Expression of A chain and B chain of beta-bungarotoxin from Taiwan banded krait: The functional implication of the interchain disulfide bond between A chain and B chain

beta -Bungarotoxin (beta -Bgt), the main presynaptic neurotoxin purified fr om the venom of Bungarus multi-cinctus, consists of two dissimilar polypept ide chains, the A chain and B chain, cross-linked by an interchain disulfid e bond. The A and B chain cDNAs were subcloned into expression vectors pT7- 7 and pET20b(+), respectively, and transformed into Escherichia coli strain BL21(DE3). The expressed protein was isolated from the inclusion bodies of E. coli and subjected to refolding into its folded structure. The yields o f the refolded A and B chains increased markedly by at least 100-fold after substituting Ser for Cys15 of A chain and Cys55 of B chain, which formed a n interchain disulfide bond. Either the A(C15S) chain or B(C55S) chain alon e or in combination cannot exhibit the phospholipase A(2) activity or synap tosome binding activity of beta -Bgt. Nevertheless, the results of competit ive enzyme-linked immunoassay, CD spectra, and fluorescence measurement rev ealed that the A(C15S) chain and B(C55S) chain possessed a native-like stru cture like the subunits of native beta -Bgt. Moreover, the interfacial inte raction between the A and B chains explored by glutaraldehyde cross-linking revealed the essential aspects of the intact interchain disulfide bond in this interaction. This suggests that the formation of the interchain disulf ide bond should not be a crucial step for the formation of folded A and B c hains in the venom glands, and that the integrity of the interchain disulfi de linkage favors the subunit interaction that consequently fulfills the fu nctional mechanism of beta -Bgt.