PubMed · 13554635
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T E CARTWRIGHT, M A LAUFFER. 1958. Temperature effects on botulinum A toxin.. https://doi.org/10.3181/00379727-98-24033
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Tetanus and botulinum neurotoxins (TeNT and BoNTs) block neuroexocytosis via specific cleavage and inactivation of SNARE proteins. Such activity is exerted by the N-terminal 50 kDa light chain (L) domain, which is a zinc-dependent endopeptidase. TeNT, BoNT/B, /D, /F and /G cleave vesicle associated membrane protein (VAMP), a protein of the neurotransmitter-containing small synaptic vesicles, at different single peptide bonds. Since the proteolytic activity of these metalloproteases is higher on native VAMP inserted in synaptic vesicles than on recombinant VAMP, we have investigated the influence of liposomes of different lipid composition on this activity. We found that the rate of VAMP cleavage with all neurotoxins tested here is strongly enhanced by negatively charged lipid mixtures. This effect is at least partially due to the binding of the metalloprotease to the lipid membranes, with electrostatic interactions playing an important role.
Fed-batch fermentation of a methanol utilization plus (Mut(+)) Pichia pastoris strain typically has a growth phase followed by a production phase (induction phase). In the growth phase glycerol is usually used as carbon for cell growth while in the production phase methanol serves as both inducer and carbon source for recombinant protein expression. Some researchers employed a mixed glycerol-methanol feeding strategy during the induction phase to improve production, but growth kinetics on glycerol and methanol and the interaction between them were not reported. The objective of this paper is to optimize the mixed feeding strategy based on growth kinetic studies using a Mut(+) Pichia strain, which expresses the heavy-chain fragment C of botulinum neurotoxin serotype C [BoNT/C(Hc)] intracellularly, as a model system. Growth models on glycerol and methanol that describe the relationship between specific growth rate ( micro ) and specific glycerol/methanol consumption rate ( nu(gly), nu(MeOH)) were established. A mixed feeding strategy with desired micro (gly)/ micro (MeOH) =1, 2, 3, 4 (desired micro (MeOH) set at 0.015 h(-1)) was employed to study growth interactions and their effect on production. The results show that the optimal desired micro (gly)/ micro (MeOH) is around 2 for obtaining the highest BoNT/C(Hc) protein content in cells: about 3 mg/g wet cells.
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