FIG. 4.

(A) SDS-PAGE of outer membranes of P. aeruginosa OCR03T carrying control vector pVLT35 (lane 1; C), native OprM-expressing plasmid pKW35TM (lane 2; TM), and OprM-encoding plasmids with malarial epitope insertions at sites ME1 to ME6 (lanes 3 to 8) and ME8 to ME13 (lanes 9 to 14). Each lane was loaded with 20 μg of protein, treated with 5% β-mercaptoethanol, and heated at 100°C for 10 min. The molecular masses (in kilodaltons) of the prestained markers in lane M are indicated on the left. The arrowhead on the right indicates the position of the oligomeric forms of the proteins. (B) SDS-PAGE of the same samples unheated (room temperature for 10 min) and loaded in the same order as in panel A. The arrowhead on the right indicates the position of the oligomeric forms of the proteins. Relative molecular masses (in kilodaltons) are indicated on the left. Expression of insertion oprM mutant forms was confirmed by Western immunoblotting with an anti-OprM antibody (C) and an anti-malarial epitope antibody (D and E). The corresponding regions of the blots are shown, with the relevant molecular mass (in kilodaltons) indicated on the left. Samples are in the same order as in the gel shown in panel A. Proteins for the Western immunoblots were untreated (E) or treated with 5% (vol/vol) β-mercaptoethanol and heated at 100°C for 10 min (C and D).