Expression of fully active ammodytoxin A, a potent presynaptically neurotoxic phospholipase A2, in Escherichia coli.
A cDNA encoding the most presynaptically neurotoxic phospholipase A2, ammodytoxin A, from the venom of the long-nosed viper (Vipera ammodytes ammodytes) has been expressed in Escherichia coli. Ammodytoxin A was produced as a fusion protein with the 81 N-terminal residues of adenylate kinase followed by the tetrapeptide recognition site for factor Xa (IEGR) just preceding the first amino acid residue of the toxin. The fusion protein was expressed under the control of tac promoter without IPTG induction in the form of insoluble inclusion bodies. It was dissolved in guanidine hydrochloride, S-sulfonated and refolded in a reoxidation mixture including a reduced/oxidized glutathione redox couple. Ammodytoxin A was fully activated by limited hydrolysis with trypsin that preferentially cleaves the fusion protein at the factor Xa recognition site and purified by cation-exchange chromatography. The correct N-terminus was confirmed by protein sequencing. Recombinant ammodytoxin A has been proved to be indistinguishable from the native toxin in its enzymatic activity and toxicity.
Liang
N S
NS
Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, Ljubljana, Slovenia.
Pungercar
J
J
Krizaj
I
I
Strukelj
B
B
Gubensek
F
F
D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000096:biosynthesis* / Q000235:genetics; D054467:Phospholipases A2; D011994:Recombinant Proteins; D012911:Snakes; D014757:Viper Venoms / Q000096:biosynthesis* / Q000235:genetics
D011994:Recombinant Proteins; D014757:Viper Venoms; D004247:DNA; C068117:ammodytoxin A; D010741:Phospholipases A; D054467:Phospholipases A2