Production of chymotrypsin-resistant Bacillus thuringiensis Cry2Aa1 delta-endotoxin by protein engineering

Cleavage of the Cry2Aa1 protoxin (molecular mass, 63 kDa) from Bacillus thu ringiensis by midgut juice of gypsy moth (Lymantria dispar) larvae resulted in two major protein fragments: a 58-kDa fragment which was highly toxic t o the insect and a 49-kDa fragment which was not toxic. In the midgut juice , the protoxin was processed into a 58-kDa toxin within 1 min, but after di gestion for 1 h, the 58-kDa fragment was further cleaved within domain I, r esulting in the protease-resistant 49-kDa fragment. Both the 58-kDa and non toxic 49-kDa fragments were also found in vivo when I-125-labeled toxin was fed to the insects. N-terminal sequencing revealed that the protease cleav age sites are at the C termini of Tyr49 and Leu144 for the active fragment and the smaller fragment, respectively. To prevent the production of the no ntoxic fragment during midgut processing, five mutant proteins were constru cted by replacing Leu144 of the toxin with Asp (L144D), Ala (L144A), Gly (L 144G), His (L144H), or Val (L144V) by using a pair of complementary mutagen ic oligonucleotides in PCR. All of the mutant proteins were highly resistan t to the midgut proteases and chymotrypsin. Digestion of the mutant protein s by insect midgut extract and chymotrypsin produced only the active 58-kDa fragment, except that L144H was partially cleaved at residue 144.