Production and purification of the heavy chain fragment C of botulinum neurotoxin, serotype A, expressed in the methylotrophic yeast Pichia pastoris.
A recombinant H(C) fragment of botulinum neurotoxin, serotype A (rBoNTA(H(C))), has been successfully expressed in a Mut(+) strain of the methylotrophic yeast Pichia pastoris for use as an antigen in a proposed human vaccine. Fermentation employed glycerol batch, glycerol-fed batch, and methanol-fed batch phases to achieve high cell density. Induction times were short to maximize rBoNTA(H(C)) production while minimizing proteolytic degradation. Concentration of rBoNTA(H(C)) in yeast cell lysates was generally 1-2% of the total protein based on ELISA analysis. The H(C) fragment was purified from cell lysates using a multistep ion-exchange (IEC) chromatographic process, including SP, Q, and HS resins. The zwitterionic detergent Chaps was included in the buffer system to combat possible interactions, such as protein-protein or protein-DNA interactions. Following IEC was a hydrophobic interaction chromatography (HIC) polishing step, using phenyl resin. The H(C) fragment was purified to >95% purity with yields up to 450 mg/kg cells based on ELISA and Bradford protein assay. The purified H(C) fragment of serotype A was stable, elicited an immune response in mice, and was protected upon challenge with native botulinum type A neurotoxin.
Potter
K J
KJ
Department of Food Science and Technology, University of Nebraska-Lincoln, Biological Process Development Facility, Lincoln, Nebraska 68583-0919, USA.
Zhang
W
W
Smith
L A
LA
Meagher
M M
MM
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D019274:Botulinum Toxins, Type A / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D002478:Cells, Cultured; D002793:Cholic Acids; D002845:Chromatography; D004306:Dose-Response Relationship, Immunologic; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005285:Fermentation; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D010843:Pichia / Q000235:genetics*; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013997:Time Factors; D014614:Vaccines, Synthetic / Q000276:immunology
D002793:Cholic Acids; D011993:Recombinant Fusion Proteins; D014614:Vaccines, Synthetic; D019274:Botulinum Toxins, Type A; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate