Author/Editor		Štrekelj, Anita
Title		Priprava mestnospecifičnega fluorescenčnega derivata nevrotoksične fosfolipaze A2
Type		monografija
Place		Ljubljana
Publisher		Medicinska fakulteta
Publication year		2004
Volume		str. 82
Language		slo
Abstract		Ammodytoxin A (AtxA), a presynaptically neurotoxic secretory phospholipase A2 (sPLA2), is the most toxic component of Vipera ammodytes ammodytes venom. Precise molecular mechanism of action of presynaptically neurotoxic sPLA2 is still under investigation, although recent experiments indicate that ammodytoxins cross the presynaptic membrane and trigger the neurotoxic effect within the neuron. To confirm this hypothesis, AtxA was labelled by two types of fluorescent probes. Fluorescent AtxA-derivates could be used in the investigation of the toxin's action on neuro-muscular preparations and/or mammalian tissue cultures. Firstly, two fusion proteins of AtxA with a mutated green fluorescent protein (mGFP) linked either to its C- or N-terminus were constructed. mGFP was prepared from wild-type GFP possessing a single mutation, S65T, which enhances fluorescence. Additional mutations were introduced by PCR-directed mutagenesis, F64L to improve protein folding at 37°C and C48V to decrease the possibility of improper disulphide bond formation. The mGFP fusion part was linked to the toxin through a 13 amino acid spacer to minimise difficulties with folding and functionality of AtxA. Both constructs were expressed in Escerichia coli and isolated as inclusion bodies. Under tested conditions, in vitro refolding and purification of the fusion proteins were not possible. On the contrary epifluorescence microscopy showed marked fluorescence of the expression cells transformed with either of the two constructed plasmids, presumably allowing localization studies of the fusion proteins in mammalian cell lines. Secondly, Texas Red CZ maleimide was chemically linked to a single free cysteine residue introduced into AtxA at position 79. The basic biochemical properties of AtxA(N79C) important for biological action, such as specific enzymatic activity, toxicity in mice and receptor binding affinities, were similar to those of the wild type. (Abstract truncated at 2000 characters).
Descriptors		PHOSPHOLIPASES A VIPER VENOMS RECOMBINANT FUSION PROTEINS MUTAGENESIS, SITE-DIRECTED NEUROTOXINS BASE SEQUENCE CLONING, MOLECULAR MICROSCOPY, FLUORESCENCE ESCHERICHIA COLI POLYMERASE CHAIN REACTION VIPERIDAE