Purification of the N- and C-terminal subdomains of recombinant heavy chain fragment C of botulinum neurotoxin serotype C.

The N-terminal and C-terminal portions of the heavy chain fragment C from botulinum neurotoxin serotype C [rBoNT(HC)] were expressed in Pichia pastoris and purified by ion-exchange chromotography (IEC). The N-terminal fragment, rBoNTC(Hc)-N, was purified in three IEC steps: a Q Sepharose Fast Flow (FF) capture step followed by a negative SP Sepharose FF step, and finally, Q Sepharose FF as a polishing step. The purification process resulted in greater than 90% pure rBoNTC(Hc)-N based on SDS-PAGE, and yielded up to 1.02 g of rBoNTC(Hc)-N/kg of cells. Alternately, the C-terminal fragment, rBoNTC(Hc)-C, was purified by using a SP Sepharose FF capture step followed by a second SP Sepharose FF step, and finally a Q Sepharose FF as a polishing step. This purification process resulted in greater than 95% pure rBoNTC(Hc)-C based on SDS-PAGE, and yielded up to 0.2 g of rBoNTC(Hc)-C/kg cells. The final protein yield is a function of protein expression level during fermentation and the purification methods, and usually final protein yield between 0.1 and 2 mg/g cells is acceptable. Another concern is protein degradation. Especially with Pichia, protease activity during cell lysis and purification is always an issue. The importance of N-terminal degradation depends on product and its function. N-terminal sequencing revealed that the purified rBoNTC(Hc)-N is missing the first eight amino acids of the N-terminus of the protein, whereas the purified rBoNTC(Hc)-C protein is intact. After a mouse bioassay test, both the intact rBoNTC(Hc)-C and the rBoNTC(Hc)-N missing the first eight amino acids of the N-terminus have vaccine potency; consequently, partial degradation did not have an impact on these protein's utility.

Transgenic cotton (Gossypium hirsutum L.) seedlings expressing a tobacco glutathione S-transferase fail to provide improved stress tolerance.

Transgenic cotton (Gossypium hirsutum L.) lines expressing the tobacco glutathione S-transferase (GST) Nt107 were evaluated for tolerance to chilling, salinity, and herbicides, antioxidant enzyme activity, antioxidant compound levels, and lipid peroxidation. Although transgenic seedlings exhibited ten-fold and five-fold higher GST activity under normal and salt-stress conditions, respectively, germinating seedlings did not show improved tolerance to salinity, chilling conditions, or herbicides. Glutathione peroxidase (GPX) activity in transgenic seedlings was 30% to 60% higher under normal conditions, but was not different than GPX activity in wild-type seedlings under salt-stress conditions. Glutathione reductase, superoxide dismutase, ascorbate peroxidase, and monodehydroascorbate reductase activities were not increased in transgenic seedlings under salt-stress conditions, while dehydroascorbate reductase activity was decreased in transgenic seedlings under salt-stress conditions. Transgenic seedlings had 50% more oxidized glutathione when exposed to salt stress. Ascorbate levels were not increased in transgenic seedlings under salt-stress conditions. Malondialdehyde content in transgenic seedlings was nearly double that of wild-type seedlings under normal conditions and did not increase under salt-stress conditions. These results show that expression of Nt107 in cotton does not provide adequate protection against oxidative stress and suggests that the endogenous antioxidant system in cotton may be disrupted by the expression of the tobacco GST.