MoA_ Bt(k)_Toxins_Oct 2010

Fermentation and crystal preparation

B. thuringiensis was cultured in half-strength tryptic soy broth medium at 29 °C for 44 h, and the crystals were purified (Carey et al., 1986).

Toxin preparation

A crystal suspension containing 100 mg of protein was centrifuged, and the pellet was suspended in 5 ml of 0.1 M-Caps buffer (Sigma), pH 10.5, containing bovine pancreatic tosylphenylalanylchloromethane ('TPCK ')- treated trypsin (I mg/ml; Sigma). After having been stirred overnight at 20 °C, the suspension was centrifuged at 10000 g for 30 min. (NH4)2SO4 (40 ",, w/v) was added to the supernatant and stirred at 4 °C for 15 min and then centrifuged. The precipitated protein was suspended in distilled water and thoroughly dialysed against distilled water at 4 °C using 50 kDa-cut-off tubing (Spectrum, Los Angeles, CA, USA). The purified toxin was collected by centrifugation.

Protein quantification

The crystal protein or toxin preparation was dissolved in KOH solution, pH 13. Estimates of protein concentration were made from u.v. absorbance at 280 nm, assuming that tryptophan and tyrosine have absorption coefficients of 5700 and 1300 M-1 cm-1 respectively (Cantor & Schimmel, 1980). Absorption coefficients of 1.37 ml/mg for the protoxin and 1.45 ml/mg for the Purified toxin were calculated. In the case of the purified toxin an absorption coefficient of 1.61 ml/mg at 280 nm was determined by quantitative amino acid analysis.

PAGE

TGels were run on a Pharmacia Phast electrophoresis system with preformed gels and other materials supplied by Pharmacia. Samples were dissolved in sample buffer (8 M-urea/2.5 % SDS/50o 2-mercaptoethanol/10 mMTris- HCl, pH 8.3), placed in a boiling-water bath for 5 min, and then applied to 10-150O gradient gels. Iso electro focusing was carried out by directly applying the native samples to gels with pH ranges from 3 to 9 and from 4 to 6.5. Iso electro focusing in urea was carried out by soaking gels in 8 M-urea with appropriate ampholytes for 30 min and then applying the same in 8 M-urea.

Amino acid analysis

Hydrolyses were carried out in 6 M-HC1 containing 0.1 M-phenol at 110 C in vacuo for 16, 24 and 48 h.

Tryptophan was determined by hydrolysis in 4 Methanesulphonic acid (Simpson et al., 1976) at 110 °C for 24 h. Serine and threonine were determined by extrapolation to zero time. Leucine, isoleucine, methionine and valine were determined from the 48 h hydrolysate. All other amino acids were determined as the average of the 24 h and 48 h hydrolysates. A Technicon TSM amino acid analyser with a ninhydrin detection system was used for quantification.

N-Terminal identification

(i)   Dansylation. A sample of toxin (0.5 mg) was allowed to react with dansyl chloride (Sigma) as described by Gray (1967). The dansyl derivative was identified by high-voltage paper electrophoresis at pH 4.5 and 2.1.

(ii)   Isolation of N-terminal peptide. The isolation procedure is based on the fact that, after acetylation of the toxin and enzymic digestion, only peptides derived from the N-terminus can be neutral at pH 2.1 and therefore readily isolated. Toxin (11 mg) was acetylated with [14C]acetic anhydride (Kaplan et al., 1982) and digested with pepsin. The neutral peptide from pH 2.1 electrophoresis was further purified by paper electrophoresis at pH 3.5 and located by autoradiography.

N-Terminal sequence determination

Toxin from SDS/polyacrylamide gels was electro blotted on to polyvinylidene difluoride membranes (Matsudaira, 1987) and the first 20 N-terminal amino acids were sequenced by using a model 470A Applied Biosystems gas-phase sequencer as described by Watson et al. (1988).

C-Terminal identification

The procedure described below is based on the rationale that, after ["4C]acetylation, the C-terminal peptide will be labelled if it contains a lysine residue. The carboxy groups of a sample (9.5 mg) of toxin were coupled with ethanolamine (Means & Feeney, 1971), dialysed and freeze-dried. The coupled protein was allowed to react with ["4C]acetic anhydride and digested with pepsin. Radioactive peptides derived from the C-terminus were identified using the diagonal electrophoretic procedure described by Duggleby & Kaplan (1975) and autoradiography.

Carbohydrate analysis

Carbohydrate content was determined by the phenol/H2S04 method (Dubois et al., 1956).

Proteolysis of purified toxin

The resistance of toxin to proteolytic cleavage was determined by incubating equimolar amounts of toxin and proteinase in 0.1 M-Caps buffer, pH 10.5, at room temperature for 1 h, followed by gel electrophoresis. Proteinases (Sigma) tested were: ac-chymotrypsin type II, elastase type III, thermolysin type X, Pronase type XIV and papain type III.

Molecular-mass determination

The molecular mass of the purified toxin was estimated in two ways:
(i) mobility on SDS/PAGE using a standard curve of log(molecular mass) versus relative mobility determined with standard molecular-mass markers;

(ii) elution position on gel-permeation chromatography, in 0.1 M-Caps, pH 10.5, employing Sephadex G-200. The elution positions of the standard molecular-mass markers, phosphorylase (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa) and carbonic anhydrase (30 kDa), were determined in 0.1 M-phosphate, pH 7.

Raman spectroscopy

Raman spectral measurements on aqueous and deuterated pellets of purified toxin were performed as previously described (Carey et al., 1986).

Toxicity assays

Toxicity assays were carried out on day-1 sixth-instar larvae of the eastern spruce budworm (Choristoneura fuminiferana). The larvae were force-fed with toxin sample dissolved in 0.2 M-Caps buffer, pH 10.5. Dose volumes were 4 or 6,ul. After the treatment the larvae were held at 25 °C, 6000 relative humidity and 16 h photoperiod. Mortality (LD50) and pupal failure (PFD50) were scored after 8 days.