3.3 Solubilizing the inclusion body in Urea
According to theSDS-PAGE analysis, inclusion bodies of FPOX were only soluble in urea 8 M with a slight solubilization in the presence of 4 M urea by traditional method while was not soluble in any of used concentrations of DMSO solvent (Fig.3). In freeze-thaw method, the inclusion bodies were not soluble in urea 0.5 M while were soluble in urea 1 and 2 M with a concentration of 1mg/mL and 1.4 mg/mL, respectively, while it was not soluble with these concentrations of urea in traditional method. However, the major part of inclusion bodies of FPOX was soluble in urea 8 M by freeze-thaw method. The dialysis method was used to refold soluble inclusion bodies; which gradually permitted the alteration from denaturing to native buffer conditions. However, considerable precipitation was observed after dilution to the refolding buffer without urea. Supernatant containing refolded FPOX was collected after centrifugation, and used for the activity assay.