Suppression of protein structure destabilizing mutations in Bacillus thuringiensis delta-endotoxins by second site mutations.
Reciprocal exchange of a small region (residues 428-450) within the specificity determining region of two Bacillus thuringiensis delta-endotoxins, CryIAa and CryIAc, resulted in two recombinant proteins that possess a decreased insecticidal activity to Bombyx mori and Manduca sexta. Site-directed mutations introduced in this region of one of the recombinant proteins, with the intent of restoring insecticidal activity, resulted in further reduction of toxicity. We determined that the loss of insecticidal activity in the mutants and the original recombinants was associated with altered toxin protein structure, as measured by sensitivity to intracellular and exogenous proteases. The structural instability of the site-directed mutant proteins could be suppressed genetically by subcloning the mutated region into cryIAc or by introducing second site mutations in defined regions of the mutated cryIAa gene. The second site mutations, by themselves, also produced unstable proteins. In the course of this study, we demonstrated that this small region does not suffice as a specificity determining region for M. sexta.
Almond
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Department of Molecular Genetics, Ohio State University, Columbus 43210.
Dean
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D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D001483:Base Sequence; D012831:Bombyx; D004277:DNA, Single-Stranded; D004731:Endotoxins / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D006460:Hemolysin Proteins; D007306:Insecticides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D008969:Molecular Sequence Data; D009036:Moths; D011487:Protein Conformation; D013489:Suppression, Genetic; D014357:Trypsin
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004277:DNA, Single-Stranded; D004731:Endotoxins; D006460:Hemolysin Proteins; D007306:Insecticides; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D014357:Trypsin