Skip to main content
Figure 6 | Microbial Cell Factories

Figure 6

From: Optimizing heterologous protein production in the periplasm of E. coli by regulating gene expression levels

Figure 6

Characterization of secretory BL1 expressed at the optimal rhamnose concentration in Lemo21(DE3). Expression of the gene encoding secretory BL1 was induced with IPTG in Lemo21(DE3) in the presence of 500 μM of rhamnose (see Figure 5). 4 h after induction a whole cell lysate was prepared and the periplasmic fraction isolated. A Nitrocellulose membranes containing increasing amounts of β-galactosidase were incubated with the whole cell lysate (top panel) and whole cell lysate that had been incubated with β-mercaptoethanol (middle panel). Binding of BL1 to the β-galactosidase spotted on the nitrocellulose membranes was detected using an α-His antibody recognizing the C-terminal His-tag of BL1. A nitrocellulose membrane containing spots with increasing amounts of BSA and incubated with the same lysate used in the top panel was included as a control (bottom panel). B The periplasmic fraction was isolated as described in Methods. SEC was used to analyze the BL1 that was isolated from the periplasmic fraction by means of IMAC. Indicated fractions (inset) from the SEC were analyzed by SDS-PAGE followed by Coomassie staining (top panel inset) or immuno-blotting using an α-His antibody (middle panel inset). The fractions representing the indicated peak were pooled and the BL1 was tested for binding to β-galactosidase (bottom panel inset). β-galactosidase concentrations correspond to the setup described in A. The bottom panel of the inset shows only the first 4 concentrations.

Back to article page