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

Research output: Contribution to journalArticle

35 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)87-92
Number of pages6
JournalBiochemical Journal
Volume334
Issue number1
DOIs
Publication statusPublished - 1998 Aug 15

Fingerprint

Bungarus
Bungarotoxins
Cloning
Taiwan
Organism Cloning
Proteins
Fusion reactions
histidylglycine
Amino Acids
Protein Sequence Analysis
Venoms
Viperidae
Amino Acid Sequence
Escherichia coli
Complementary DNA
RNA
Resins
Membranes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

@article{8b6cdfa31ee942c4ba5b4049908459b5,
title = "Cloning and functional expression of B chains of β-bungarotoxins from Bungarus multicinctus (Taiwan banded krait)",
abstract = "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.",
author = "Wu, {Pei Fung} and Sheng-Nan Wu and Chang, {Chun Chang} and Chang, {Long Sen}",
year = "1998",
month = "8",
day = "15",
doi = "10.1042/bj3340087",
language = "English",
volume = "334",
pages = "87--92",
journal = "Biochemical Journal",
issn = "0264-6021",
publisher = "Portland Press Ltd.",
number = "1",

}

Cloning and functional expression of B chains of β-bungarotoxins from Bungarus multicinctus (Taiwan banded krait). / Wu, Pei Fung; Wu, Sheng-Nan; Chang, Chun Chang; Chang, Long Sen.

In: Biochemical Journal, Vol. 334, No. 1, 15.08.1998, p. 87-92.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Cloning and functional expression of B chains of β-bungarotoxins from Bungarus multicinctus (Taiwan banded krait)

AU - Wu, Pei Fung

AU - Wu, Sheng-Nan

AU - Chang, Chun Chang

AU - Chang, Long Sen

PY - 1998/8/15

Y1 - 1998/8/15

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0032528860&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032528860&partnerID=8YFLogxK

U2 - 10.1042/bj3340087

DO - 10.1042/bj3340087

M3 - Article

VL - 334

SP - 87

EP - 92

JO - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

IS - 1

ER -