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The four salt bridges (Asp222–Arg281, Arg233–Glu288, Arg234–Glu274, and Asp242–Arg265) linking domains I and II in Cry1Aa were abolished individually in α-helix 7 mutants D222A, R233A, R234A, and D242A. Two additional mutants targeting the fourth salt bridge (R265A) and the double mutant (D242A/R265A) were rapidly degraded during trypsin activation. Mutations were also introduced in the corresponding Cry1Ac salt bridge (D242E, D242K, D242N, and D242P), but only D242N and D242P could be produced. All toxins tested, except D242A, were shown by light-scattering experiments to permeabilize Manduca sextalarval midgut brush border membrane vesicles. The three active Cry1Aa mutants at pH 10.5, as well as D222A at pH 7.5, demonstrated a faster rate of pore formation than Cry1Aa, suggesting that increases in molecular flexibility due to the removal of a salt bridge facilitated toxin insertion into the membrane. However, all mutants were considerably less toxic to M. sextalarvae than to the respective parental toxins, suggesting that increased flexibility made the toxins more susceptible to proteolysis in the insect midgut. Interdomain salt bridges, especially the Asp242–Arg265bridge, therefore contribute greatly to the stability of the protein in the larval midgut, whereas their role in intrinsic pore-forming ability is relatively less important.

Cooperation of two crystal proteins from Bacillus thuringiensissubsp. thompsoni. strain HnC was shown to be essential for the formation of inclusion bodies. Expression of the operon containing the 34‐kDa and 40‐kDa protein genes from HnC in a B. thuringiensiscrystal minus strain resulted in the formation of inclusion bodies identical to those from strain HnC. Interruption of one of the genes in the operon led to the lack of inclusion body and to low production of the remaining protein. Absence of inclusion body and low rate of protein production were also observed when both genes were simultaneously expressed but on different vectors. To show a cooperative effect in the formation of the inclusion body, both proteins must be produced from the same transcript.

The potential role of a molecular chaperone on the rate of production of extensively altered Bacillus thuringiensis Cry 1C proteins was investigated. Analysis of the proteins produced by the recombinant B. thuringiensis strains showed that the truncated proteins were produced at a low rate. Expression of the 20-kDa protein gene from B. thuringiensis ssp. israelensis in tandem with the truncated-cry1C genes led to the production of a greater amount of proteins. The formation of inclusion bodies, however, did not occur even when the 20-kDa protein gene was expressed.