Cloning and Functional Expression of Neurotoxin cDNA from Naja naja atra

Three new cDNAs named NL1, NL2 and NL3 were cloned from the total RNA of Naja naja atra by RT-PCR. The protein sequences encoded by them showed 77%, 72% and 98% structure identity to cobrotoxin which is a postsynaptic neurotoxin from Taiwan cobra (Naja naja atra), respectively. The five conservative residues, Tyr(25), Lys(27), Trp(29), Arg(33) and Lys(47), essential for the function of cobrotoxin were also found in the three cDNAs. The NL3, the most homologous to cobrotoxin, was expressed in E.coli BL21(DE3) by cloning into pET28b+. The expressed product was insoluble inclusion bodies and could be purified up to 90% purity in the range of 6 mg per liter culture cells by a single affinity step. The purified protein was refolded in vitro and the toxicity was assessed to be less than the native cobrotoxin in mice.