title: Purification of the N- and C-terminal subdomains of recombinant heavy chain fragment C of botulinum neurotoxin serotype C.
abstract: 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.
authors.affiliation: Department of Chemical Engineering, University of Nebraska, Lincoln, NE, USA.
authors.forename: Jicai
authors.initials: J
authors.lastname: Huang
authors.forename: Rick
authors.initials: R
authors.lastname: Barent
authors.forename: Mehmet
authors.initials: M
authors.lastname: Inan
authors.forename: Mark
authors.initials: M
authors.lastname: Gouthro
authors.forename: Virginia P
authors.initials: VP
authors.lastname: Roxas
authors.forename: Leonard A
authors.initials: LA
authors.lastname: Smith
authors.forename: Michael M
authors.initials: MM
authors.lastname: Meagher
mesh_terms: D001905:Botulinum Toxins / Q000737:chemistry* / Q000302:isolation & purification*; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation; D010843:Pichia / Q000235:genetics; D010957:Plasmids; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification*; D012685:Sepharose
chemical_list: D011994:Recombinant Proteins; D012685:Sepharose; D001905:Botulinum Toxins; C096324:botulinum toxin type C