Comparison and optimization of the method for Cry1Ac protoxin preparation in HD73 strain

Zhou, Zishan; Yang, Sicheng; Shu, Changlong; Song, Fuping; Zhou, Xin; Zhang, Jie

doi:10.1016/s2095-3119(14)60950-3

Cited by 19 publications

(16 citation statements)

References 24 publications

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“…He obtained Cry1Ac protoxin by growing the HD73 strain of Bt subsp. kurstaki in 1/2 Luria-Bertani medium using the repeated crystal solubilization method 59 . In the first and second sets of bioassays, we compared Cry1Ac protoxin with trypsin-activated Cry1Ac purchased from Marianne Pusztai-Carey that she produced as described above.…”

Section: Methodsmentioning

confidence: 99%

Dual mode of action of Bt proteins: protoxin efficacy against resistant insects

Tabashnik

Zhang

Fabrick

et al. 2015

Sci Rep

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Transgenic crops that produce Bacillus thuringiensis (Bt) proteins for pest control are grown extensively, but insect adaptation can reduce their effectiveness. Established mode of action models assert that Bt proteins Cry1Ab and Cry1Ac are produced as inactive protoxins that require conversion to a smaller activated form to exert toxicity. However, contrary to this widely accepted paradigm, we report evidence from seven resistant strains of three major crop pests showing that Cry1Ab and Cry1Ac protoxins were generally more potent than the corresponding activated toxins. Moreover, resistance was higher to activated toxins than protoxins in eight of nine cases evaluated in this study. These data and previously reported results support a new model in which protoxins and activated toxins kill insects via different pathways. Recognizing that protoxins can be more potent than activated toxins against resistant insects may help to enhance and sustain the efficacy of transgenic Bt crops.

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Section: Methodsmentioning

confidence: 99%

Dual mode of action of Bt proteins: protoxin efficacy against resistant insects

Tabashnik

Zhang

Fabrick

et al. 2015

Sci Rep

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“…The Cry1Ab/1Ac fusion gene was driven by the promoter of Cry1Ac gene and the construct was expressed in Bt HD73-strain. The recombinant protein was extracted as described previously by Zhou et al (2015). The protoxin was incubated with trypsin (2 mg per 100 mg of protoxin) at 37 C for 3 h, and the 66 kDa fusion protein was generated.…”

Section: Test Materialsmentioning

confidence: 99%

A 52-week safety study in cynomolgus macaques for genetically modified rice expressing Cry1Ab/1Ac protein

Mao

Sun

Cheng

et al. 2016

Food and Chemical Toxicology

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“…B. thuringiensis strain Biot1Ah, carrying the cry1Ah1 gene (21), was grown in 0.5ϫ LB medium at 220 rpm and 30°C until sporulation. Cry1Ah protoxin was prepared using repeated crystal solubilization methods as described previously (42). Cry1Ah protoxin was digested by trypsin at a 20:1 mass ratio (protein to trypsin) at 37°C for 2 h. The activated Cry1Ah toxin was purified by size exclusion chromatography (Superdex 75 10/300, AKTA Avant; GE Healthcare, Uppsala, Sweden) using phosphate-buffered saline (PBS) buffer at a 1-ml/min flow rate.…”

Section: Methodsmentioning

confidence: 99%

Insecticidal Specificity of Cry1Ah to Helicoverpa armigera Is Determined by Binding of APN1 via Domain II Loops 2 and 3

Zhou

Liu

Liang

et al. 2017

Appl Environ Microbiol

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Bacillus thuringiensis Cry1Ah protein is highly toxic against Helicoverpa armigera but shows no toxicity against Bombyx mori larvae. In contrast, the closely related Cry1Ai toxin showed the opposite phenotype: high activity against B. mori but no toxicity against H. armigera. Analysis of binding of Cry1Ah to brush border membrane vesicle (BBMV) proteins from H. armigera and B. mori by surface plasmon resonance revealed association of toxin binding with insect specificity. Pulldown experiments identified aminopeptidase N1 (APN1) as a Cry1Ah binding protein that was not observed in the assays using B. mori BBMV proteins. The APN1 Cry1Ah binding region was narrowed to the region from A 548 to S 798 (fragment H3) by expressing four different APN1 fragments in Escherichia coli and analyzing Cry1Ah binding by ligand blot. Binding competition experiments of Cry1Ah to APN1 fragment H3 using synthetic peptides corresponding to four predicted domain II loop regions showed that loop 2 and loop 3 have additive effects on binding to APN1 fragment H3. Moreover, switching of loop 2 and loop 3 regions from Cry1Ah to Cry1Ai toxins showed that loop 2 and loop 3 are both involved in specificity and toxicity against H. armigera. IMPORTANCE Domain II loop regions have been shown to be involved in binding to larval gut proteins mediating insect specificity. The modification of loop regions is a direct and effective method to construct new Cry toxin variants to increase toxicity or modify specificity. Our results show that the exchange of loop regions from one toxin into another is a successful scheme for modification of B. thuringiensis Cry toxin specificity.

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Comparison and optimization of the method for Cry1Ac protoxin preparation in HD73 strain

Cited by 19 publications

References 24 publications

Dual mode of action of Bt proteins: protoxin efficacy against resistant insects

Dual mode of action of Bt proteins: protoxin efficacy against resistant insects

A 52-week safety study in cynomolgus macaques for genetically modified rice expressing Cry1Ab/1Ac protein

Insecticidal Specificity of Cry1Ah to Helicoverpa armigera Is Determined by Binding of APN1 via Domain II Loops 2 and 3

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