Treated trypsin from bovine pancreas

African green monkey kidney cells (VERO, ATCC-American Type Culture Collection CCL-81) and Madin Darby Canine Kidney cells (MDCK, ATCC^® CRL-2936™) were propagated in Dulbecco’s modified Eagle medium (DMEM; Euroclone), supplemented with 10% fetal bovine serum (FBS; Euroclone), 2 mM l-glutamine, 1 mM sodium pyruvate, 100 U/mL penicillin, and 100 μg/mL streptomycin sulfate.
Acyclovir-sensitive herpes simplex virus 1 (HSV-1) clinical isolate (kindly provided by V. Ghisetti, Amedeo di Savoia Hospital, Turin, Italy) was propagated and titrated by plaque assay on VERO cells as described previously [31 (link)]. The influenza A virus strain A/Puerto Rico/8/34 (IAV) was a generous gift from Arianna Loregian (University of Padua, Italy), and was propagated and titrated by plaque assay on MDCK cells. Infections with IAV were performed in the presence of 2 µg/mL TosylPhenylalanylChloromethylKetone -treated trypsin from bovine pancreas (Sigma-Aldrich, Saint Louis, USA) and 0.14% bovine serum albumin (BSA) (Sigma-Aldrich, Saint Louis, USA) in serum-free complete medium [32 (link)].

B. thuringiensis strains expressing Cry1Ab, Cry1Ab F371A [8 (link),28 (link)] or Cry1AbMod [29 (link)] were grown for 3 days at 30°C in nutrient broth sporulation medium, supplemented with erythromycin at 10 μg·ml⁻¹. After complete sporulation, the harvested products were washed twice in 300 mM NaCl and 10 mM EDTA, and crystal inclusions were purified using discontinuous sucrose gradients [30 (link)]. Protoxins were produced by solubilizing crystal inclusions in an alkaline buffer (50 mM Na₂CO₃ and 0.2% 2-mercaptoethanol, pH 10.5) for 2 h at 37°C. The soluble protoxins were recovered after centrifugation at ~18400 g for 20 min). Trypsin-activated toxins were obtained by treatment of soluble protoxin with trypsin [TPCK (tosylphenylalanylchloromethane)-treated trypsin from bovine pancreas (Sigma–Aldrich)] at a mass ratio of 1:50 (trypsin/toxin) for 2 h at 37°C after lowering the pH to 8.5 by adding 1 M Tris buffer (pH 8.5) at a 1:4 (v/v) ratio. 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) in an ÄKTA FPLC System (GE Healthcare), using a 50 mM Tris/HCl and 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 BSA as a standard.