Cloning and Functional
Expression of Neurotoxin cDNA from Naja naja atra
ZHONG Xiao-Yan, PAN Hua, WU Jun, YANG Guang-Zheng, WU
Xiang-Fu, ZHOU Yuan-Cong*
( Shanghai Institute of Biochemistry, the Chinese Academy of Sciences,
Shanghai 200031, China )
Abstract 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, Tyr25,
Lys27, Trp29, Arg33 and Lys47, 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.
Key words neurotoxin； expression； clone
Received: June 25,
1999 Accepted: August 30, 1999
* Corresponding author: Tel, 86-21-64374430; Fax, 86-21-64338357; e-mail, [email protected]