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.