Analysis of non-active engineered Bacillus thuringiensis crystal proteins.
Crystal proteins of Bacillus thuringiensis are known for their insecticidal specificity. This specificity is, to a large extent, determined by the interaction of the proteins with high-affinity binding sites on the epithelial membrane of the midgut of sensitive insects. In particular, domain II of the three domains of the toxic moiety has been implicated in specificity. To determine which sequences of the protein are involved in binding, loops of domain II which terminate in the molecular apex of CryIA(b) were replaced by the corresponding regions of CryIE, a protein with different binding characteristics and insect specificity. In contrast to expression of the wild-type genes, expression of the mutant alleles in Escherichia coli resulted in the formation of biologically inactive, insoluble aggregates. Although these aggregates could be solubilized in vitro using urea, in contrast to the wild-type CryIA(b), the mutant proteins did not correctly refold as is shown by their increased protease sensitivity and lack of biological activity. The results indicate that engineering CryI proteins, based on the CryIIIA structure, is likely to prove difficult, particularly since the conformation of CryIIIA and CryI proteins might differ in domain II.
Bosch
D
D
Department of Molecular Biology, Centre for Plant Breeding and Reproduction Research (CPRO-DLO), Wageningen, The Netherlands.
Visser
B
B
Stiekema
W J
WJ
D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis / Q000737:chemistry*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D001427:Bacterial Toxins; D001483:Base Sequence; D004731:Endotoxins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D015870:Gene Expression; D005798:Genes, Bacterial / Q000235:genetics; D006460:Hemolysin Proteins; D002479:Inclusion Bodies / Q000737:chemistry; D007814:Larva; D007915:Lepidoptera; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010572:Pest Control, Biological; D011487:Protein Conformation; D017510:Protein Folding; D016415:Sequence Alignment
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis