Efficient Production of Bacillus thuringiensis Cry1AMod Toxins under Regulation of cry3Aa Promoter and Single Cysteine Mutations in the Protoxin Region
| Autor: | Alejandra Bravo, Blanca I. García-Gómez, Jorge E. Ibarra, Jorge Sánchez, Diana L. Martínez de Castro, Mario Soberón |
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| Rok vydání: | 2013 |
| Předmět: |
DNA
Bacterial Bacterial Toxins Bacillus thuringiensis Mutagenesis (molecular biology technique) Biology medicine.disease_cause Applied Microbiology and Biotechnology Serine Hemolysin Proteins chemistry.chemical_compound Bacterial Proteins Escherichia coli Invertebrate Microbiology medicine Animals Cysteine Protein Precursors Pest Control Biological Promoter Regions Genetic Mutation Bacillus thuringiensis Toxins Ecology fungi Promoter Hydrogen-Ion Concentration biology.organism_classification Molecular biology Endotoxins Lepidoptera Biochemistry chemistry Larva Mutagenesis Site-Directed DNA Food Science Biotechnology |
| Zdroj: | Applied and Environmental Microbiology. 79:6969-6973 |
| ISSN: | 1098-5336 0099-2240 |
| DOI: | 10.1128/aem.02546-13 |
| Popis: | Bacillus thuringiensis Cry1AbMod toxins are engineered versions of Cry1Ab that lack the amino-terminal end, including domain I helix α-1 and part of helix α-2. This deletion improves oligomerization of these toxins in solution in the absence of cadherin receptor and counters resistance to Cry1A toxins in different lepidopteran insects, suggesting that oligomerization plays a major role in their toxicity. However, Cry1AbMod toxins are toxic to Escherichia coli cells, since the cry1A promoter that drives its expression in B. thuringiensis has readthrough expression activity in E. coli , making difficult the construction of these CryMod toxins. In this work, we show that Cry1AbMod and Cry1AcMod toxins can be cloned efficiently under regulation of the cry3A promoter region to drive its expression in B. thuringiensis without expression in E. coli cells. However, p3A-Cry1Ab(c)Mod construction promotes the formation of Cry1AMod crystals in B. thuringiensis cells that were not soluble at pH 10.5 and showed no toxicity to Plutella xylostella larvae. Cysteine residues in the protoxin carboxyl-terminal end of Cry1A toxins have been shown to be involved in disulfide bond formation, which is important for crystallization. Six individual cysteine substitutions for serine residues were constructed in the carboxyl-terminal protoxin end of the p3A-Cry1AbMod construct and one in the carboxyl-terminal protoxin end of p3A-Cry1AcMod. Interestingly, p3A-Cry1AbMod C654S and C729S and p3A-Cry1AcMod C730S recover crystal solubility at pH 10.5 and toxicity to P. xylostella . These results show that combining the cry3A promoter expression system with single cysteine mutations is a useful system for efficient expression of Cry1AMod toxins in B. thuringiensis . |
| Databáze: | OpenAIRE |
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