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Figure 2 | Microbial Cell Factories

Figure 2

From: A generalised module for the selective extracellular accumulation of recombinant proteins

Figure 2

Monitoring cellular integrity of the E. coli host strain expressing AT chimeras. (A) SDS-PAGE analysis of OM (M) and culture supernatant (S) fractions derived from E. coli TOP10 expressing Pet*, mCherry*and ESAT6*. Non cleaved species are denoted by arrows. Molecular weight markers (MWM, kDa) are indicated to the right of the panel. OM fractions demonstrating the presence and absence of the β-domain are shown in Additional file 6: Figure S5. (B) E. coli cells expressing empty vector, Pet*, mCherry*, ESAT6* and their cleaved parents were harvested 2 h after induction and subjected to indirect immunofluorescence using the indicated antibody. For each population expressing a cleavage deficient variant, a sample was divided in two: one half was probed with an antibody to the periplasmic protein BamD, while the other half was permeabilised (−P) and subsequently probed with the same antibody. Corresponding fields are also shown by phase contrast microscopy. For the mCherry constructs panels showing mCherry derived fluorescence are shown. (C) The integrity of E. coli host cells expressing wild type Pet and secreted ESAT6-Pet fusions were assessed by staining with BOX and PI prior to and 2 h post induction. Q3 represents the population that is viable and healthy and did not label with either stain. Q2 represents cells that stain with both stains and are no longer viable. Q4 (BOX-positive) represents cells with impaired membrane potential suggesting compromised membrane integrity. (D) Periplasmic leakage from E. coli TOP10 cultures secreting Pet or ESAT6-Pet fusion proteins was assessed by measuring (2 h after induction) the activity in the culture medium of the periplasmic enzyme alkaline phosphatase. Clarified whole cell lysate was used as a positive control and the wild-type plasmid-free strain as a negative control. There is no significant difference between the negative control and culture medium derived from strains expressing Pet or ESAT6-Pet-BB. In contrast, culture medium from both constructs displayed activity significantly less than the positive control. Absorbance measurements are derived from equal volumes of culture and are normalised relative to positive control (in %). The error bars represent standard error for two independent data sets.

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