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. 2011 Oct 17;286(49):42283–42291. doi: 10.1074/jbc.M111.306118

FIGURE 3.

FIGURE 3.

A rigid linker interferes with OM translocation. A, SDS-PAGE analysis of TCA-precipitated culture supernatant fractions harvested after growth of TOP10 and TOP10 ΔdsbA expressing Pet, Pet1HA (1HA), Pet2HA (2HA), and Pet3HA (3HA). Intramolecular disulfide bond formation was prevented through growth in the presence of β-ME (top panel) or in a dsbA− background (bottom panel). A schematic description of the HA epitope tag insertions is shown on the right. M, molecular mass markers. B, SDS-PAGE analysis of TCA-precipitated culture supernatant fractions harvested after growth of TOP10 expressing empty vector (EV), Pet, and Pet9G in the presence or absence of β-ME. C, mPEG-Mal labeling of Pet9G and PetC12G/C17G in the presence and absence of TCEP. Supernatants were harvested and concentrated after growth of TOP10 cells expressing Pet9G and PetC12G/C17G. Samples were resolved on a gradient 4–20% Tris-HEPES-SDS-PAGE gel, and Pet was localized by Western immunoblotting using anti-Pet passenger antibody. The arrow indicates labeled/pegylated Pet, and the asterisk shows unlabeled/unpegylated Pet.