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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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Pichia Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 389))

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.

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Author information

Authors and Affiliations

  1. Biological Process Development Facility, Department of Chemical Engineering, University of Nebraska, Lincoln, NE

    Jicai Huang, Rick Barent & Mark Gouthro

  2. Department of Chemical Engineering, University of Nebraska, Lincoln, NE

    Mehmet Inan & Michael M. Meagher

  3. United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD

    Virginia P. Roxas & Leonard A. Smith

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  1. Jicai Huang
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  3. Mehmet Inan
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  4. Mark Gouthro
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  6. Leonard A. Smith
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  1. Keck Graduate Institute of Applied Life Sciences, Claremont, CA

    James M. Cregg

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© 2007 Humana Press Inc., Totowa, NJ

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Huang, J. et al. (2007). Purification of the N- and C-Terminal Subdomains of Recombinant Heavy Chain Fragment C of Botulinum Neurotoxin Serotype C. In: Cregg, J.M. (eds) Pichia Protocols. Methods in Molecular Biology, vol 389. Humana Press. https://doi.org/10.1007/978-1-59745-456-8_6

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  • DOI: https://doi.org/10.1007/978-1-59745-456-8_6

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-429-6

  • Online ISBN: 978-1-59745-456-8

  • eBook Packages: Springer Protocols

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