Author/Editor		Barlič, Ariana
Title		Izolacija in karakterizacija cisteinskih mutant ekvinatoksina II
Translated title		Isolation and characterization of equinatoxin II cysteine mutants
Type		monografija
Place		Ljubljana
Publisher		Medicinska fakulteta
Publication year		1998
Volume		str. 53
Language		slo
Abstract		Equinatoxin II (Eqt II), a basic protein of 179 amino isolated from sea anemone (Actinia equina L.), is a member of large group of cytolytic txins that are capagle to form pores in biological membranes and artificial lipid bilayers. Although this molecule is relatively well characterised, its detailed mechanism of insertion into membrane and pore formation is not known yet. To better explain this process we must understand the structure and function relationship. Besides other approaches this is attainable also by cysteine mutants. This study is based upon isolation and characterisation of mutants S1C, K77C, R126C and A179C. Expression of heterologous protein was induced in the middle log phase of bacterial growth in the strain Esherichia coli BL21(DE3). After multiple consecutive toxin extraction from bacterial cells we purified it by a simple two-step procedure that included cation-exchange and size-exclusion chromatographies. All the mutants isolated were pure. For each isolated mutant, the N-terminal sequence was determined. It is obvious that inaccurate processing accured in mutants S1C and A179C (deletion of the first amino acid residue). THe N-terminal Cys is present only in 25% of mutants S1C and N-terminal Ser in 65% of mutants A179C. Formation of covalent dimers in the case of mutants K77C, R126C and A179C is an interesting feature that tells a lot about exosure of a particular amino acid residue. In native Eqt II (lacking Cys residues) covalent dimers never occur. Biological activity of isolated mutants was not significantly altered compared to native Eqt II, except for mutant K77C which was approximately 100 times less active. Changed conformation of the molecule can not be the reason for reduced activity because all the mutants exhibit the same conformation as native toxin. Some samples of mutant K77C (probably due to frequent freezing and thawing) show completely different type of hemolysis.(Abstract truncated at 2000 characters)
Descriptors		CNIDARIAN VENOMS CYSTEINE SEA ANEMONES ESCHERICHIA COLI AMPICILLIN CHROMATOGRAPHY, ION EXCHANGE CHROMATOGRAPHY, GEL MUTATION PLASMIDS AMINO ACID SEQUENCE HEMOLYSIS