Tpck treated trypsin from bovine pancreas

Bt HD73 expressing Cry1Ac or Bt 407 expressing Cry1AcMod^{36 (link)} were grown at 30 °C until complete sporulation (3 to 4 days) in nutrient broth sporulation medium. In the case of Cry1AcMod the medium was supplemented with erythromycin at 10 μg ml⁻¹. Spores/crystals were washed twice in 300 mM NaCl, 10 mM EDTA. Crystal inclusions were solubilized in an alkaline buffer (50 mM Na₂CO₃, 0.2% β-mercaptoethanol, pH 10.5) for 2 h at 37 °C. Trypsin activated toxins were obtained by treatment of soluble protoxin with trypsin (TPCK treated trypsin from bovine pancreas, SIGMA Aldrich, St. Louis, MO) in a mass ratio of 1:50 (trypsin: toxin) for 2 h at 37 °C after lowering the pH to 8.5 by adding 1:4 (w/w) of 1 M Tris buffer pH 8.5. Phenylmethylsulfonyl fluoride (PMSF) (1 mM final concentration) was added to stop proteolysis. Activated proteins were purified by anion exchange chromatography Mono Q-Sepharose fast flow (GE Healthcare, Little Chalfont, UK) in an AKTA FPLC System (GE Healthcare, Little Chalfont, UK), using a 50 mM Tris-HCl, 50 mM NaCl, pH 8.5 buffer, and a linear NaCl concentration gradient from 50 to 300 mM. Protein concentrations were determined by the method of Bradford, using bovine serum albumin as a standard.

Bt HD73 expressing Cry1Ac or Bt 407 expressing Cry1AcMod [31 (link)] were grown at 30°C until complete sporulation (3 to 4 days) in nutrient broth sporulation medium. In the case of the Bt strain producing Cry1AcMod the medium was supplemented with erythromycin at 10 μg ml^-1. Spores/crystals were washed twice in 300 mM NaCl, 10 mM EDTA. Crystal inclusions were solubilized in an alkaline buffer (50 mM Na₂CO₃, 0.2% β-mercaptoethanol, pH 10.5) for 2 h at 37°C. Trypsin activated toxins were obtained by treatment of soluble protoxin with trypsin (TPCK treated trypsin from bovine pancreas, SIGMA Aldrich, St. Louis, MO) in a mass ratio of 1:50 (trypsin: toxin) for 2 h at 37°C after lowering the pH to 8.5 by adding 1:4 (w/w) of 1 M Tris buffer pH 8.5. Phenylmethylsulfonyl fluoride (PMSF) (1 mM final concentration) was added to stop proteolysis. Activated proteins were purified by anion exchange chromatography Mono Q-Sepharose fast flow (GE Healthcare, Little Chalfont, UK) in an AKTA FPLC System (GE Healthcare, Little Chalfont, UK), using a 50 mM Tris-HCl, 50 mM NaCl, pH 8.5 buffer, and a linear NaCl concentration gradient from 50 to 300 mM. Protein concentrations were determined by the method of Bradford, using bovine serum albumin as a standard.

Microsomes were prepared from a culture of strain BY4741 grown to mid-log phase in YPD broth at 20°C as described in Brodsky et al. [71] (link). The microsomes were resuspended in B88 buffer (20 mM HEPES, pH 6.8, 250 mM sorbitol, 150 mM KOAc, 5 mM MgOAc) to an OD₂₈₀ of 10. Sodium carbonate extractions were performed by incubating 50 µl of microsomes with 1 ml extraction buffer [200 mM Na₂Co₃ (pH 11.5), 10 mM DTT, Complete Mini, EDTA-free protease inhibitor cocktail (Roche), 0.5 M sucrose, 2% glycerol] on ice for 30 min. Samples were centrifuged at 230,000× g at 4°C for 1 hr, and the pellet was dissolved in 1× SDS loading buffer. Proteins in the supernatant were precipitated by incubation on ice for 30 min with trichloroacetic acid (TCA) added to a final concentration of 10%, followed by a 10-min centrifugation at 16,060× g at 4°C. The pellet was solubilized in 1× SDS loading buffer, and proteins separated on 10% SDS-PAGE gels were analyzed by western blot analysis, as described above.
Protease protection assays were performed by incubating 50 µl of microsomes with and without 1% Triton X-100 with 0.2 mg/ml TPCK-treated trypsin from bovine pancreas (Sigma) for 15 min on ice. The reactions were stopped by the addition of 5× SDS loading buffer. Ty1 Gag and Kar2-TAP were detected by western blot analysis, as described above.