Table I.
Predicted molecular characteristics of recombinant PcF Toxin Family members
| Recombinant plasmid | Expressed protein | CNBr-digestion | N. of AA | pIa | ɛ280nma (M−1 cm−1) | Average massb |
|---|---|---|---|---|---|---|
| pET32b/PcF | Trx-PcF | No | 212 | 5.08 | 17460 | 22863.7 |
| PcF | Yes | 52 | 4.40 | 3355 | 5606.2 | |
| Mature wild-type PcF | – | 52 | 4.40 | 3355 | 5622.1c | |
| pET32b/SCR91 | Trx-SCR91 | No | 230 | 5.26 | 18950 | 24847.9 |
| SCR91 | Yes | 67 | 4.56 | 4845 | 7204.9d | |
| Predicted mature wild-type SCR91 | – | 70 | 4.78 | 4845 | 7590.3 | |
| pET32b/SCR74-B10 | Trx-SCR74-B10 | No | 213 | 5.49 | 17585 | 22897.9 |
| SCR74-B10 | Yes | 53 | 5.55 | 3480 | 5640.4 | |
| Predicted mature wild-type SCR74-B10 | – | 53 | 6.69 | 3480 | 5639.4 | |
| pET32b/SCR74-D5 | Trx-SCR74-D5 | No | 213 | 5.58 | 17585 | 22913.9 |
| SCR74-D5 | Yes | 53 | 6.03 | 3480 | 5656.4 | |
| Predicted mature wild-type SCR74-D5 | – | 53 | 6.85 | 3480 | 5655.4 | |
| pET32b/SCR74-C25E | Trx-SCR74-C25E | No | 213 | 5.59 | 17460 | 23101.1 |
| SCR74-C25E | Yes | 46 | 5.41 | 3355 | 5136.7d |
Bacterially-expressed recombinant proteins from the indicated plasmid constructs are presented either as thioredoxin (Trx) fusion proteins or as species resulting from CNBr cleavage (see corresponding sequences in Fig. 1, bottom). The characteristics of their wild-type counterparts are also shown for comparison. Molecular characteristics were predicted from sequences by using
ProtParam (http://expasy.org/cgi-bin/protparam) and
PeptideMass (http://expasy.org/tools/peptide-mass.html), assuming that all pairs of Cys residues form cystines. Wild-type PcF mass value
takes into account proline-49 posttranslational hydroxylation.16 Mass values of C-terminally CNBr-cleaved protein isoforms
take into account the conversion of methionine in homoserine lactone.