TABLE 1.
Characteristics of ACT constructs with MalE epitope
| ACT proteina | Insertion point/flanking sequencesb | Binding activityc | Invasive AC activity c,d | Hemolytic activityc |
|---|---|---|---|---|
| ACT wild type | None | 100 ± 11 | 100 ± 12 | 100 ± 15 |
| ACT108/MaIE | AHG107VLNGKLIAYPIAVEALSVH108HTA | 97 ± 12 | 99 ± 10 | 98 ± 13 |
| ACT133/MaIE | TGM132VYGNGKLIAYPIAVEALSYT134DGV | 96 ± 11 | 98 ± 12 | 95 ± 17 |
| ACT233/MaIE | TGG232VLNGKLIAYPIAVEALSVH233LDR | 100 ± 8 | 98 ± 13 | 99 ± 8 |
| ACT336/MaIE | YIG335VLNGKLIAYPIAVEALSVH336QQR | NDe | ND | 96 ± 9 |
| ACT424/MaIE | SDM423VYGNGKLIAYPIAVEALSYT425AAV | 90 ± 15 | 99 ± 6 | 111 ± 20 |
| ACT594/MaIE | LLA593CTNGKLIAYPIAVEALSGT594RGV | 75 ± 23 | <2 | 20 ± 5 |
| ACT607/MaIE | GAS606VYGNGKLIAYPIAVEALSYT608GAA | 61 ± 18 | 15 ± 8 | 80 ± 34 |
| ACT751/MaIE | EQL750VYGNGKLIAYPIAVEALSYT752NS | 83 ± 11 | 93 ± 8 | 99 ± 15 |
| ACT926/MaIE | NAS925CTNGKLIAYPIAVEALSGT926RIH | 0 | 0 | 0 |
| ACT1334/MaIE | WFG1333VLNGKLIAYPIAVEALSVH1334NTQ | 88 ± 15 | 90 ± 11 | 99 ± 8 |
| ACT1648/MaIE | WYR1647VLNGKLIAYPIAVEALSVH1648DAD | 100 ± 9 | 98 ± 12 | 88 ± 7 |
Three double-stranded synthetic oligonucleotides in the required reading frames were inserted into the unique BsrGI sites located at different positions within the cyaA gene on a set of pT7CACT1-BsrGI plasmids (12). The orientation and exact sequence of all inserted oligonucleotides were verified by DNA sequencing, and the proteins were produced and purified as previously described (8, 16).
The MaIE epitope sequence is underscored, and the flanking residues are indicated in boldface.
Samples were tested using sheep erythrocytes as model target cells and expressed as percent wild-type ACT activity. The activities of the proteins were determined prior to ablation of the AC activity (insertion of a dipeptide insert between residues 188 and 189) as described previously (12). The activities are expressed in percent wild-type ACT activity and represent average values with standard deviations from at least three independent determinations performed in duplicates with three different toxin preparations (n = 6).
Invasive activity was determined as the AC activity translocated into sheep erythrocytes and protected against digestion by extracellularly added trypsin (12).
ND, not determined because of no measurable AC activity.