Individual clones with high expression levels for boophilin or D1 were selected (data not shown). A single P. pastoris colony (Mut+) expressing high levels of boophilin or D1 was selected and used to inoculate 120 mL BMGY medium in a 1 L sterile flask, and incubated at 30 °C and 250 rpm for 24 h. Expression was performed as described above and the culture supernatant was stored at 4 °C prior to purification. Recombinant boophilin or D1-containing yeast culture supernatant was loaded onto an affinity trypsin-Sepharose column previously equilibrated with 50 mM Tris–HCl buffer pH

8.0 (buffer A). Weakly bound proteins were washed out with buffer A supplemented with 0.15 M NaCl. The bound material was C59 wnt cell line eluted with 0.5 M KCl pH 2.0 and the collected fractions were immediately neutralized with 1 M Tris–HCl buffer pH 8.0. Absorbance at 280 nm was also monitored. The inhibitory activity of the fractions was analyzed in protease activity assays (see below). The fractions containing inhibitory activity and displaying one main protein band in SDS-PAGE were pooled and concentrated

using a 5000 MWCO membrane (Millipore, Billerica, MA, USA). The concentration of active trypsin find more was determined by active site titration with p-nitrophenyl-p′-guanidino-benzoate as previously described ( Chase and Shaw, 1969). The equilibrium dissociation constants of complexes formed by boophilin or D1 with bovine trypsin or neutrophil elastase were determined using the method described by Bieth (1980). Briefly, the serine proteases were incubated at 37 °C with different concentrations of inhibitors in 0.1 M Tris–HCl buffer pH 8.0 containing 0.15 M NaCl and DNA ligase 0.1% Triton X-100. The residual enzyme activity was measured after the addition of the chromogenic substrate tosyl-Gly-Pro-Arg-pNA or elastase substrate I (MeOSuc-Ala-Ala-Pro-Val-pNA) for trypsin and neutrophil elastase, respectively. Apparent Ki values were calculated by fitting the steady-state velocities to

the equation (Vi/Vo = 1 − Et + It + Ki − [(Et + It + Ki)2 − 4EtIt]1/2/2Et) for tight-binding inhibitors and using a non-linear regression analysis ( Morrison, 1969). Boophilin (1.2 and 2.4 μM) was pre-incubated with α-thrombin (0.025 U) or γ-thrombin (1 μg) for 10 min at 37 °C in 100 mM Tris–HCl buffer pH 8.0 containing 150 mM NaCl and 0.1% Triton X-100. The residual thrombin activity against the fluorogenic substrate Benzoyl-Phe-Val-Arg-AMC (200 μM) was measured, after incubation in the same conditions for 20 min. The fluorescence was monitored at λem = 460 nm and λex = 380 nm in a Synergy HT microplate reader (BioTek, Winooski, VT, USA) for 20 min. As a control, the same assay was performed in the absence of boophilin.