Structural and functional analysis of a cloned delta endotoxin of Bacillus thuringiensis berliner 1715

Höfte, Monica; Greve, Henri De; Seurinck, Jef; Jansens, Stefan; Mahillon, Jacques; Ampè, Christophe; Vandekerckhove, Joël; Vanderbruggen, Hilde; Montagu, Marc Van; Zabeau, Marc; Vaeck, Mark

doi:10.1111/j.1432-1033.1986.tb10443.x

Cited by 200 publications

(126 citation statements)

References 30 publications

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“…Activity of protein batch J6B significantly decreased 16-fold during one week to four months storage at 4 • C. This could be caused by further proteolysis of a partly incorrectly refolded protein with remaining trypsin after trypsinization of Cry1Ab protein, and is consistent with the SDS-PAGE analysis, which showed the degradation of a part of this protein from 60 to 59 kDa at 4 • C. The loss of activity of Cry1Ab protein was also observed by Höfte et al (1986) and Martens et al (1995) when a few amino acids from either the N-terminus or the C-terminus of the Cry1Ab trypsin-resistant core were removed. The potential impact of residual trypsin activity on batch J6B is further supported by a previous study, when no significant difference in the activity of an ultra-filtrated Cry1Ab protein batch could be observed during 4.5 months of storage at 4 • C (Nguyen et al, 2004).…”

Section: Discussionsupporting

confidence: 78%

Stability of Cry1Ab protein during long-term storage for standardization of insect bioassays

Nguyen

Jehle

2009

Environ. Biosafety Res.

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The reliable use of purified Cry1Ab protein standards is a prerequisite for ecological studies and resistance monitoring programs of Cry1Ab-expressing transgenic corn. In this study the stability and activity of different Cry1Ab protein batches expressed in and purified from Escherichia coli were determined during two-year storage at different temperature conditions (4 • C, -20 • C, and -80 • C). SDS-Polyacrylamide gel electrophoresis showed degradation of the protein stored at 4 • C over four months, whereas no difference in the band intensity of the Cry1Ab proteins stored at -20 • C and -80 • C was observed. Bioassays with neonate larvae of Ostrinia nubilalis indicated that the biological activity of Cry1Ab varied from batch to batch, depending on the production process. Cry1Ab protein stored at 4 • C for four months showed a significantly decreasing activity measured as median lethal concentration (LC 50 ), whereas the protein activity declined less than 11-fold after two years storage at -20 • C. When stored at -80 • C the toxin activity remained relatively stable for at least 30 months, as indicated by low LC 50 values of 7-10 ng Cry1Ab per cm 2 diet. These experiments demonstrate that appropriate long-term storage conditions of Cry1Ab protein standards are crucial for resistance monitoring programs of Bt corn, and storage at -80 • C is recommended.

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Section: Discussionsupporting

confidence: 78%

Stability of Cry1Ab protein during long-term storage for standardization of insect bioassays

Nguyen

Jehle

2009

Environ. Biosafety Res.

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“…The inclusion bodies were solubilized and purified as described [20]. The monomeric structure of the toxins was produced by activation of protoxins with trypsin in a mass ratio of 1:20 for 1 h at 25°C.…”

Section: Preparation Of Insecticidal Crystal Proteinsmentioning

confidence: 99%

Permeability Changes of Manduca sexta Midgut Brush Border Membranes Induced by Oligomeric Structures of Different Cry Toxins

et al. 2006

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Abstract. The pore-formation activity of monomeric and oligomeric forms of different Cry1 toxins (from Cry1A to Cry1G) was analyzed by monitoring ionic permeability across Manduca sexta brush border membrane vesicles. The membrane vesicles were isolated from microvilli structures, showing a high enrichment of apical membrane markers and low intrinsic K + permeability. A fluorometric assay performed with 3,3¢-dipropylthiodicarbocyanine fluorescent probe, sensitive to changes in membrane potential, was used. Previously, it was suggested that fluorescence determinations with this dye could be strongly influenced by the pH, osmolarity and ionic strength of the medium. Therefore, we evaluated these parameters in control experiments using the K + -selective ionophore valinomycin. We show here that under specific ionic conditions changes in fluorescence can be correlated with ionic permeability without effects on osmolarity or ionic strength of the medium. It is extremely important to attenuate the background response due to surface membrane potential and the participation of the endogenous permeability of the membrane vesicles. Under these conditions, we analyzed the pore-formation activity induced by monomeric and oligomeric structures of different Cry1 toxins. The Cry1 toxin samples containing oligomeric structures correlated with high pore activity, in contrast to monomeric samples that showed marginal pore-formation activity, supporting the hypothesis that oligomer formation is a necessary step in the mechanism of action of Cry toxins.

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“…Particular attention was focused on the 5'-terminal region of the cryIF gene, since this region has been shown to encode the active toxin moiety of other Cryl ICPs (2,19,35).…”

Section: Resultsmentioning

confidence: 99%

Isolation and characterization of a novel insecticidal crystal protein gene from Bacillus thuringiensis subsp. aizawai

et al. 1991

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Bacillus thuringiensis subsp. aizawai EG6346, a novel grain dust isolate, was analyzed by Southern blot hybridization for its insecticidal crystal protein (ICP) gene profile. Strain EG6346 lacks previously characterized cryIA ICP genes yet does possess novel cryI-related gene sequences. A recombinant genomic plasmid library was constructed for strain EG6346 in Escherichia coli. One recombinant plasmid, pEG640, isolated from the library contained a novel ICP gene on a 5.7-kb Sau3A insert. The sequence of this gene, designated cryIF, was related to, but distinct from, the published sequences for other cryI genes. A second novel cryI-related sequence was also located on pEG640, approximately 500 bp downstream from cryIF. Introduction of cryIF into a Cry- B. thuringiensis recipient strain via electroporation enabled sufficient production of CryIF protein for quantitative bioassay analyses of insecticidal specificity. The CryIF crystal protein was selectively toxic to a subset of lepidopteran insects tested, including the larvae of Ostrinia nubilalis and Spodoptera exigua.

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Structural and functional analysis of a cloned delta endotoxin of Bacillus thuringiensis berliner 1715

Cited by 200 publications

References 30 publications

Stability of Cry1Ab protein during long-term storage for standardization of insect bioassays

Stability of Cry1Ab protein during long-term storage for standardization of insect bioassays

Permeability Changes of Manduca sexta Midgut Brush Border Membranes Induced by Oligomeric Structures of Different Cry Toxins

Isolation and characterization of a novel insecticidal crystal protein gene from Bacillus thuringiensis subsp. aizawai

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