Cloning and functional expression of B chains of beta-bungarotoxins from Bungarus multicinctus (Taiwan banded krait).
The cDNA species encoding the B chains (B1 and B2) of beta-bungarotoxins (beta-Bgt) were constructed from the cellular RNA isolated from the venom glands of Bungarus multicinctus (Taiwan banded krait). The deduced amino acid sequences of the B chains were different from those determined previously by a protein sequencing technique. One additional Arg residue is inserted between Val-19 and Arg-20 of the B1 chain. Similarly the insertion of one additional Val residue between Val-19 and Arg-20 of the B2 chain is noted. Thus the B chains should comprise 61 amino acid residues. Moreover, the residues at positions 44-46 are Gly-Asn-His, in contrast with a previous result showing the sequence His-Gly-Asn. Instead of Asp, the residues at positions 41 and 43 are Asn. The B chain was subcloned into the expression vector pET-32a(+) and transformed into Escherichia coli strain BL21(DE3). The recombinant B chain was expressed as a fusion protein and purified on a His-Bind resin column. The yield of affinity-purified fusion protein was increased markedly by replacing Cys-55 of the B chain with Ser. However, the isolated B(C55S) chain became insoluble in aqueous solution after removal of the fused protein from the affinity-purified product, suggesting that protein-protein interactions might be crucial for stabilizing the structure of the B chain. The B(C55S) chain fusion protein showed activity in blocking the voltage-dependent K+ channel, but did not inhibit the binding of beta-Bgt to synaptosomal membranes. These results, together with the finding that modification of His-48 of the A chain of beta-Bgt caused a marked decrease in the ability to bind toxin to its acceptor proteins, suggest that the B chain is involved in the K+ channel blocking action observed with beta-Bgt, and that the binding of beta-Bgt to neuronal receptors is not heavily dependent on the B chain.
Wu
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PF
Department of Biochemistry, Kaohsiung Medical College, Kaohsiung, Taiwan.
Wu
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Chang
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Chang
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002038:Bungarotoxins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D017838:Bungarus; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D015221:Potassium Channels / Q000187:drug effects / Q000502:physiology; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism / Q000494:pharmacology; D013574:Synaptosomes / Q000378:metabolism*
D002038:Bungarotoxins; D017931:DNA Primers; D018076:DNA, Complementary; D046911:Macromolecular Substances; D015221:Potassium Channels; D011994:Recombinant Proteins