Maximal toxicity of cloned CytA δ-endotoxin from Bacillus thuringiensis subsp. israelensis requires proteolytic processing from both the N- and C-termini

S. A S Al-yahyaee, D. J. Ellar

Research output: Contribution to journalArticle

38 Citations (Scopus)

Abstract

Unlike the CytA toxin from native Bacillus thuringiensis subsp. israelensis (Bti) crystals, the inclusions of cloned CytA produced by Bti IFS 78/11 in the presence of the 20 kDa 'helper' protein require a reducing agent in addition to a highly alkaline pH for complete solubilization. Activation of the solubilized CytA with a range of proteases produced 25-22 kDa products. SDS-PAGE analysis and N-terminal amino acid sequencing revealed that CytA was processed very similarly at both termini by proteinase K or by Anopheles or Culex gut extracts. Trypsin, by contrast, processed CytA predominantly at the N-terminus. In vitro cytolytic assays against Aedes aegypti cells, and haemolytic assays against rat erythrocytes, showed that CytA processed at both termini by proteinase K was the most active form. Thus CytA, like other Bt δ-endotoxins, is processed to a well-defined protease-resistant product and this processing enhances the toxicity in vitro and possibly in vivo.

Original languageEnglish
Pages (from-to)3141-3148
Number of pages8
JournalMicrobiology
Volume141
Issue number12
Publication statusPublished - 1995

Fingerprint

Endopeptidase K
Bacillus thuringiensis
Endotoxins
Peptide Hydrolases
Culex
Anopheles
Aedes
Reducing Agents
Protein Sequence Analysis
Trypsin
Polyacrylamide Gel Electrophoresis
Erythrocytes
Proteins
In Vitro Techniques

Keywords

  • δ-endotoxins
  • Bacillus thuringiensis
  • CytA
  • Haemolytic assay

ASJC Scopus subject areas

  • Microbiology

Cite this

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title = "Maximal toxicity of cloned CytA δ-endotoxin from Bacillus thuringiensis subsp. israelensis requires proteolytic processing from both the N- and C-termini",
abstract = "Unlike the CytA toxin from native Bacillus thuringiensis subsp. israelensis (Bti) crystals, the inclusions of cloned CytA produced by Bti IFS 78/11 in the presence of the 20 kDa 'helper' protein require a reducing agent in addition to a highly alkaline pH for complete solubilization. Activation of the solubilized CytA with a range of proteases produced 25-22 kDa products. SDS-PAGE analysis and N-terminal amino acid sequencing revealed that CytA was processed very similarly at both termini by proteinase K or by Anopheles or Culex gut extracts. Trypsin, by contrast, processed CytA predominantly at the N-terminus. In vitro cytolytic assays against Aedes aegypti cells, and haemolytic assays against rat erythrocytes, showed that CytA processed at both termini by proteinase K was the most active form. Thus CytA, like other Bt δ-endotoxins, is processed to a well-defined protease-resistant product and this processing enhances the toxicity in vitro and possibly in vivo.",
keywords = "δ-endotoxins, Bacillus thuringiensis, CytA, Haemolytic assay",
author = "Al-yahyaee, {S. A S} and Ellar, {D. J.}",
year = "1995",
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journal = "Microbiology",
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T1 - Maximal toxicity of cloned CytA δ-endotoxin from Bacillus thuringiensis subsp. israelensis requires proteolytic processing from both the N- and C-termini

AU - Al-yahyaee, S. A S

AU - Ellar, D. J.

PY - 1995

Y1 - 1995

N2 - Unlike the CytA toxin from native Bacillus thuringiensis subsp. israelensis (Bti) crystals, the inclusions of cloned CytA produced by Bti IFS 78/11 in the presence of the 20 kDa 'helper' protein require a reducing agent in addition to a highly alkaline pH for complete solubilization. Activation of the solubilized CytA with a range of proteases produced 25-22 kDa products. SDS-PAGE analysis and N-terminal amino acid sequencing revealed that CytA was processed very similarly at both termini by proteinase K or by Anopheles or Culex gut extracts. Trypsin, by contrast, processed CytA predominantly at the N-terminus. In vitro cytolytic assays against Aedes aegypti cells, and haemolytic assays against rat erythrocytes, showed that CytA processed at both termini by proteinase K was the most active form. Thus CytA, like other Bt δ-endotoxins, is processed to a well-defined protease-resistant product and this processing enhances the toxicity in vitro and possibly in vivo.

AB - Unlike the CytA toxin from native Bacillus thuringiensis subsp. israelensis (Bti) crystals, the inclusions of cloned CytA produced by Bti IFS 78/11 in the presence of the 20 kDa 'helper' protein require a reducing agent in addition to a highly alkaline pH for complete solubilization. Activation of the solubilized CytA with a range of proteases produced 25-22 kDa products. SDS-PAGE analysis and N-terminal amino acid sequencing revealed that CytA was processed very similarly at both termini by proteinase K or by Anopheles or Culex gut extracts. Trypsin, by contrast, processed CytA predominantly at the N-terminus. In vitro cytolytic assays against Aedes aegypti cells, and haemolytic assays against rat erythrocytes, showed that CytA processed at both termini by proteinase K was the most active form. Thus CytA, like other Bt δ-endotoxins, is processed to a well-defined protease-resistant product and this processing enhances the toxicity in vitro and possibly in vivo.

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