Molecular Cloning and Functional Expression of α-Bungarotoxin (V31) From Chinese Continental Banded Krait

The cDNA encoding a variant of α-bungarotoxin was cloned from the venom glands of Bungarus multicinctus by RT-PCR.The deduced protein precursor contained a 21 amino acid signal peptide and a following 74 amino acid mature protein.The signal peptide is very similar to those of short chain neurotoxins,κ-neurotoxins and cardiotoxins.The amino acid sequence of the mature protein is identical to α-bungarotoxin (V31),a minor variant of α-bungarotoxin identified by protein sequencing technique.Furthermore,the cDNA encoding the deletion precursor of α-bungarotoxin was also cloned.By use of pMAL-p2,the variant was overexpressed in E.coli as a soluble fusion protein and purified by sepharose 6B-amylose affinity chromatography,which was confirmed by western blotting with the antisera against α-bungarotoxin.The recombinant variant was achieved after digestion by factor Xa.It displayed about 1/6 in vivo toxicity of natural α-bungarotoxin.The successful cloning and functional expression of α-bungarotoxin provided a basis for the future study of structure-function of long neurotoxins.