Cloning and characterization of a unique cytotoxic protein parasporin-5 produced by Bacillus thuringiensis A1100 strain

Abstract

Parasporin is the cytocidal protein present in the parasporal inclusion of the non-insecticidal Bacillus thuringiensis strains, which has no hemolytic activity but has cytocidal activities, preferentially killing cancer cells. In this study, we characterized a cytocidal protein that belongs to this category, which was designated parasporin-5 (PS5). PS5 was purified from B. thuringiensis serovar tohokuensis strain A1100 based on its cytocidal activity against human leukemic T cells (MOLT-4). The 50% effective concentration (EC₅₀) of PS5 to MOLT-4 cells was approximately 0.075 μg/mL. PS5 was expressed as a 33.8-kDa inactive precursor protein and exhibited cytocidal activity only when degraded by protease at the C-terminal into smaller molecules of 29.8 kDa. Although PS5 showed no significant homology with other known parasporins, a Position Specific Iterative-Basic Local Alignment Search Tool (PSI-BLAST) search revealed that the protein showed slight homology to, not only some B. thuringiensis Cry toxins, but also to aerolysin-type β-pore-forming toxins (β-PFTs). The recombinant PS5 protein could be obtained as an active protein only when it was expressed in a precursor followed by processing with proteinase K. The cytotoxic activities of the protein against various mammalian cell lines were evaluated. PS5 showed strong cytocidal activity to seven of 18 mammalian cell lines tested, and low to no cytotoxicity to the others.

Figure 1

Cytopathic effect of crystal protein produced from B. thuringiensis A1100 on MOLT-4 cells. The parasporal inclusions were solubilized in alkali buffer and treated with proteinase K. (A) Mock-inoculated cells; (B) The cells were incubated with the solubilized protein at 37 °C for 1 h. Arrows indicate ballooned cells caused by administration of the cytotoxic protein.

Figure 2

Protein profiles of the purified cytotoxic protein from B. thuringiensis A1100. Lane 1, molecular mass standards. Lane 2, purified 30-kDa protein.

Figure 3

Dose-response curves for the purified 30-kDa protein from B. thuringiensis A1100 against MOLT-4 cells. The cell survival rate was evaluated using the MTT assay 20 h post-administration. The data are the means ± SDs of triplicate analyses.

Figure 4

Molecular mass of the purified 30-kDa protein was determined by MALDI-TOF mass spectrometry. Arbitrary index a.i.; charge-to-mass ratio, m/z.

Figure 5

Analysis of the recombinant PS5 protein expressed by E. coli BL21 (DE3) harboring the plasmid pET-33k. (A) Lane 1, uninduced soluble fraction (control). Lane 2, uninduced insoluble fraction (control). Lane 3, IPTG-induced soluble fraction. Lane 4, IPTG-induced insoluble fraction. Lane 5, alkali-solubilized insoluble 33-kDa protein; (B) Lane 1, alkali-solubilized insoluble 33-kDa protein purified by a nickel-chelating column. Lane 2, the protein of lane 1 treated with proteinase K. Lane 3, molecular mass standards.