Figure 2
Expression and secretion of recombinant mannan endo-1,4-β-mannosidase (ManB) from B. licheniformis. A) The pFLAG-CTS system (Sigma) was used for the expression of recombinant β-mannanase. Enzyme expression was under the control of tac promoter, which can be induced for overexpression by IPTG. The vector contains the ampicillin resistant gene, and a hexahistidine tag was incorporated C-terminally. The mature enzyme was fused to the E. coli OmpA signal peptide (shown in a box) for secretion into the periplasmic space. B) Cells were grown until OD600 reach ~1.0 before IPTG was added to a final concentration of 1 mM, and incubation continued at 28°C. Samples were taken at 0, 4 h, and overnight (20 h, o/n) after induction with IPTG. Culture supernantant, periplasmic and cytosolic fractions were prepared as described in Material and Method. Approximately equal amounts of total protein in the periplasmic and cytosolic fraction were loaded onto each lane.