of BthTx-1 (2 kbar, 16 h) was performed in the presence of refolding buffer at various pHs (5.5–9.0) and pH of 7.5 was chosen. A low value of intensity of band was obtained at the sample compressed at pH 8.5, probably due to protein precipitation at this pH near the pI value for BthTx-1 (pI = 8.2).

Presence of additives at concentrations usually reported in the literature for protein refolding [25] (0.1% polyethylene glycol 6000, 0.5 M arginine, 2.5 M glycerol, 1 M sucrose, 1 M glucose, 0.1 M Tween-20, 0.5 mM Triton X-100, and 0.15 M NaCl) did not improve the yields of soluble BthTx-1 (data not shown).

Presence of non-denaturing levels of GdnHCl was previously shown to improve the yields of refolding of aggregated proteins subjected to HHP. Pressurized BthTx-1 IBs were not solubilized in the absence of GdnHCl, as can be seen in the analysis of the SDS-PAGE bands (Table 1). Higher yields of soluble BthTx-1 were obtained in the presence of 2 and 4 M GdnHCl. The band of BtxTh-1 corresponding to the sample compressed in the presence of 1 M GdnHCl presented a slightly lower intensity. The lowest concentration (1 M) was used in the following experiments due to a higher probability of maintaining the secondary and tertiary native structures of BtxTh-1, which were previously shown to exist in IBs produced by E. coli in the absence or in presence of low concentration of denaturing reagent [26, 27]. At 6 M GdnHCl the yield of soluble BthTx-1 diminishes, indicating that the protein was solubilized but did not fold correctly, thus being more prone to aggregation during dialysis.

The concentration of the aggregated protein can also be an important parameter, since the correct refolding pathway competes with the misfolding and aggregation ones. Usually, low protein concentrations (typically 50–100 μg/ml) are required to prevent reaggregation and thus higher yields of refolded protein are obtained when traditional, chemically induced solubilization of aggregated proteins are used in refolding. Table 1 shows that the yields of soluble BthTx-1 obtained after compression (2 kbar) and dialysis were proportional to the dilutions of IBs. Thus, we choose a low BthTX-1 concentration (O.D. of 0.5), which corresponds to a protein concentration of approximately 50 μg/ml, for the HHP refolding.

A SDS-PAGE image showing the phases in the process of obtaining soluble BthTx-1 is shown in Fig. 1. Expression of BthTx-1 by activated E. coli cells is low (lane 3). However, enrichment of insoluble BthTx-1 occurred after the cells were sonicated and IBs were washed, as shown in lane 4. The sample treated with compression at optimal conditions is shown in lane 7 as a very strong and nearly unique band. As can be observed in lane 8 (sample maintained at atmospheric pressure), the BthTx-1 band is very faint, indicating that the protein was not solubilized/refolded under this condition.

The yield of BthTx-1 solubilization was 32%. A total amount of 7.6 mg of BthTx-1 was obtained from 1 l of bacterial culture.

The immunological identity of BthTx-1 was confirmed by western blot analysis (not shown).

Insoluble samples of BthTx-1, non-treated (A) or treated (B) with 2 kbar pressure (16 h) in refolding buffer (50 mM Tris–HCl, pH 7.5, 1 mM EDTA, 3 mM glutathione, 2:3 ratio of GSH/GSSG, and 1 M GdnHCl), are shown in Fig. 2. The IBs were mostly regular spherical particles with an average diameter of 0.27 ± 0.032 μm. Although less dense material was observed, the morphology and size of the insoluble particles remaining after 2.0 kbar application was similar to the non-treated IBs, suggesting that the high pressure was not able to induce solubilization of all the IBs particles.Fig. 2

Field scanning electron microscopy (SEM) of insoluble BthTx-1. a IBs; b insoluble aggregates after compression. Scale bars: 10 μm (original magnification ×1000), 1 μm (original magnification ×10,000)

BthTx-1 is a myotoxic protein, which shows lytic activity on differentiated muscle cells [24]. BthTx-1 cytotoxicity was estimated by the release of LDH to supernatant after incubation with the toxin for 4 h. Figure 3 shows LDH activity in the supernatant of C2C12 muscle cells treated with the refolded toxin and with the natural one, purified from B. jararacussu venom. The cytotoxic effect induced by the recombinant protein at the concentration of 30–120 μg/ml was comparable to the activity of the natural toxin thus validating our refolding procedure. Dose-dependent cytolytic activity of BthTx-1 refolded under HHP is shown for C2C12 cells.Fig. 3

Cytotoxicity was determined by the release of LDH to cell supernatants, 4 h after exposure of the cells to recombinant BthTx-1 refolded under high pressure or to BthTx-1from crude venom. Each bar represents mean ± SD of triplicate cultures