Cloning and functional expression of B chains of β-bungarotoxins from Bungarus multicinctus (Taiwan banded krait)

Pei Fung Wu, Sheng Nan Wu, Chun Chang Chang, Long Sen Chang

研究成果: Article同行評審

35 引文 斯高帕斯(Scopus)

摘要

The cDNA species encoding the B chains (B1 and B2) of β-bungarotoxins (β-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 B1 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 β-Bgt to synaptosomal membranes. These results, together with the finding that modification of His-48 of the A chain of β-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 β-Bgt, and that the binding of β-Bgt to neuronal receptors is not heavily dependent on the B chain.

原文English
頁(從 - 到)87-92
頁數6
期刊Biochemical Journal
334
發行號1
DOIs
出版狀態Published - 1998 八月 15

All Science Journal Classification (ASJC) codes

  • 生物化學
  • 分子生物學
  • 細胞生物學

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