Purification of the Insecticidal Toxin in Crystals of Bacillus thuringiensis

Abstract

Crystals were purified from four serotypes of the insect pathogen Bacillus thuringiensis. Crystals from these serotypes were similar in amino acid and N-terminal analyses, but differed in their toxicity to two species of Lepidoptera and in their immunological properties. Toxic polypeptides were obtained following trypsin digestion of solutions of the crystals. In two strains (serotypes 3 and 9) this fraction contained only one polypeptide. Similar results were obtained when dissolved crystals were digested with other proteolytic enzymes or with gut contents from Pieris brassicae. The trypsin-resistant polypeptide was further purified by gel and ion-exchange chromatograhy and had a molecular weight of about 70,000, estimated by gel chromatogrpahy and gel electrophoresis. No evidence was obtained for a toxin of lower molecular weight. This purified toxin accounted for most, if not all, of the toxic activity originally present in the crystal solution and was active by injection and ingestion. The purified toxic fraction from serotype 1 appeared to contain two polypeptides, one of which corresponded to that found with serotypes 3 and 9. There were no major differences in the composition of crystals from different serotypes of B. thuringiensis and it is concluded that the trypsin-resistant polypeptide represents the active insecticidal toxin of the crystal.

Active Form of Dipteran-Specific Insecticidal Protein Cry11A Produced byBacillus thuringiensissubsp.israelensis

“…8,9) The 130-kDa lepidopteran speciˆc protoxins, for example the Cry1A family, are cleaved into the activated forms of about 60 kDa.…”

Isolation and characterization of a novel insecticidal crystal protein gene from Bacillus thuringiensis subsp. aizawai

“…Upon ingestion, ICPs are solubilized and, in some cases, proteolytically processed by insect gut proteases to yield an active truncated toxin moiety (28). This active toxin moiety disrupts the osmotic balance of midgut epithelial cells, eventually resulting in cell lysis.…”

“…The ICPs (also termed delta endotoxins) can comprise up to 20 to 30% of the total dry weight of sporulated cells (28) and form crystalline inclusions which are toxic when ingested by susceptible insects. The crystalline inclusions may be of various morphologies which reflect the differences in the nature of the ICPs that comprise them.…”

Organisms with potential to assist in the control of Helicoverpa armigera in Australian cotton production systems

“…Insecticidal activity of the Cry proteins requires that the protein be ingested by the target insect pest. In the insect gut, the protein is solubilized due to the high pH of the insect gut and is proteolytically cleaved to the active core of the protein, which is resistant to further degradation by the insect gut proteases (Lilley et al, 1980;English and Slatin, 1992). The core protein binds to specific receptors on the mid-gut epithelium cells of susceptible insects, inserts into the membrane, and forms ion-specific pores (English and Slatin, 1992).…”

“…This result was expected because protease-resistant core proteins of B.t. insecticidal proteins are known to be resistant to further trypsin digestion (Lilley et al, 1980). In vivo, the Cry2Ab2 protein would be exposed to gastric conditions prior to entering the intestinal lumen.…”