Author/Editor		Ivanovski, Gabriela
Title		Študij nevrotoksičnega mesta na molekuli fosfolipaz A2
Type		monografija
Place		Ljubljana
Publisher		Medicinska fakulteta
Publication year		1999
Volume		str. 79
Language		slo
Abstract		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 U/mg, although its LD50 is slightly lower, 0.16 mg/kg 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-times less toxic atxB differ. Using polymerase chain reaction, we prepared four mutants of atxA: the quadruple mutant atxA (Y115K/I16K/R118M/N119L) (atxA KKML), and three double mutants atxA (Y115K/I116K) (atxA KK), atxA (R118M/M119L) (atxA ML) and atxA (R118M/N119F) (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.
Descriptors		VIPER VENOMS PHOSPHOLIPASES A NEUROTOXINS POLYMERASE CHAIN REACTION RECOMBINANT PROTEINS RECOMBINATION, GENETIC TRYPSIN PLASMIDS ESCHERICHIA COLI RECOMBINANT FUSION PROTEINS BASE SEQUENCE LETHAL DOSE 50