Figure 1.
Schematic linear representation of the expression plasmids and SDS-PAGE analysis of Cry11A, Cry4Aa and Cyt2Ba expression.
Panels A, B and C: cyt2BaHis, cry4Aa and cry11A genes inserted, respectively, into the BamHI cloning site of the pSVP27A plasmid. The recombinant genes are under the control of a sporulation phase promoter, Pcyt. The genes for positive clone selection in E. coli (Amp-r) and in B. thuringiensis (CAT-r) are shown. Selected restriction enzyme sites used to confirm cloning are also shown. Panels D and E: SDS-PAGE of purified protoxins (Cry11A, Cry4Aa and Cyt2Ba) from recombinant B. thuringiensis strains. As a control, a sporulated acrystaliferous B. thuringiensis strain (4Q7) extract was used. Selected molecular masses of the protein markers are shown (ColorPlus™ Prestained Marker-NEB, panel D and BenchMark™ Protein Ladder-Invitrogen, Panel E) on the left side of both panels.
Figure 2.
Cytotoxicity assays in insect cell cultures and western blot analysis of Cyt2Ba activation.
Panel A: 20 µg/mL of each activated toxin, Cry4Aa and Cry11A, were incubated with different insect cell lines for 30 min. Untreated cells (control) were not incubated with any of the toxins. The percentage of cytotoxicity (cell mortality) was determined by luminometric readings based on luciferase activity. Panel B: Trypsin activation of Cyt2BaHis was proceeded and both protoxin and the activated toxin samples were analyzed by western blot with an anti-His antibody. The arrow shows a band of 27 kDa expected for the non-activated protein. The proteolytic cleavage of Cyt2BaHis resulted in the 6xHis tag loss, making the activated toxin undetectable in western blot analysis using anti-His antibody. Panel C: The activated Cyt2Ba toxin was incubated with insect cells at two different concentrations (5 and 20 µg/mL). Cytotoxicity was determined as for panel A. Panel D: Combinations of Cyt2Ba (5 µg/mL) and the two Cry toxins (20 µg/mL for each toxin) were incubated with insect cell lines. Cytotoxicity percentages were measured according to the same method employed in panel A and C. The toxins concentrations are shown in brackets. One type of dipteran cells (C6/36) and three types of lepidopteran cells (Ld, Sf-21 and Bm) are shown on the cytotoxicity assays graphics.
Table 1.
Cytotoxicity of mixtures of Cry and Cyt toxins of Bacillus thuringiensis subsp. israelensis to insect cell lines.
Table 2.
Observed and expected cytotoxicity of Cry and Cyt toxin mixtures to insect cell lines and the calculated synergism factor.
Figure 3.
SDS-PAGE analysis of Cry4Aa and Cry11A proteolytic activation.
The solubilized Cry4Aa and Cry11A protoxins were activated either by standard incubation using trypsin (T) or by using S. frugiperda’ gastric juice (GJ). The processed proteins were stained with Coomassie blue. Molecular masses in kDa are shown on the left (Prestained Protein Molecular Weight Marker, Fermentas).
Table 3.
Toxicity of individual Cry proteins to second-instar larvae of A.aegypti.
Figure 4.
In vitro analysis of Cry and Cyt toxicity to MCF-7 human cells.
The percentage of cytotoxicity (cell mortality) was obtained by luminometry for all assays. Individual trypsin-activated Cry4Aa or Cry11A as well as the mixture of these two toxins were incubated with MCF-7 cell cultures for 30 min. Cyt2Ba and isolated crystals of Bti were activated by trypsin and assayed against MCF-7 cells for 30 min. Combinations of the three trypsin activated toxins (Cry4Aa, Cry11A and Cyt2Ba) were incubated with MCF-7 cells for two distinct periods of time (30 and 60 min) and cytotoxicity was measured to verify any variation of toxic activity related to time of incubation. Untreated cells (control) were not incubated with any of the toxins. Concentrations of toxins are in parentheses.