Display of Multimeric Antimicrobial Peptides on the Escherichia coli Cell Surface and Its Application as Whole-Cell Antibiotics
Figure 4
Confirmation of LO-Bn fusion proteins displayed on the cell surface.
Cells harboring pLO-B0, -B1, -B2, and -B3 were incubated with anti-His-FITC antibody diluted (1∶200) in NAPB for 1 h at room temperature. Confocal images show the localization of LO-B0 (Lpp-OmpA, A), LO-B1 (B), LO-B2 (C), and LO-B3 fusion proteins (D), respectively. No fluorescence signal was detected when the same cells were not induced by IPTG (E–H). The z-stack confocal image (D, inset) clearly shows the surface location of LO-B3 fusion proteins as a concentrated fluorescent ring along the cell surface. (I) SDS-PAGE analysis of the outer membrane proteins and inclusion bodies. Lanes 1, 3, 5, and 7 represent inclusion bodies in the cytoplasm of E. coli BL21 (DE3) harboring pLO-B0, -B1, -B2, and -B3, respectively. Lanes 2, 4, 6, and 8 represent the outer membrane proteins isolated from E. coli BL21 (DE3) harboring pLO-B0, -B1, -B2, and -B3, respectively. Lane M represents molecular weight markers. The closed triangles indicate the LO-Bn fusion proteins. (J) The ratio of surface-displayed LO-Bn fusion proteins to total expressed LO-Bn fusion proteins.