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

Abstract

Cleavage of the Cry2Aa1 protoxin (molecular mass, 63 kDa) from Bacillus thuringiensis by midgut juice of gypsy moth (Lymantria dispar) larvae resulted in two major protein fragments: a 58-kDa fragment which was highly toxic to 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 digestion for 1 h, the 58-kDa fragment was further cleaved within domain I, resulting in the protease-resistant 49-kDa fragment. Both the 58-kDa and nontoxic 49-kDa fragments were also found in vivo when (125)I-labeled toxin was fed to the insects. N-terminal sequencing revealed that the protease cleavage 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 nontoxic fragment during midgut processing, five mutant proteins were constructed by replacing Leu144 of the toxin with Asp (L144D), Ala (L144A), Gly (L144G), His (L144H), or Val (L144V) by using a pair of complementary mutagenic oligonucleotides in PCR. All of the mutant proteins were highly resistant to the midgut proteases and chymotrypsin. Digestion of the mutant proteins by insect midgut extract and chymotrypsin produced only the active 58-kDa fragment, except that L144H was partially cleaved at residue 144.

FIG. 1

SDS–10% polyacrylamide gel electrophoresis of the products of Cry2Aa1 and Cry1Aa1 processed by gypsy moth larval midgut extract at different periods of incubation. The protein solutions were mixed with the gut extract at a ratio of 10:1 (vol/vol) and incubated at room temperature. The reactions were stopped by adding protease inhibitors, as described in Methods and Materials. Lanes: 1, molecular size markers (phosphorylase b, 97 kDa; bovine serum albumin, 66 kDa; aldolase, 39 kDa; triosephosphate isomerase, 26 kDa); 2, Cry2Aa1 protoxin; 3, Cry2Aa1 digested for 1 min; 4, Cry2Aa1 digested for 1 h; 5, Cry1Aa1 protoxin; 6, Cry1Aa1 digested for 1 h; 7, Cry1Aa1 digested for 16 h. The arrowhead indicates a trace amount of the 58-kDa fragment.

FIG. 2

Autoradiography of L. dispar larval midgut and digested Cry2Aa1 after 1 h of feeding on diet contaminated with ¹²⁵I-labeled Cry2Aa1. (A) The peritrophic membrane with its contents (image 1) and midgut tissue after removal of peritrophic membrane (image 2). Note that the anterior regions of both the peritrophic sac and the midgut tissue were arranged upward. (B) Lanes: 1, ¹²⁵I-labeled Cry2Aa1 before it was applied to the insect; 2, ¹²⁵I-labeled Cry2Aa1 extracted from the midgut fluid; 3, ¹²⁵I-labeled Cry2Aa1 extracted from the midgut membrane of the same insect as in lane 2.

FIG. 3

Digestion of Cry2Aa1 protoxin and the mutant proteins by gut extract (A) and chymotrypsin (B). Samples (10 μg) of the protein were incubated with midgut extract at a 10:1 ratio (vol/vol) of protein solution to gut extract or with chymotrypsin (20% by mass). Lanes 1, molecular size markers. Even-numbered lanes show protoxins, as follows: lanes 2, Cry2Aa1; lanes 4, L144D; lanes 6, L144H; lanes 8, L144V; lanes 10, L144A; and lanes 12, L144G. Odd-numbered lanes show digestion products of the proteins after 1 h of incubation, as follows: lanes 3, Cry2Aa1 wild-type protein; lanes 5, L144D; lanes 7, L144H; lanes 9, L144V; lanes 11, L144A; and lanes 13, L144G.