Cloning, sequencing, and expression of equinatoxin II.
Equinatoxin II (EqtII), a basic protein of 179 amino acids lacking cysteine residues, is the most abundant cytolysin isolated from the sea anemone Actinia equina. Its mode of action is still poorly understood. In order to initiate further structure-function studies by protein engineering, cDNA library was prepared from the whole animal and hybridized with a PCR-derived probe, deduced from the EqtII primary structure. The longest positive clone of 899 bp was shown to encode a 214 residue precursor of EqtII. The mature protein region was amplified by PCR, cloned into a T7 RNA polymerase-based expression vector and expressed in Escherichia coli. Recombinant toxin was isolated by a simple, two-step isolation procedure including separation on CM-cellulose and gel filtration using an FPLC system. Its biochemical properties and hemolytic activity were practically indistinguishable from those of native toxin.
Anderluh
G
G
Department of Biology, Biotechnical Faculty, University of Ljubljana, Slovenia.
Pungercar
J
J
Strukelj
B
B
Macek
P
P
Gubensek
F
F
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003064:Cnidarian Venoms / Q000737:chemistry* / Q000235:genetics*; D003603:Cytotoxins / Q000737:chemistry / Q000235:genetics*; D015342:DNA Probes; D018076:DNA, Complementary / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry; D012615:Sea Anemones
D003064:Cnidarian Venoms; D003603:Cytotoxins; D015342:DNA Probes; D018076:DNA, Complementary; D011994:Recombinant Proteins; C012307:equinatoxin