Activation process of dipteran-specific insecticidal protein produced by Bacillus thuringiensis subsp israelensis

Dipteran-specific insecticidal protein Cry4A is produced as a protoxin of 1 30 kDa in Bacillus thuringiensis subsp. israelensis. Here we performed the in vitro processing of Cry4A and showed that the 130-kDa protoxin of Cry4A was processed into the two protease-resistant fragments of 20 and 45 kDa th rough the intramolecular cleavage of a 60-kDa intermediate. The processing into these two fragments was also observed in vivo. To investigate function al properties of the two fragments, GST (glutathione S-transferase) fusion proteins of the 60-kDa intermediate and the 20- and 45-kDa fragments mere c onstructed. Neither the GST-20-kDa fusion protein (GST-20) nor the GST-45-k Da fusion protein (GST-45) was actively toxic against mosquito larvae of Cu lex pipiens, whereas the GST-60-kDa intermediate fusion protein (GST-60) ex hibited significant toxicity. However, when the two fusion proteins GST-20 and GST-45 coexisted, significant toxicity was observed. The coprecipitatio n experiment demonstrated that the two fragments associated with each other . Therefore, it is strongly suggested that the two fragments formed an acti ve complex of apparently 60 kDa. A mutant of the 60-kDa protein which was a pparently resistant to the intramolecular cleavage with the midgut extract of C. pipiens larvae had toxicity slightly lower than that of GST-60.