Purification of Vip3Aa from Bacillus thuringiensis HD-1 and its contribution to toxicity of HD-1 to spruce budworm (Choristoneura fumiferana) and gypsy moth (Lymantria dispar) (Lepidoptera).
We developed a protocol for obtaining high yields (10-15 mg per 1100 ml of culture supernatant) of highly purified (up to 95%) Vip3Aa protein from HD-1 cultures. The protocol is based on acetone precipitation of supernatant protein, followed by HPLC fractionation (DEAE-5PW column) and several concentration steps. Our protocol resulted in higher yields and purity of Vip3Aa than a previously published method [Estruch, J.J., Warren, G.W., Mullins, M.A., Nye, G.J., Craig, J.A., Koziel, M.G., 1996. Vip3A, a 353 novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of 354 activities against lepidopteran insects. Proc. Nat. Acad. Sci. USA 93, 5389-5394.]. This was achieved by using acetone rather than ammonium sulfate for precipitation of proteins from culture supernatants, and a shallow rather than a steep NaCl gradient for elution of the toxin, and by conducting all the purification steps at low temperature to prevent toxin degradation. In bioassays of the purified protein, Choristoneura fumiferana and Lymantria dispar larvae were less susceptible than Spodopteraexigua (10- and approximately 100-fold, respectively). A B. thuringiensis var. kurstaki strain HD-1 from which the vip3Aa gene had been deleted (EG12414) showed reduced toxicity to S. exigua relative to the unmodified parental strain (EG2001), but not to L. dispar or C. fumiferana. We interpret these results as indicating that the Vip3Aa toxin does not contribute measurably to pathogenicity of HD-1 in these species.
Milne
Ross
R
Great Lakes Forestry Centre, Canadian Forest Service, Natural Resources Canada, 1219 Queen Street East, Sault Ste. Marie, Ont., Canada P6A 2E5.
Liu
Yuehong
Y
Gauthier
Debbie
D
van Frankenhuyzen
Kees
K
D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics / Q000472:pathogenicity*; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000633:toxicity*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D009036:Moths / Q000382:microbiology*; D010572:Pest Control, Biological
D001426:Bacterial Proteins; C447139:Vip3A protein, Bacillus thuringiensis