2.3 Purification of soluble and inclusion bodies of recombinant FPOX
For purification of soluble FPOX, the supernatant from the cell lysiswas applied onto a 15 mL column containing 1 mL of Ni–NTA agarose (Qiagen) equilibrated with lysis buffer (20 mMTris–HCl pH 8.0, 40 mM NaCl) and washed with wash buffer (20 mM Tris–HCl pH 8.0, 40 mM NaCl, and 20 mM imidazole).His-tagged FPOX was eluted using the elution buffer containing 300 mM imidazole [7]. In order to achieve a purified state of inclusion bodies, after cell lysis by lysozyme and sonication, it centrifuged at low speed (4000g for 10 min) for sedimenting the bacterial cells that are not broken by sonication and also separating the large-size particels of inclusion bodies. The presence of large-size particels of inclusion bodies in the pellet was analyzedby SDS-PAGE. The supernatant of this stage was again centrifuged at high speed (13000g for 20 min) and the supernatant was removed and the pellet containing the small-size particels of inclusion body was stored at -20°C for analysis with SDS-PAGE. The pellet from centrifugation at low speed contained intact cells was lyzed and centrifuged at high speed (13000g for 20 min) and the supernatant was analyzed.