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        Purification of the N- and C-Terminal Subdomains of Recombinant Heavy Chain Fragment C of Botulinum Neurotoxin Serotype C

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        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.
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