Figure 3

Modification of the Pet-AT secretion platform. (A) SapA, Pmp17, Pet and a Pet derivative (PetΔD1) lacking the serine protease domain were modified to add a His₆-Tag to the N-terminus of the secreted passenger domain. The His-tagged Pmp17 protein was expressed in an E. coli TOP10 dsbA strain and the rest of proteins were produced in the wild-type E. coli TOP10. In all cases the proteins are well secreted. The cysteine-containing Pmp17 was expressed in E. coli TOP10 (wt) and a dsbA^- derivative. A full length protein is present in the E. coli TOP10 dsbA^- derivative but not the E. coli TOP10 parent strain. Break down products are apparent and correspond to proteins with a truncated N-terminus. A multicomponent construct was created by fusing DNA encoding Ag85 to ESAT-6 and Pet (see Figure 1A) to encode a single polypeptide chain contiguous with the AT-translocation unit. This latter chimera was detected in the culture supernatant with antibodies directed at Pet and ESAT-6. Equivalent amounts of culture supernatant fractions were analysed by SDS-PAGE. (B) Secretion of heterologous fusions from S. Typhimurium. Culture medium from S. enterica SL1344 strains expressing ESAT6-Pet-BP and ESAT6-PetΔ*6 (see Figure 4) were harvested and analysed by SDS-PAGE and detected by immunoblotting with a polyclonal antibody to ESAT-6. In all SDS-PAGE gels the positions of the molecular weight markers (MWM, kDa) are depicted at the right side of the panel. The equivalent OM fractions demonstrating the presence of the cleaved β-barrel are shown in Additional file 5: Figure S4.