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Figure 3 | Microbial Cell Factories

Figure 3

From: A structurally informed autotransporter platform for efficient heterologous protein secretion and display

Figure 3

Cell surface exposure of HbpD-ESAT6 fusions. (A-B) Expression of Hbp display constructs analyzed by Coomassie staining (A) or immunoblotting (B) as described in the legend to Figure 1. (C) Proteinase K accessibility of HbpD-ESAT6 fusions at the cell surface. Part of the cells described under A was collected and resuspended in 50 mM Tris–HCl, pH 7.4, 1 mM CaCl. For Hbp(Δβ-cleav) and HbpD(Δd1), half of the cell suspension was lysed by sonication (son) on ice using a tip sonicator (Branson Sonifier 250). Subsequently, all samples were incubated with Proteinase K (pk; 100 μg/ml) at 37°C for 1 h. The reaction was stopped by addition of 0.1 mM phenylmethanesulfonylfluoride (PMSF) and incubation on ice for 5 min. Samples were TCA precipitated before analysis by SDS-PAGE and Coomassie staining. Non-cleaved Hbp species (*) are indicated. Molecular mass (kDa) markers are indicated at the left side of the panels.

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