Page 151 - Demo

SECTION I: MOLECULAR GENETIC ENGINEERING AND BIOCHEMICAL TECHNOLOGY 155 3.2. Expression of ncsS53The expression of nscS53 gene is followed the lightly modified standard protocol [8]. E. coli BL21(DE3) harboring pncsS53, the expression vector, was grown in 10 ml LB medium supplemented with ampicillin (100 %u03bcg.ml-1) at 37 %u00b0C and 250 rpm for 8 h. The cultures were transferred to 600 ml of fresh LB medium then incubated at 37 %u00b0C and 250 rpm. At an OD600 of 0.6, isopropyl-b-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.4 mM and continued the incubation at 20 oC for 20 h leaded excessive formation of soluble proteins. The cells were harvested by centrifugation at 6000 x g for 10 min, washed twice with phosphate buffer (50 mM, pH 7.5), suspended in 12 ml of the same buffer and stored at %u201320%u00b0C for 6 h. The molecular weights of denatured proteins observed in SDS-PAGE analyses were in good agreement with that of calculated values (Figure 2). The bands observed approximately at 52 kDa correspond to the expression products of ncsS53 as the 6%u00d7histidine- and thioredoxin-tagged fusion proteins at N-terminal.Figure 3. SDS- PAGE analyses for Ncs53 expressed in E. coli BL21-DE3 showing the molecular weight of protein in the denature form 52kDa): 1 and 2 protein expressed in soluble form; 3: Protein expressed in insoluble form; M: Protein marker