Figure 4.

Digestion of bovine β-casein (A) and human cationic trypsinogen (B) with wild-type and mutant N52S CTRC produced in HEK 293T cells and wild-type CTRC produced in E.coli. At the indicated times samples were precipitated with 10% trichloroacetic acid (final concentration) and analyzed by 15% SDS-PAGE and Coomassie blue staining. A. Casein at 0.2 mg/mL concentration was incubated with 5 nM CTRC at 37 °C in 0.1 M Tris-HCl (pH 8.0), 1 mM CaCl₂ (final concentrations) in 100 μL final volume. B. Human cationic trypsinogen was incubated at 2 μM concentration with 20 nM CTRC at 37 °C in 0.1 M Tris-HCl (pH 8.0), 0.14 mM CaCl₂, and 20 nM human SPINK1 trypsin inhibitor in 100 μL final volume. Note that for these experiments a stable trypsinogen mutant was used in which the activation site was destroyed by a K23Q mutation.