| Autor: |
Nicholson Gregory, Fernandez-Salas Ester, Brown Meenakshi, Okawa Yumiko, Francis Joseph, Sun Sarah, Lance E. Steward, K. Roger Aoki, Hieu T. Arpawong, Satorius Alan, Maria C. Ardila, Joseph V. Ordas, Wang Joanne, Marcella A. Gilmore |
| Rok vydání: |
2008 |
| Předmět: |
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| Zdroj: |
Toxicon. 51:11-12 |
| ISSN: |
0041-0101 |
| DOI: |
10.1016/j.toxicon.2008.04.035 |
| Popis: |
Both rHis6-BoNT/E and rBoNT/E-His6 were produced from a codon optimized gene within Escherichia coli. Similar to native BoNT/E isolated from Clostridium botulinum, the recombinant proteins were isolated as single-chain molecules. The His6 affinity tag facilitated a two-step IMAC-IEX purification. While reasonable yields were obtained, single-chain rHis6-BoNT/E was produced less efficiently than was rBoNT/E-His6. Treatment with recombinant trypsin efficiently converted both purified rBoNT/E constructs to the activated dichain forms, with concomitant removal of the His6 tag, and the nicking profile for each rBoNT/E molecule matched that of native BoNT/E. Following nicking with trypsin, it was observed that the resultant rHis6-BoNT/E dichain displayed reduced in vivo potency (mouse potency assay) compared to activated native BoNT/E dichain. In contrast, activated rBoNT/E-His6 displayed a specific potency equivalent to that of trypsin-nicked native BoNT/E in the mouse potency assay. These results demonstrate that rBoNT/E, containing a C-terminal His6 affinity tag, can be expressed and purified in high yield, with the activated molecule displaying equivalent in vivo potency to activated native BoNT/E. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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Full Text from ScienceDirect