PMC

Amino acid sequences of Cry1Ac-pIII fusion proteins as derived from their DNA sequences. W indicates a tryptophan substituted in place of a stop codon by the amber suppressor present in the host JM109 cells. Underlining indicates protein sequence which is present in neither the native Cry1Ac nor the cpIII protein. | indicates the predicted signal sequence protease cleavage site.

Immunoblot detection of phage-displayed Cry1Ac fusion proteins. Phages (10⁹ TU for fUSE5 constructs and 10¹⁰ TU for SurfZAP constructs) were boiled for 4 min in 30 μl of Laemmli denaturing sample buffer and size separated by SDS–8% PAGE. The proteins were transferred to nitrocellulose and detected by a rabbit polyclonal anti-Cry1Ac antibody and then by an alkaline phosphatase-conjugated goat anti-rabbit antibody. Lane 1, 1Ac-fUSE5 phage; lane 2, WT-fUSE5 phage; lane 3, 10 ng of purified trypsin-activated HD-73 Cry1Ac; lane 4, SurfZAP-FAb phage; lane 5, SurfZAP-1Ac phage; lane 6, 10 ng of purified trypsin-activated HD-73 Cry1Ac. The blot was divided through the molecular mass markers between lanes 3 and 4, and the two sides were developed separately. The SurfZAP side of the blot was allowed to develop for twice as long as the fUSE5 side, since SurfZAP-1Ac phage produced a lower-intensity signal. The effect of the longer development time can be observed by comparing the 10-ng toxin bands on each side of the blot (lanes 3 and 6).

ELISA detection of phage-displayed Cry1Ac fusion proteins. Cry1Ac-expressing phage, 1Ac-fUSE5 and SZ-1Ac, and their respective control phages, WT-fUSE5 (no insert) and SZ-FAb (antibody insert), were compared with purified, trypsin-activated HD-73 Cry1Ac in an ELISA. The numbers on the horizontal axis refer to different units per well for the fUSE5 phage, SurfZAP phage, and purified Cry1Ac toxin as indicated on the figure. Ten times more SurfZAP (SZ) phage than fUSE5 phage was applied per well per dilution in order to obtain absorbance readings in the same range. For SurfZAP phage, the results from one trial are shown (n = 2). For fUSE5 phage and HD-73 Cry1Ac, the means of results from two different trials are plotted (n = 4). Mean A₄₅₀ (n = 2) values for the individual trials for purified Cry1Ac were (trial 1) 0.021, 0.112, 0.366, 1.137, and 2.422 and (trial 2) 0.029, 0.109, 0.333, 1.170, and 3.426, for 0.1, 0.3, 1.1, 3.3 and 11.0 ng of toxin, respectively. Mean A₄₅₀ (n = 2) values for the individual trials for 1Ac-fUSE5 phage were (trial 1) 0.173, 0.506, 1.243, and 1.521 and (trial 2) 0.213, 0.308, 1.04, and 2.255 for 3.3 × 10⁷, 1.1 × 10⁸, 3.3 × 10⁸, and 1 × 10⁹ TU, respectively. All A₄₅₀ values for WT-fUSE5 phage in both trials were below 0.050.

Immunoblot detection of Cry1Ac and Cry1Ac phage binding to BBMV in the presence and absence of proteinase inhibitors. Cry1Ac-fUSE5 phage (3 × 10⁹ TU) or purified and trypsin-activated HD-73 Cry1Ac toxin (50 ng) was incubated with M. sexta BBMV (100 μg of total protein) in a total volume of 25 μl for 90 min at 22°C. Equal aliquots of phage, toxin, and BBMV were also held separately at 22°C for 90 min, after which all the tubes containing BBMV were microcentrifuged for 5 min to pellet the vesicles. Supernatants were removed to new tubes and mixed with equal volumes of denaturing sample buffer as were the samples containing phage or toxin alone. BBMV pellets were resuspended in 200 μl of TBS and repelleted, and the wash supernatants were discarded. Sample buffer (20 μl) was added to the pellets, and all samples were boiled for 3 min before electrophoresis on an SDS–8% (A) or 10% (B) polyacrylamide gel. The proteins were then electrophoretically transferred to nitrocellulose membrane, and Cry1Ac and Cry1Ac fusion protein were detected by immunoblotting as described for Fig. . Experiments shown in panels A and B are identical except that in panel B proteinase inhibitors (500 μM phenylmethylsulfonyl fluoride and 5 mM benzamidine) were added to the BBMV prior to addition of toxin or phage. Lanes 1, prestained standard molecular mass markers (stds; sizes are indicated on the left); lanes 2, HD-73 Cry1Ac toxin (50 ng); lanes 3, HD-73 Cry1Ac toxin plus BBMV supernatant; lanes 4, HD-73 Cry1Ac toxin plus BBMV pellet; lanes 5, 1Ac-fUSE5 phage; lanes 6, 1Ac-fUSE5 phage plus BBMV supernatant; lanes 7, Ac-fUSE5 phage plus BBMV pellet; lanes 8, BBMV alone (100 μg of total protein). Phg, phage; S, supernatant; P, pellet.