Overexpression and purification of recombinant proteins.

For biosynthesis of pro-Per, pro-Per^S355A, pro-PerΔCa, pro-PerΔCa^S355A, pro-PerΔIS, and pro-PerΔIS^S355A, competent E. coli BL21(DE3) cells were transformed with the pMD204 expression constructs as described above. The cells were grown at 37°C in 2× yeast extract tryptone broth (Carl Roth) supplemented with chloramphenicol (34 μg/ml). When an optical density at 600 nm (OD₆₀₀) of ∼1.5 was reached, protein expression was induced with 0.1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG), followed by incubation at 23°C with agitation at 225 rpm for 16 h. For biosynthesis of the propeptide, competent E. coli BL21(DE3) cells were transformed with pMB_Pro and grown at 37°C in Luria–Bertani broth (Carl Roth) supplemented with ampicillin (110 μg/ml). Propeptide expression was induced at an OD₆₀₀ of ∼0.8, using 1 mM IPTG. After addition of the IPTG, the cells were incubated at 23°C with agitation at 225 rpm for 16 h.

All of the chromatographic steps were carried out using an NGC chromatography system (Bio-Rad). The His₁₀-tagged pernisine variants (i.e., pro-Per, pro-Per^S355A, pro-PerΔCa, pro-PerΔCa^S355A, pro-PerΔIS, and pro-PerΔIS^S355A) were isolated from the E. coli periplasm using immobilized metal affinity chromatography (IMAC). The periplasmic fractions were obtained as described previously (39) with some modifications. Briefly, the cells were harvested by centrifugation at 3,000 × g for 20 min at 4°C and resuspended in TSE buffer (100 mM Tris-HCl, pH 8.0, 20% sucrose, 0.5 mM EDTA). The cell suspensions were incubated on ice for 15 min and centrifuged at 4,000 × g for 15 min at 4°C. The pellets were resuspended in 10 mM Tris (pH 8.0), incubated on ice for 60 min, and centrifuged at 8,000 × g for 20 min at 4°C. The collected supernatants were regarded as the periplasmic fractions. For purification of pro-Per^S355A, pro-PerΔCa^S355A, and pro-PerΔIS^S355A, the periplasmic fractions were supplemented with 300 mM NaCl and 10 mM imidazole and passed through HisTrap HP columns (GE Healthcare), which were preequilibrated with IMAC buffer (50 mM NaH₂PO₄, 300 mM NaCl, pH 8.0) containing 10 mM imidazole. The columns were then washed with 25 column volumes of the same buffer. Finally, pro-Per^S355A, pro-PerΔCa^S355A, and pro-PerΔIS^S355A were eluted with IMAC buffer containing 250 mM imidazole and dialyzed against 10 mM Tris (pH 8.0) overnight. After dialysis, the proteins were incubated with 5 mM CaCl₂ at 90°C for 30 min and then centrifuged at 18,000 × g for 15 min at 4°C to remove heat-labile protein contaminants. The remaining protein impurities in the supernatants were hydrolyzed by addition of activated pernisine (2 μg/ml), with incubation at 90°C for 30 min. After centrifugation at 18,000 × g for 15 min at 4°C, the supernatants containing the purified pro-Per^S355A, pro-PerΔCa^S355A, and pro-PerΔIS^S355A were treated with 10 mM EDTA and dialyzed against 10 mM Tris-HCl (pH 8.0) for 30 h, with two buffer changes, to obtain the proteins in their Ca²⁺-free forms. After the final dialysis, the purified proteins were concentrated using 10-kDa-cutoff centrifuge filters (Ultra-15; Amicon) and then stored at −80°C. The purification procedures for pro-Per, pro-PerΔCa, and pro-PerΔIS were the same as for pro-Per^S355A, pro-PerΔCa^S355A, and pro-Per-ΔIS^S355A, except that the proteins eluted from the HisTrap HP columns were directly stored at −80°C after the first dialysis against 10 mM Tris (pH 8.0). The concentrations of the purified proteins were determined with a combination of bicinchoninic acid assays and SDS-PAGE using 12% polyacrylamide gels.

The propeptide was isolated from the E. coli cytoplasm. The cells were harvested with centrifugation at 4,000 × g for 25 min at 4°C, resuspended in 50 mM Tris (pH 7.0), and sonicated on ice. Cellular debris was removed by centrifugation at 15,000 × g for 15 min at 4°C. To remove the heat-labile proteins, the clarified lysate was incubated at 80°C for 20 min and then cooled on ice and centrifuged at 18,000 × g for 15 min at 4°C. The supernatants were collected and combined with 1.5 M (NH₄)₂SO₄ (final concentration). After centrifugation at 18,000 × g for 15 min at 4°C, the supernatants were loaded onto HiTrap phenyl (LS) columns (GE Healthcare) that were preequilibrated with 1.5 M (NH₄)₂SO₄ in 50 mM Tris (pH 7.0). The columns were then washed with 30 column volumes of the same buffer. The propeptide was eluted with 0.6 M (NH₄)₂SO₄ in 50 mM Tris (pH 7.0) and dialyzed against 10 mM Tris (pH 8.0) overnight. After dialysis, the propeptide was loaded onto a HiTrap Q HP column (GE Healthcare) and eluted with a linear gradient from 0 mM to 500 mM NaCl in 20 mM Tris-HCl (pH 8.0) as a single peak at ∼180 mM NaCl. The purified propeptide was dialyzed against 10 mM Tris (pH 8.0) and stored at −80°C. The concentrations of the propeptide were calculated using absorbance at 280 nm and an extinction coefficient of 1,490 M⁻¹cm⁻¹. This extinction coefficient was obtained using the online ProtParam tool (ExPASy). The purity of the propeptide was analyzed using SDS-PAGE with 15% polyacrylamide gels.