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Študij nevrotoksičnega mesta na molekuli fosfolipaz A2 : magistrsko delo
Ivanovski, Gabriela
Vipera a. ammodytes venom contains three presynaptically neurotoxic PLA2s, ammodytoxins (atx) A, B and C, among which atxA is the most lethal. Snake venom PLA2s have been effectively produced in the ...Escherichia coli expression system by using a plasmid with strong T7 promotor. Recombinant toxins can be produced in the bacterial cytoplasm either as mature proteins or as fusion proteins. The latter must be activated in vitro by cleaving the N-terminal propeptide. For this purpose trypsin is usually used. Since ammodytoxins and trypsin have similar isoelectric points, isolation of ammodytoxins is difficult. We therefore carried out the activation with acetylated trypsin, which is an affective activating enzyme, easily separated from recombinant atxC by cation exchange chromatography. The enzymatic activity against egg yolk substrate was used to monitor correct folding. The specific enzymatic activity atxC is the same as that of naturally occuring atxC, 286 Užmg, although its LD50 is slightly lower, 0.16 mgžkg of mause. The structure-function relationships of presynaptically neurotoxic PLA2s have not yet been elucidated. We have used site-directed mutagenesis to determine at least part of the molecule that is involved in the process of neurotoxicity. Attention has been focussed on the region 115-119, where atxA and the 28-timesless toxic atxB differ. Using polymerase chain reaction, we prepared four mutants of atxAČ the quadruple mutant atxA (Y115KžI16KžR118MžN119L) (atxAKKML), and three double mutants atxA (Y115KžI116K) (atxA KK), atxA (R118MžM119L) (atxA ML) and atxA (R118MžN119F) (atxA MF). All were expressed as fusion proteins. After isolation and renaturation they were activated with acetylated trypsin and purified by cation exchange chromatography. Abstract truncated at 2000 characters.
Type of material - master's thesis
Publication and manufacture - Ljubljana : [G. Ivanovski], 1999
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