Skip to main content
Figure 6 | Microbial Cell Factories

Figure 6

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

Figure 6

Display of ESAT6 by attenuated Salmonella typhimurium. (A-B) Expression of HbpD(Δd1)-ESAT6. S. typhimurium SL3261 (-) and a derivative expressing HbpD(Δd1)-ESAT6 were grown to mid-log phase in LB medium at 37°C. The equivalent of 0.03 OD660 units cells (c) and corresponding culture medium (m) samples was analyzed by SDS-PAGE and Coomassie staining (A) or immunoblotting (B). (C-E) Exposure of HbpD(Δd1)-ESAT6 at the S. typhimurium cell surface. (C) Cells from A were collected and resuspended in icecold reaction buffer (50 mM Tris HCl, pH 7.4, 1 mM CaCl2). The samples were treated with 100 μg/ml Proteinase K (+ pk) or mock-treated (- pk) at 37°C for 1 h. The reaction was stopped by incubation with PMSF (0.1 mM) for 10 min on ice. Samples were TCA precipitated and analyzed as described under A. (D-E) Samples described under C were analyzed by immunoblotting. Cell integrity during the procedure was demonstrated by showing the inaccessibility of the periplasmic chaperone SurA towards Proteinase K using anti-SurA (cf. lanes 1, 3, 5 and 2, 4, 6, resp.). Non-cleaved Hbp species (*) are indicated. Molecular mass (kDa) markers are shown at the left side of the panels.

Back to article page