2.2.2. Separation and Purification of LEL CD81-SA

Bacterial cells were separated from the culture medium by centrifugation for 30 min at 3500 rpm. The deposited cells were resuspended in PBS and centrifuged at a speed of 4000 rpm for 15 min, and then destroyed using an ultrasonic disintegrator under cooling conditions with the addition of glass beads (GlassBeads, 500 µm, Sigma-Aldrich, Moscow, Russia). After removing the glass beads, the supernatant and the cell mass were centrifuged at a speed of 13,500 rpm for 15 min. The supernatant filtered through a paper filter was subjected to chromatographic purification on a Ni-agarose column (column volume 1.5 mL), according to the standard protocol of the sorbent manufacturer (Invitrogen, Waltham, MS, USA). The column was loaded with 30 mL of supernatant in 10 mM imidazole, 0.3 M NaCl, 50 mM Tris-HCl, pH 8.0. Desorption was carried out by adding 5 mL of a solution containing 200 mM imidazole, 0.3 M NaCl, 50 mM Tris-HCl, pH 8.0. The mobile phase flow rate was 0.3 mL/min. The target protein was detected using polyacrylamide gel-electrophoresis (PAGE).

The precipitate of destroyed cells remaining after isolation from the soluble cell fraction was washed with PBS, dissolved in solution of 8 M urea or 6 M guanidine chloride (pH 1.5) and left for 1 h. The precipitate was then centrifuged at 12,000 rpm for 15 min and purified as it was described above.

Fractions resulting from protein purification on the Ni-agarose were analyzed by gel-electrophoresis in 5–12% polyacrylamide gel in a presence of sodium dodecyl sulfate (SDS PAGE). The Coomassie staining was applied for protein visualization. The standard protocol can be found elsewhere [24,25].