Display of biologically functional insecticidal toxin on the surface of lambda phage

Abstract

The successful use of Bacillus thuringiensis insecticidal toxins to control agricultural pests could be undermined by the evolution of insect resistance. Under selection pressure in the laboratory, a number of insects have gained resistance to the toxins, and several cases of resistance in the diamondback moth have been reported from the field. The use of protein engineering to develop novel toxins active against resistant insects could offer a solution to this problem. The display of proteins on the surface of phages has been shown to be a powerful technology to search for proteins with new characteristics from combinatorial libraries. However, this potential of phage display to develop Cry toxins with new binding properties and new target specificities has hitherto not been realized because of the failure of displayed Cry toxins to bind their natural receptors. In this work we describe the construction of a display system in which the Cry1Ac toxin is fused to the amino terminus of the capsid protein D of bacteriophage lambda. The resultant phage was viable and infectious, and the displayed toxin interacted successfully with its natural receptor.

FIG. 1.

Cloning strategy for the translational fusion cry1Ac1::gpD (A) and its cloning into EMBL3 phage (B). Primers are shown as horizontal arrows pointing in the 5′-3′ direction. Primers with MluI (M) and NotI (N) sites are indicated.

FIG. 2.

Coomassie staining (A) and immunodetection (B and C) of Cry1Ac1 (lanes 1 to 4) and Cry1Ac1-D (lanes 5 to 8) expressed from plasmids pSVC7 and pSVC1-31A. Lanes 1, 3, 5, and 7 show the supernatant fraction of the cultures, and lanes 2, 4, 6, and 8 show the pellet fraction of cultures under noninduced conditions (lanes 1, 2, 5, and 6) and under induced conditions (lanes 3, 4, 7, and 8). Lanes M, molecular size markers. Panels B and C show immunodetection with anti-Cry1Ac1 and anti-D antibodies, respectively.

FIG. 3.

Cry1Ac1 and Cry1Ac1-D ligand blot of brush border membrane vesicles samples. Lane M, molecular size markers (in kilodaltons). Lane 1: overlay of gut extract-treated Cry1Ac1 protein over brush border membrane vesicles (40 μg). Lane 2: overlay of gut extract-treated Cry1Ac1-D fusion protein over brush border membrane vesicles (40 μg). Lane 3: overlay of nontreated Cry1Ac1-D fusion protein over brush border membrane vesicles (40 μg).

FIG. 4.

Results of the diet surface contamination assay with M. sexta, comparing insecticidal activities of Cry1Ac1 (open circles) and Cry1Ac1-D (solid circles) proteins. The average mass change of eight larvae after 5 days of growth is represented as a function of the toxin concentration used in the assay.

FIG. 5.

Immunoblot detection of Cry1Ac1-D and Cry1Ac1-phage with anti-D (A) and anti-Cry1Ac1 (B) antibodies. Lane M: molecular size markers (in kilodaltons). Lanes 1: Cry1Ac1-D fusion protein expressed in E. coli BLR. Lanes 2: E. coli LE392 lysates obtained by infection with V_c phage. Lanes 3: E. coli LE392 lysates obtained by infection with V₁₃ phage.