3.2 Purification of inclusion bodies
Recombinant FPOX was expressed as both soluble form and inclusion
bodies. FPOX contained a His-tag in the C-terminal of the protein to
enable protein purification by Ni-NTA column. Purified soluble FPOX was
evaluated by SDS-page and has been shown in Fig.1A with a band of 47
KDa. For inclusion bodies, after cell lysis with sonication, it was
centrifuged in low speed (4000g for 10 min) and the supernatant was
separated and then the supernatant was centrifuged in high speed (13000g
for 20 min) and in each case, the pellets were investigated for the
presence of inclusion bodies. The pellet from low speed stage was
sonicated to lyze the intact cells in the pellet. As shown in Fig.2 B
FPOX protein has been expressed around 47 KDa. The soluble protein in
the supernatant from the first and second stages of cell lyses was low
and was mainly formed as inclusion bodies. The results showed that
inclusion bodies of FPOX have been consisted of small and large
particles. The large particles were sedimented with centrifugation at
4000g for 10 min that is corresponding with the sedimentation of the
bacterial cells in the same speed, while the small particels were
sedimented in 13000g for 20 min. The main part of FPOX inclusion bodies
were particles with large size.A band of insoluble FPOX with purity
about 95 % was observed in analysis with SDS-PAGE that was related to
the small particles of inclusion bodies that sedimented in high speed of
centrifugation of supernatant obtained in low speed stage.