Production of Chymotrypsin-Resistant Bacillus thuringiensis Cry2Aa1 δ-Endotoxin by Protein Engineering
| Autor: | Algimantas P. Valaitis, Donald H. Dean, Oscar Alzate, Mongkon Audtho |
|---|---|
| Rok vydání: | 1999 |
| Předmět: |
Insecticides
Proteases medicine.medical_treatment Proteolysis Bacterial Toxins Moths Biology Protein Engineering Applied Microbiology and Biotechnology Hemolysin Proteins Structure-Activity Relationship Bacterial Proteins Bacillus thuringiensis Invertebrate Microbiology medicine Animals Chymotrypsin Protease Bacillus thuringiensis Toxins Ecology medicine.diagnostic_test fungi Midgut Protein engineering Apical membrane biology.organism_classification Molecular biology Endotoxins Biochemistry Mutagenesis Site-Directed biology.protein Food Science Biotechnology |
| Zdroj: | Applied and Environmental Microbiology. 65:4601-4605 |
| ISSN: | 1098-5336 0099-2240 |
| Popis: | During sporulation, Bacillus thuringiensis, a group of gram-positive bacteria, produces crystalline (Cry) proteins which are toxic against a range of insect groups. Recently, over 120 cry genes have been reported and a new nomenclature system classifying Cry proteins based on the amino acid homology of the proteins has been proposed (5). The mechanism of action of most Cry proteins consists of three major steps: solubilization and activation of protoxin in the insect midgut (34), binding of the activated fragment to midgut receptor (2, 13), and insertion of the toxin into the midgut apical membrane, which causes destruction of membrane potential (9, 12). The molecular sizes of most Cry proteins are around 130 to 140 kDa (14). After digestion by trypsin-like enzymes in the midgut (23, 34), the active fragment is produced. In general, the protease-resistant core protein is in the range of 60 to 70 kDa (1, 14), covering three domains of the active toxin (10, 19). Deletion of the cry genes beyond the active sequences completely abolished the toxic activity of the gene products (33, 35). Proteolytic processing of Cry protein by midgut proteases is reported to generate active toxins of varying potency and specificity (11). However, digestion of Cry1A by diamondback moth (Plutella xylostella) midgut extract generated a core that lacks α-helix 1 in domain I of Cry1A (27). Similarly, digestion of Cry3A with chymotrypsin caused a nick at the region between α-helix 3 and α-helix 4 of domain I (3). The protease cleavage inside domain I was also found in Cry9Ca1, and more-stable protein was produced after removal of the trypsin-cleaved residue (16). Cry2Aa1, a 633-amino-acid toxin with molecular mass of 63 kDa, was originally described by Yamamoto as a dipteran- and lepidopteran-active protein (6, 36, 37). Even though the sequence of this Cry protein shows rather limited homology to those of the other Cry proteins, its structure has recently been determined and observed to be similar to those of Cry1Aa and Cry3A, consisting of three distinct domains (24). English et al. (7) reported the distinct binding and ion channels formed by Cry2Aa1 in Helicoverpa zea, indicating a mode of action unique among the Cry proteins. Here, we report that Cry2Aa1 is rapidly cleaved at a single position in domain I by midgut enzymes of gypsy moth (Lymantria dispar). The cleavage product is not toxic to the insect. Several mutant proteins were constructed by removing the amino acid targeted by the proteases. All of these mutants were able to release the active fragment, which is more resistant to protease digestion than the wild-type toxin. |
| Databáze: | OpenAIRE |
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