2.2 Cloning and Expression of recombinant protein
The recombinant plasmid pET22b-fpox was transformed intoE.coli BL21 (DE3) and shuffle strains using calcium chloride-heat
shock method [18]. Transformants were inoculated in the LB medium
supplemented with 100 μg/mL ampicilin and incubated at 37 °C overnight.
Protein expression was conducted by the inoculation of 1 mL into 100 mL
LB medium containing antibiotic and incubated at 37 °C to achieve an
optical density at 600 nm (OD600) of 0.6. The FPOX
protein expression was induced by adding a final concentration of 0.1 mM
isopropyl β-D-1-thiogalactopyranoside (IPTG) and incubation at 16 °C for
24 h. Cells were harvested by centrifugation at 4000g for 12 min and
were washed twice with PBS buffer (1 X). The pellets were stored at -20
°C. The pellets were suspended in lysis buffer containing 20 mM
Tris–HCl pH 8.0, 40 mM NaCl [7]and lysozyme 1 % w/v and then
incubated in 37 °C for 1 h followed by sonication, and then centrifuged
at 13000g for 20 min. The insoluble inclusion body was washed twice with
PBS buffer and in order to remove cell debris from insoluble protein; it
re-suspended in washing buffer (Tris 20 mM, NaCl 300 mM, EDTA 1 mM,
Triton X-100 1%, urea1 M, pH 8.0) and was washed three times with this
buffer. Lastly, the inclusion bodies was washed with PBS buffer to
eliminate contaminating detergent and then centrifuged at 12,000 g for
20 minutes and it was applied for following solubilization [19].