Bacterial lysis and plasmid recovery

The bacterial cell paste (typically 200 g) was re-suspended in 2.4 L of a buffer containing 61 mM glucose, 10 mM Tris–HCL, and 50 mM EDTA, at pH 8. Bacteria were lysed using a previously described procedure.⁴ The clarified lysate was concentrated five times on a tangential flow filtration (TFF) system (nominal molecular weight cutoff 100,000 kDa, 0.1 m² ). All experiments were conducted keeping a constant transmembrane pressure (TMP) of 0.8 bar at 4 °C, using Sartocon Slice equipment (Sartorius-Stedim Biotech). To remove RNA, the concentrated clarified lysate was loaded on Sepharose CL 4B in a BPG 113 column equilibrated with 20 mM Tris–HCL, 3 mM EDTA, and 1.5 M (NH4)₂ SO₄, at pH 7.2.

Plasmid capture and elution using the Sartobind membrane was carried out after buffer exchange on a G25 matrix using 25 mM Tris, 10 mM EDTA, and 0.5 M NaCl, at pH 8 and a rate of 50 mL/min, to reach a conductivity of 45 ms/cm. The pDNA fraction was pumped at 150 mL/min to the membrane holder 91-D-01K-15-03. After loading the feeding solution, the membrane was rinsed with a pH 8 buffer containing 25 mM Tris, 10 mM EDTA, and 0.7 M NaCl. Bound plasmid DNA was eluted with a buffer solution containing 25 mM Tris, 10 mM EDTA, and 1.2 M NaCl at pH 8. Finally, the membrane was regenerated using 0.5 M NaOH. The pDNA polishing was achieved on Sephacryl S-1000 media on a formulation buffer of 20 mM NaH₂PO₄–Na₂HPO₄, 1 mM EDTA, and 30 mM NaCl at pH 6.7, using a 50X K column (Amersham BioSciences) at 4.8 mL/min. Fractions containing more than 89% supercoiled pDNA were processed by TFF until reaching a concentration of 2 mg/mL as determined by the absorbance at 260, using a VivaFlow 200 system (nominal molecular weight cutoff 100,000 kDa, 200 cm² ). The final pDNA was filtered (0.22 μm) before further analyses were performed.

Flow-through and eluted fractions were precipitated by adding one volume of 2-propanol for 15 min on ice and then centrifuged at 14,000g for 10 min at 4 °C. The pellet was washed with 70% ethanol and centrifuged again at 4,000g for 10 min. The pellet was air-dried for 5–10 min and dissolved in 50 μl 10 mM Tris–HCl at pH 8.5. Fractions prepared as described were tested for restriction digestion analysis, using transformation experiments, and analyzed by electrophoresis on 0.8% agarose gels. Binding capacity and pDNA purity were determined by measuring absorbance at 260 (A260) and 280 (A280) and expressed as the ratio A260/A280 in a low-salt buffer. The pDNA purified as reported was used as a positive control.⁴ Identity was evaluated by restriction endonuclease digestion using a panel of enzymes followed by analysis on agarose gels.

Mice immunization and biological activity

Fifteen female Balb/c mice (6 to 8 weeks old), purchased from CENPALAB (Havana, Cuba), were immunized intramuscularly three times every second week with 50 μg pIDKE2 + 5 μg Co.120, obtained using a method previously reported,⁴ or following the method described in the present paper. The presence of antibodies against recombinant HCV structural antigens were determined by ELISA, as previously described.¹¹ The induction of antibody titers of above 1:50 against HCV structural antigens in >25% of the mice on day 42 was considered a positive response.