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 from the venom of Bungarus multicinctus, consists of two dissimilar polypeptide chains, the A chain and B chain, cross-linked by an interchain disulfide 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 of the refolded A and B chains increased markedly by at least 100-fold after substituting Ser for Cys1S of A chain and Cys55 of B chain, which formed an interchain disulfide bond. Either the A(C15) chain or B(C55S) chain alone or in combination cannot exhibit the phospholipase A2 activity or synaptosome binding activity of beta-Bgt. Nevertheless, the results of competitive enzyme-linked immunoassay, CD spectra, and fluorescence measurement revealed that the A(C15S) chain and B(C55S) chain possessed a native-like structure like the subunits of native beta-Bgt. Moreover, the interfacial interaction 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 disulfide bond should not be a crucial step for the formation of folded A and B chains in the venom glands, and that the integrity of the interchain disulfide linkage favors the subunit interaction that consequently fulfills the functional mechanism of beta-Bgt.
Wu
P F
PF
Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan.
Chang
L S
LS
D000595:Amino Acid Sequence; D000818:Animals; D002038:Bungarotoxins / Q000737:chemistry* / Q000235:genetics; D017838:Bungarus; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004220:Disulfides; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
D002038:Bungarotoxins; D018076:DNA, Complementary; D004220:Disulfides; D011994:Recombinant Proteins