2.4 Solubility of FPOX inclusion bodies
Two different methods including traditional urea-denatured method and
freeze-thawing method were used to solubilize inclusion bodies of FPOX
in urea. In the urea-denatured method, purified inclusion body was
suspended in buffer
(NaH2PO4.2H2O/Na2HPO4100
mM, tris-HCl 100 mM, DTT 5 mM) with different concentrations of urea (1,
2, 4, 8 M) and stirred for 30 min at room temperature followed by a
centrifugation at 12000g for 20 min, and supernatant was analyzed by
SDS-PAGE. The solubility of purified inclusion body also studied in
different concentrations of DMSO (5, 10, and 20 %). In the second
method, the purified inclusion body of FPOX was suspended in PBS buffer
at different concentrations of urea (0.5, 1, 2, 8 M) and the suspension
was frozen at -20°C and thawed at room temperature, finally, the
supernatant was obtained by a centrifugation at 12000 g for 20 min and
analyzed by SDS–PAGE[19]. At the end of this experiments the
supernatant was dialysis against refolding buffer (20 mMTris, 100 mM
NaCl, 10 mM CaCl2, 0.2mM ZnCl2,pH 7.5) at 4°C for 3 hours.