Advertisement

Antonie van Leeuwenhoek

, Volume 54, Issue 3, pp 257–265 | Cite as

Affinity purification of a 65-kilodalton parasporal protein from Bacillus thuringiensis PG-14 that shows mosquitocidal activity

  • Yong Man Yu
  • Michio Ohba
  • Keio Aizawa
General Papers

Abstract

By using antibody-mediated affinity chromatography, a highly mosquito larvicidal but nonhemolytic fraction was obtained from alkali-solubilized, silkworm (Bombyx mori) larval gut juice-treated parasporal inclusions of Bacillus thuringiensis strain PG-14 (serotype 8a : 8b). This fraction contained a 65-kDa protein only but not a 25-kDa protein, the main component in the flow through fraction unbound to the affinity column. The 25-kDa protein purified from the unbound fraction by CM-cellulose chromatography demonstrated a high hemolytic activity against sheep red blood cells but very low mosquito larvicidal activity.

Key words

Bacillus thuringiensis PG-14 mosquitocidal activity 65-kDa protein affinity purification 25-kDa protein 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Bourgouin, C., A. Klier & G. Rapoport (1986) Characterization of the genes encoding the hemolytic toxin and the mosquitocidal delta-endotoxin of Bacillus thuringiensis israelensis. Mol. Gen. Genet. 205: 390–397Google Scholar
  2. Cheung, P.Y.K., D. Buster & B.D. Hammock (1987) Lack of mosquitocidal activity by the cytolytic protein of the Bacillus thuringiensis subsp. israelensis parasporal crystal. Curr. Microbiol. 15: 21–23Google Scholar
  3. Held, G.A., Y.S. Huang & C.Y. Kawanishi (1986) Effect of removal of the cytolytic factor of Bacillus thuringiensis subsp. israelensis on mosquito toxicity. Biochem. Biophys. Res. Commun. 141: 937–941Google Scholar
  4. Hurley, J.M., L.A. BullaJr. & R.E. AndrewsJr. (1987) Purification of the mosquitocidal and cytolytic proteins of Bacillus thuringiensis subsp. israelensis. Appl. Environ. Microbiol. 53: 1316–1321Google Scholar
  5. Hurley, J.M., S.G. Lee, R.E. AndrewsJr., M.J. Klowden & L.A. BullaJr. (1985) Separation of the cytolytic and mosquitocidal proteins of Bacillus thuringiensis subsp. israelensis. Biochem. Biophys. Res. Commun. 126: 961–965Google Scholar
  6. Ibarra, J.E. & B.A. Federici (1986a) Parasporal bodies of Bacillus thuringiensis subsp. morrisoni (PG-14) and Bacillus thuringiensis subsp. israelensis are similar in protein composition and toxicity. FEMS Microbiol. Lett. 34: 79–84Google Scholar
  7. Ibarra, J.E. & B.A. Federici (1986b) Isolation of a relatively nontoxic 65-kilodalton protein inclusion from the parasporal body of Bacillus thuringiensis subsp. israelensis. J. Bacteriol. 165: 527–533Google Scholar
  8. Kim, K.-H., M. Ohba & K. Aizawa (1984) Purification of the toxic protein from Bacillus thuringiensis serotype 10 isolate demonstrating a preferential larvicidal activity to the mosquito. J. Invertebr. Pathol. 44: 214–219Google Scholar
  9. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685Google Scholar
  10. Lowry, O.H., N.J. Rosebrough, A.L. Farr & R.J. Randall (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265–275Google Scholar
  11. Padua, L.E. (1982) Studies on the isolates of Bacillus thuringiensis with preferential and high toxicities against mosquito larvae. Ph. D. Thesis, Kyushu UniversityGoogle Scholar
  12. Padua, L.E., M. Ohba & K. Aizawa (1984) Isolation of a Bacillus thuringiensis strain (serotype 8a: 8b) highly and selectively toxic against mosquito larvae. J. Invertebr. Pathol. 44: 12–17Google Scholar
  13. Pfannenstiel, M.A., G.A. Couche, E.J. Ross & K.W. Nickerson (1986) Immunological relationship among proteins making up the Bacillus thuringiensis subsp. israelensis crystalline toxin. Appl. Environ. Microbiol. 52: 644–649Google Scholar
  14. Sekar, V. (1986) Biochemical and immunological characterization of the cloned crystal toxin of Bacillus thuringiensis var. israelensis. Biochem. Biophys. Res. Commun. 137: 748–751Google Scholar
  15. Thomas, W.E. & D.J. Ellar (1983) Bacillus thuringiensis var. israelensis crystal δ-endotoxin: Effects on insect and mammalian cells in vitro and in vivo. J. Cell Sci 60: 180–197Google Scholar
  16. Tojo, A., W. Samasanti, N. Yoshida & K. Aizawa (1986) Effects of the three proteases from gut juice of the silkworm, Bombyx mori, on the two morphologically different inclusions of δ-endotoxin produced by Bacillus thuringiensis kurstaki HD-1 strain. Agric. Biol. Chem. 50: 575–580Google Scholar
  17. Wu, D. & F.N. Chang (1985) Synergism in mosquitocidal activity of 26 and 65 kDa proteins from Bacillus thuringiensis subsp. israelensis crystal. FEBS Lett. 190: 232–236Google Scholar
  18. Yamamoto, T., T. Iizuka & J.N. Aronson (1983) Mosquitocidal protein of Bacillus thuringiensis subsp. israelensis: Identification and partial isolation of the protein. Curr. Microbiol. 9: 279–284Google Scholar
  19. Yu, Y.M., M. Ohba, K. Aizawa & L.E. Padua (1987) Mosquito larvicidal and hemolytic proteins purified from parasporal inclusions produced by Bacillus thuringiensis strain PG-14 (serotype 8a: 8b). System. Appl. Microbiol. 9: 320–323Google Scholar
  20. Zalunin, I.A., L.I. Kostina, G.G. Chestukhina, M.E. Eormatova, S. Klepikova, O.M. Khodova & V.M. Stepanov (1986) A comparative study of Bacillus thuringiensis subsp. israelensis proteins forming entomocidal crystals. Biokhimiya 51: 449–457Google Scholar

Copyright information

© Kluwer Academic Publishers 1988

Authors and Affiliations

  • Yong Man Yu
    • 1
  • Michio Ohba
    • 1
  • Keio Aizawa
    • 1
  1. 1.Institute of Biological Control, Faculty of AgricultureKyushu UniversityFukuokaJapan

Personalised recommendations