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.