M. Yamagiwa et al., Activation process of dipteran-specific insecticidal protein produced by Bacillus thuringiensis subsp israelensis, APPL ENVIR, 65(8), 1999, pp. 3464-3469
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.