Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1989 May 15;260(1):87–91. doi: 10.1042/bj2600087

Facile preparation and characterization of the toxin from Bacillus thuringiensis var. kurstaki.

H Bietlot 1, P R Carey 1, C Choma 1, H Kaplan 1, T Lessard 1, M Pozsgay 1
PMCID: PMC1138629  PMID: 2549961

Abstract

We report a simple three-step method of generating a homogeneous toxic fragment (toxin) in high yield from B. thuringiensis var. kurstaki. Purified crystals were digested with trypsin at pH 10.5, followed by (NH4)2SO4 precipitation and dialysis. For the HD73 strain the preparation is toxic to eastern-spruce-budworm (Choristoneura fuminiferana) larvae. It gives a single 66 kDa band on polyacrylamide-gel electrophoresis and a single band with an isoelectric point of 5.5 on an isoelectric-focusing gel. A single isoleucine N-terminus was detected, and the first 20 amino acids were found to be identical with those predicted from the gene nucleotide sequence. A single lysine C-terminus was detected, and the amino acid composition was in excellent agreement with tryptic cleavages at arginine-28 and lysine-623 of the protoxin. Raman spectroscopic analysis gave values of 20% alpha-helix, 35% beta-sheet and 45% unordered structure. The resistance of the toxin to most proteinases and its susceptibility to proteolysis by papain and Pronases indicates a compact multidomain structure.

Full text

PDF
87

Images in this article

89Fig. 1.
on p.89

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adang M. J., Staver M. J., Rocheleau T. A., Leighton J., Barker R. F., Thompson D. V. Characterized full-length and truncated plasmid clones of the crystal protein of Bacillus thuringiensis subsp. kurstaki HD-73 and their toxicity to Manduca sexta. Gene. 1985;36(3):289–300. doi: 10.1016/0378-1119(85)90184-2. [DOI] [PubMed] [Google Scholar]
  2. Andrews R. E., Jr, Bibilos M. M., Bulla L. A., Jr Protease activation of the entomocidal protoxin of Bacillus thuringiensis subsp. kurstaki. Appl Environ Microbiol. 1985 Oct;50(4):737–742. doi: 10.1128/aem.50.4.737-742.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Aronson A. I., Beckman W., Dunn P. Bacillus thuringiensis and related insect pathogens. Microbiol Rev. 1986 Mar;50(1):1–24. doi: 10.1128/mr.50.1.1-24.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Aronson J. N., Arvidson H. C. Toxic Trypsin Digest Fragment from the Bacillus thuringiensis Parasporal Protein. Appl Environ Microbiol. 1987 Feb;53(2):416–421. doi: 10.1128/aem.53.2.416-421.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chou P. Y., Fasman G. D. Conformational parameters for amino acids in helical, beta-sheet, and random coil regions calculated from proteins. Biochemistry. 1974 Jan 15;13(2):211–222. doi: 10.1021/bi00699a001. [DOI] [PubMed] [Google Scholar]
  6. Duggleby R. G., Kaplan H. A general method for the determination of the carboxyl-terminal sequence of proteins. Anal Biochem. 1975 May 12;65(1-2):346–354. doi: 10.1016/0003-2697(75)90518-7. [DOI] [PubMed] [Google Scholar]
  7. Garnier J., Osguthorpe D. J., Robson B. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J Mol Biol. 1978 Mar 25;120(1):97–120. doi: 10.1016/0022-2836(78)90297-8. [DOI] [PubMed] [Google Scholar]
  8. Haider M. Z., Ellar D. J. Analysis of the molecular basis of insecticidal specificity of Bacillus thuringiensis crystal delta-endotoxin. Biochem J. 1987 Nov 15;248(1):197–201. doi: 10.1042/bj2480197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kaplan H., Hamel P. A., Chan A. M., Oda G. Chemical properties of the N-termini of human haemoglobin. Biochem J. 1982 May 1;203(2):435–443. doi: 10.1042/bj2030435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kronstad J. W., Whiteley H. R. Inverted repeat sequences flank a Bacillus thuringiensis crystal protein gene. J Bacteriol. 1984 Oct;160(1):95–102. doi: 10.1128/jb.160.1.95-102.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Lippert J. L., Tyminski D., Desmeules P. J. Determination of the secondary structure of proteins by laser Raman spectroscopy. J Am Chem Soc. 1976 Oct 27;98(22):7075–7080. doi: 10.1021/ja00438a057. [DOI] [PubMed] [Google Scholar]
  12. Matsudaira P. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem. 1987 Jul 25;262(21):10035–10038. [PubMed] [Google Scholar]
  13. Simpson R. J., Neuberger M. R., Liu T. Y. Complete amino acid analysis of proteins from a single hydrolysate. J Biol Chem. 1976 Apr 10;251(7):1936–1940. [PubMed] [Google Scholar]
  14. Watson D. C., Yaguchi M., Bisaillon J. G., Beaudet R., Morosoli R. The amino acid sequence of a gonococcal growth inhibitor from Staphylococcus haemolyticus. Biochem J. 1988 May 15;252(1):87–93. doi: 10.1042/bj2520087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Yamamoto T., Iizuka T. Two types of entomocidal toxins in the parasporal crystals of Bacillus thuringiensis kurstaki. Arch Biochem Biophys. 1983 Nov;227(1):233–241. doi: 10.1016/0003-9861(83)90366-1. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES