Structural stability of Bacillus thuringiensis δ-endotoxin homolog- scanning mutants determined by susceptibility to proteases

Abstract

Forty homolog-scanning (double-reciprocal-crossover) mutant proteins of two Bacillus thuringiensis δ-endotoxin genes (cryIAa and cryIAc) were examined for potential structural alterations by a series of proteolytic assays. Three groups of mutants could be identified. Group 1, consisting of 13 mutants, showed no δ-endotoxin present during overexpression conditions in Escherichia coli (48 h at 37°C, with a p(tac) promoter). These mutants produced full-sized δ-endotoxin detectable by polyacrylamide gel electrophoresis with Coomassie blue staining or Western immunoanalysis after 24 h of growth but not after 48 h, suggesting sensitivity to intracellular proteases. Group 2 consisted of 13 mutants that produced stable δ-endotoxins that were completely digested by 2% bovine trypsin. In contrast, native δ- endotoxin produces a 65,000-Da trypsin-resistant peptide, which is the active toxin. Group 3 mutants expressed δ-endotoxin and trypsin-stable toxins, similar to the wild type. In this study, 12 group 3 mutant toxins were compared with wild type toxins by thermolysin digestion at a range of temperatures. The two wild-type toxins exhibited significant differences in thermolysin digestion midpoints. Among the group 3 mutants, most possessed significantly different protein stabilities relative to their parental toxins. Two of the group 3 mutants were observed to have exchanged the thermolysin sensitivity properties of the parental toxins.