Cloning and expression of 130-kd mosquito-larvicidal δ-endotoxin gene of Bacillus thuringiensis var. Israelensis in Escherichia coli

Angsuthanasombat, Chanan; Chungjatupornchai, Wipa; Kertbundit, Sunee; Luxananil, Plernpis; Settasatian, Chatri; Wilairat, Prapon; Panyim, Sakol

doi:10.1007/bf00328128

Cited by 58 publications

(37 citation statements)

References 29 publications

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“…The recombinant plasmid pMEx-B4A encoding the 130-kDa Bt Cry4A larvicidal protein, which has been reconstructed in the pMEx8 expression vector (Buttcher et al, 1990) under control of the tac promoter together with the cry4B promoter (Angsuthanasombat et al, 1987), was used as a template for the site-directed mutagenesis. Mutant plasmids were generated by a polymerase chain reaction (PCR) using a pair of mutagenic primers ( Table 1) that were purchased from Proligo Inc. (Singapore) and Pfu DNA polymerase, following the procedure of the QuickChange TM Mutagenesis Kit (Stratagene, La Jolla, USA).…”

Section: Construction Of Mutant Toxinsmentioning

confidence: 99%

Aromaticity of Tyr-202 in the α4-α5 Loop Is Essential for Toxicity of the Bacillus thuringiensis Cry4A Toxin

Pornwiroon

Katzenmeier²,

Panyim³

et al. 2004

BMB Reports

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Section: Construction Of Mutant Toxinsmentioning

confidence: 99%

Aromaticity of Tyr-202 in the α4-α5 Loop Is Essential for Toxicity of the Bacillus thuringiensis Cry4A Toxin

Pornwiroon

Katzenmeier²,

Panyim³

et al. 2004

BMB Reports

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“…The complex crystal is composed of at least three major proteins of approximately 130 kDa, 70 kDa, and 28 kDa. The genes for the 130-kDa and the 28-kDa crystal proteins have been cloned and their nucleotide sequences have been reported (1,25,30,31). These cloning experiments have indicated that the 130-kDa and the 28-kDa B. thuringiensis subsp.…”

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confidence: 99%

Molecular characterization of a gene encoding a 72-kilodalton mosquito-toxic crystal protein from Bacillus thuringiensis subsp. israelensis

1988

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A gene encoding a 72,357-dalton (Da) crystal protein of Bacillus thuringiensis var. israelensis was isolated from a native 75-MDa plasmid by the use of a gene-specific oligonucleotide probe. Bacillus megaterium cells harboring the cloned gene (cryD) produced significant amounts of the 72-kDa protein (CryD), and the cells were highly toxic to mosquito larvae. In contrast, cryD-containing Escherichia coli cells did not produce detectable levels of the 72-kDa CryD protein. The sequence of the CryD protein, as deduced from the sequence of the cryD gene, was found to contain regions of homology with two previously described B. thuringiensis crystal proteins: a 73-kDa coleopteran-toxic protein and a 66-kDa lepidopteran-and dipteran-toxic protein of B. thuringiensis subsp. kurstaki. A second gene encoding the B. thuringiensis subsp. israelensis 28-kDa crystal protein was located approximately 1.5 kilobases upstream from and in the opposite orientation to the cryD gene.Certain varieties of Bacillus thuringiensis synthesize parasporal crystals composed of proteins that have been shown to be toxic to the larvae of specific insects. B. thluringiensis subsp. kurstaki as well as other varieties produces a bipyramidal crystal composed of one or more related proteins of approximately 130 kilodaltons (kDa) which are toxic to lepidopterans (caterpillars) (for recent reviews, see references 2 and 34) and also a cuboidal crystal composed of a 66-kDa protein that is toxic to both lepidopteran and dipteran (mosquito, black fly) insects (6,37). Other subspecies of B. thuringiensis have been identified which produce rhomboid crystals composed of a 73-kDa protein that is specifically toxic to coleopteran (beetle) larvae (12, 16; W. P. Donovan, J. M. Gonzalez, Jr., M. P. Gilbert, and C. Dankocsik, Mol. Gen. Genet., in press).B. thuringiensis subsp. israelensis synthesizes an irregularly shaped parasporal crystal that is highly toxic to certain dipteran larvae (8). The complex crystal is composed of at least three major proteins of approximately 130 kDa, 70 kDa, and 28 kDa. The genes for the 130-kDa and the 28-kDa crystal proteins have been cloned and their nucleotide sequences have been reported (1,25,30,31). These cloning experiments have indicated that the 130-kDa and the 28-kDa B. thuringiensis subsp. israelensis crystal proteins are mosquito toxic. However, other researchers have reported that the 28-kDa protein has little or no mosquitocidal activity (4,5,11,14,15,28

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“…Toxin expression, inclusion solubilization, and proteolytic activation The cloned Cry4B toxin (Angsuthanasombat et al, 1987), its non-toxic R158A mutant (Sramala et al, 2001), and the cloned Cry1Aa toxin (a generous gift of Dr. J-L Schwartz, National Research Council of Canada) were over-expressed in E. coli as inclusion bodies upon IPTG (isopropyl-β-D-thiogalactopyranoside) induction at 37 o C. After disruption in a French Press Cell, protoxin inclusions in the crude lysates were obtained by centrifugation, as described earlier (Uawithya et al, 1998). Protein concentrations were determined by using a dye-binding method (BioRad, Hercules, USA), with bovine serum albumin (Sigma, St. Louis, USA) as a standard.…”

Section: Methodsmentioning

confidence: 99%

Ex vivo Cytotoxicity of the Bacillus thuringiensis Cry4B δ-Endotoxin to Isolated Midguts of Aedes aegypti Larvae

Barusrux¹,

Sramala²,

Katzenmeier³

et al. 2003

BMB Reports

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The pathological effect of the Bacillus thuringiensis Cry δ-endotoxins on susceptible insect larvae had extensive damage on the midgut epithelial cells. In this study, an ex vivo assay was devised for assessing the insecticidal potency of the cloned Cry4B mosquito-larvicidal protein that is expressed in Escherichia coli. Determination of toxicity was carried out by using a cell viability assay on the midguts that were dissected from 5-day old Aedes aegypti mosquito larvae. After incubation with the toxin proteins, the number of viable epithelial cells was determined photometrically by monitoring the quantity of the bioreduced formazan product at 490 nm. The results showed that the 65-kDa trypsin-activated Cry4B toxin exhibited toxic potency ca. 3.5 times higher than the 130-kDa Cry4B protoxin. However, the trypsin-treated products of the non-bioactive Cry4B mutant (R158A) and the lepidopteran-specific Cry1Aa toxin displayed relatively no ex vivo activity on the mosquito-larval midguts. The ex vivo cytotoxicity studies presented here confirms data that was obtained in bioassays.

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Cloning and expression of 130-kd mosquito-larvicidal δ-endotoxin gene of Bacillus thuringiensis var. Israelensis in Escherichia coli

Cited by 58 publications

References 29 publications

Aromaticity of Tyr-202 in the α4-α5 Loop Is Essential for Toxicity of the Bacillus thuringiensis Cry4A Toxin

Aromaticity of Tyr-202 in the α4-α5 Loop Is Essential for Toxicity of the Bacillus thuringiensis Cry4A Toxin

Molecular characterization of a gene encoding a 72-kilodalton mosquito-toxic crystal protein from Bacillus thuringiensis subsp. israelensis

Ex vivo Cytotoxicity of the Bacillus thuringiensis Cry4B δ-Endotoxin to Isolated Midguts of Aedes aegypti Larvae

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