Expression of the p20 gene from Bacillus thuringiensis H-14 increases Cry11A toxin production and enhances mosquito-larvicidal activity in recombinant gram-negative bacteria

Experimental analyses with recombinant Escherichia coli and Pseudomonas put ida transformed with plasmids bearing genes coding for the Cry11A toxin and P20 protein from Bacillus thuringiensis H-14 showed that cells producing b oth proteins were more toxic when fed to third-instar Aedes aegypti larvae than were cells expressing cry11A alone; the 50% lethal concentrations were in the range of 10(4) to 10(5) cells/ml. Western blots revealed a higher p roduction of Cry11A when the p20 gene was coexpressed, Cry11A was detected primarily in insoluble form in recombinant cells. Cry11A was not detected i n P, putida when P20 was not coproduced, and these recombinants were not to xic to larvae, whereas P, putida recombinants producing both proteins were toxic at concentrations similar to those for E, coli, A coelution experimen t was conducted, in which a p20 gene construct producing the P20 protein wi th an extension of six histidines on the C terminus was mixed with the Cry1 1A protein. The results showed that Cry11A bound to the P20 (His,) on a nic kel chelating column, whereas Cry11A produced without the P20(His(6)) prote in was washed through the column, thus indicating that Cry11A and P20 physi cally interact. Thus, P20 protein either stabilizes Cry11A or helps it atta in the folding important for its toxic activity.