Legends to the figures
Fig.1. Different expression condition in Shuffle and BL21 strains of E.coli . M : Marker, A ) Expression with IPTG 1 mM at 37 °C in BL21, 1: negative control, 2: expressed protein, 3:supernatant, 4: inclusion body solution in urea 8 M.B ) expression with IPTG 1 mM at 30 °C in BL21, 1:before induction of IPTG, 2: expressed protein, 3: negative control4: supernatant.C ) Expression with IPTG 0.5 mM at 30 °C in BL21, 1: negative control, 2: expressed protein, 3: supernatant.D ) Expression with IPTG 0.5 mM at 26 °C in BL21, 1: before induction by IPTG, 2: expressed protein, 3:negative control, 4: supernatant (cell lysis using lysozyme and sonication), 5: supernatant (cell lysis using sonication and without lysozyme), 6: inclusion body in urea 8 M.E ) Expression with IPTG 0.1 mM at 16 °C in BL21, 1:before induction by IPTG, 2 ,3: expressed protein, 4:negative control, 5: supernatant. F ) Expression with IPTG 0.5 mM at 26 °C in Shuffle, 1: expressed protein, 2: negative control, 3: supernatant.G ) Expression with IPTG 0.1 mM at 26 °C in Shuffle, 1: before induction by IPTG, 2: expressed protein, 3: negative control, 4: supernatant, 5: inclusion body in urea 8 M. H ) Expression with IPTG 0.1 mM at 16 °C in Shuffle, 1: negative control, 2: inclusion body in urea 8 M, 3:expressed protein, 4: supernatant.
Fig.2. The purification of soluble and inclusion bodies of FPOX. A) Purification of FPOX B) The purification of inclusion body of FPOX by the centrifugation at low and high speeds.M : marker,1 : negative control, 2 : expression of FPOX before cell lysis, 3 : supernatant from centrifugation at high speed,4 : supernatant after pellet lysis from the stage with low speed centrifugation, 5 : inclusion bodyobtained at low speed of centrifugation, 6 : purified inlcusion body from the centrifugation of supernatant at high speed,7 : inlcusion body after pellet lysis from the stage with low speed centrifugation.
Fig.3. SDS–PAGE analysis of solubilized FPOX from inclusion bodies in different concentrations of urea by two different methods and also the solubility of FPOX in DMSO solvent. A) M :marker,1 : urea 1M, 2 : urea 2M, 3 : urea 4 M,4 : urea 8 M, 5 : DMSO 5% ,6 : DMSO 10 %,7 : DMSO 20%, B)M : marker, 1 : urea 0.5 M,2 : urea 1M, 3 : urea 2 M, 4 : urea 8 M,
Fig.4. TheSDS-PAGE analysis of the effect of sorbitol and arginine on the solubility and expression of FPOX. M: marker, 1: negative control, 2: expressed FPOX in control(without sorbitol and arginine), 3: expressed FPOX in the presnce of sorbitol, 4: expressed FPOX in the presence of arginine, 5: supernatant from control, 6: supernatant from cells growed in the presence of sorbitol, 7: supernatant from cells growed in the presence of arginine.
Fig.5. Periplasmic extraction of recombinant FPOX produced inE. coli BL21 and Shuffle strains.M: marker, 1: negative control, 2: expression of FPOX in shuffle, 3: expression of FPOX in BL21,4: periplasmic fraction in shuffle, 5: periplasmic fraction in BL21, 6: supernatant from lysis of cells after periplasmic extraction in shuffle, 7: supernatant from lysis of cells after periplasmic extraction in BL21, 8: periplasmic fraction in shuffle treated by sonication, 9: periplasmic fraction in BL21 treated by sonication.10: periplasmic fraction in shuffle after adding lysozyme, 11: periplasmic fraction in BL21 after adding lysozyme.
Fig. 6. The effect of different proteases on hemoglobin as substrate. 1: Control, 2: Alkaline protease, 3: Neutral protease, 4: Pepsin, 5: Papain, 6: Chymotrypsin.