TABLE 2.
Expression and characterization of OprM epitope insertion mutant proteins in P. aeruginosa
| Plasmida | Insertion site (amino acid)b | Western immunoblot resultc obtained with:
|
|
|---|---|---|---|
| Anti-OprM antibody | Antimalarial epitope antibody | ||
| pVLT35 | None (control vector) | − | − |
| pKW35TMd | None (wild-type OprM) | +++ | − |
| pKWIN1 | ME1 (23) | ++ | +++ |
| pKWIN2e | ME2 (37) | +++ | +++ |
| pKWIN3 | ME3 (72) | − | − |
| pKWIN4 | ME4 (77) | − | − |
| pKWIN5e | ME5 (103) | +++ | +++ |
| pKWIN6 | ME6 (130) | − | − |
| pKWIN7 | ME7 (143) | − | − |
| pKWIN8 | ME8 (159) | − | − |
| pKWIN9e | ME9 (241) | ++ | +++ |
| pKWIN10 | ME10 (251) | ++ | +++ |
| pKWIN11 | ME11 (315) | + | ++ |
| pKWIN12 | ME12 (377) | − | − |
| pKWIN13e | ME13 (393) | ++ | − |
a
pKWIN1 to pKWIN12 contain insertion mutant gene oprM cloned into pVLT35, and pKWIN13 contains insertion mutant gene oprM cloned into pVLT31 (a derivative of pVLT35).
b
Position 1 is the N-terminal amino acid of the mature OprM amino acid sequence.
c
These results were obtained using outer membranes of P. aeruginosa OprM-deficient strain OCR03T containing the various plasmids. Expression levels ranged from undetectable (−) to strong (+++).
d
pKW35TM contains wild-type oprM cloned into pVLT35.
e
This plasmid contains the native oprM sequence at the 3′ end, and the other plasmids contain an insertion into oprM*, the previously published oprM sequence.