Fig. 5

Large-scale soluble expression of recombinant AaLT-NL zymogen protease grown in TB media at 10 °C (induced with 0.1 mM IPTG). Plasmid construct was transformed into SHuffle® T7 Express Competent E. coli cells (NEB). Samples were collected at the given time points (in hours). The MW ladder is in kilo-Daltons (kDa). Gel analysis of samples collected from the growth of AaLT was first visualized using InVision™ His-Tag In-Gel Stain (Invitrogen), which specifically chelates to and enhances the fluorescence of poly his-tagged proteins (top figure). The His-Tag stain is the positive identification that the bands expressed in the gel below are indeed the expression of soluble AaLT-zymogen (MW ~ 27.6 kDa, red arrows). The growth was extended beyond 24 h due to the 10 °C growth conditions, which helped in solubly expressing the protease, but also to increase bacterial cell density in order to obtain a large quantity of cell paste for purification