TABLE 1.
Expression and characterization of OprM epitope insertion mutant proteins in E. coli
| Plasmida | Insertion site (amino acid)b | Amino acids inserted | Protein expression (by SDS-PAGE)c | Surface reactivityd (antimalarial) |
|---|---|---|---|---|
| pVLT35 | None (control vector) | − | − | |
| pKW35TMe | None (wild-type OprM) | +++ | − | |
| pKWIN1 | ME1 (23) | GPAPNA(NPNA)2GHAGP | +++ | +++ |
| pKWIN2f | ME2 (37) | KRK(NPNA)2NPN | +++ | +++ |
| pKWIN3 | ME3 (72) | GPAPNA(NPNA)2GHAGP | + | − |
| pKWIN4 | ME4 (77) | None (stop) | − | − |
| pKWIN5f | ME5 (103) | KRKNPNANPN | +++ | +++ |
| pKWIN6 | ME6 (130) | TC(NPNA)3CRS | +++ | +++ |
| pKWIN7 | ME7 (143) | None (stop) | − | − |
| pKWIN8 | ME8 (159) | GPAPNA(NPNA)2GHAGP | ++ | +++ |
| pKWIN9f | ME9 (241) | KRK(NPNA)2NPN | + | + |
| pKWIN10 | ME10 (251) | GTC(NPNA)3CRS | ++++ | +++ |
| pKWIN11 | ME11 (315) | GTC(NPNA)3CRS | +++ | +++ |
| pKWIN12 | ME12 (377) | DLQ(NPNA)2NALDVQV | ++ | + |
| pKWIN13f | ME13 (393) | KRKNPNAPNANPN | + | − |
Plasmids pKWIN1 to pKWIN12 contain insertion mutation oprM cloned into pVLT35; pKWIN13 contains insertion mutation oprM cloned into pVLT31 (a derivative of pVLT35).
Position 1 is the N-terminal amino acid of the mature OprM amino acid sequence.
These results were obtained using outer membranes of E. coli CE1248 containing the various plasmids. Expression levels ranged from undetectable (−) to strong (++++). Expression was confirmed by Western immunoblot assay using a murine monoclonal antibody against OprM.
Fluorescence levels ranged from no (−) to strong (+++) fluorescence as assessed by indirect immunofluorescence assay.
pKW35TM contains wild-type oprM with the native sequence cloned into pVLT35.
This plasmid contains the native oprM sequence at the 3′ end; the other plasmids contain an insertion into the previously published oprM sequence (termed oprM*).