Expression and mutagenesis studies of cobrotoxin from Taiwan cobra

The cDNA encoding cobrotoxin was constructed from the cellular RNA isolated from the venom glands of Naja naja atra (Taiwan cobra). The cDNA was subcl oned into the expression vector pET20b(+) and transformed into BL21(DE3) Es cherichia coli strain. Expressed cobrotoxin was isolated from inclusion bod ies off. coli and subjected to refolding into its folded structure. The ref olded cobrotoxin was purified by high-performance liquid chromatography and exhibited a neurotoxicity in inhibiting acetylcholine-induced muscle contr actions. Recombinant cobrotoxin showed a tendency to isomerize its disulfid e bonds as that observed with native cobrotoxin. An appreciable decrease in the rate of isomerization reaction was observed when Glu-38 was replaced w ith Gln-38 or Lys-47 was replaced with Glu-47 or Gln-47. These results refl ect that the element in controlling the disulfide isomerization of cobrotox in is closely associated with the charged side chains in the cobrotoxin mol ecule. (C) 1999 Academic Press.