Table 1.
Purification of RgpB-6His from 3 representative purifications from 1 liter of culture
| Purification Stepa | Protein | Activity | Specific activity | Purification | Yield |
|---|---|---|---|---|---|
|
| |||||
| mg | Units b | Units/ODA280 | Fold | % | |
| Culture fluid (range) | 2353 (2033–2611) | 6980 (6000–8439) | 3.0 (2.48–3.2) | - | - |
| Acetone precipitation | 406.5 (94.5–777) | 3895 (3640–4195) | 24.8 (5.4–38.5) | 6.0 (1.7–11.9) | 56 (43–65) |
| DE-52 | 12.2 (7.9–18.0) | 2317 (1065–4730) | 217.6 (54–441) | 69.3 (22–137) | 33 (16–56) |
| Ni-Sepharose/elution | 2.4 (1.1–3.1) | 1634 (1041–2755) | 737.3 (359–992) | 259.6 (112–400) | 22 (16–33) |
| Ni-Sepharose/flow through | 5 (3.5–6.61) | 215 (74–450) | 37.8 (21–68) | 12.5 (6.6–21.1) | 3 (1–5) |
1L of early stationary phase cultures were clarified by centrifugation (18,000 × g, 30 min) and soluble proteins in the medium were precipitated by the slow addition (50 ml/min) of 1.5 L of chilled acetone (−20°C) with vigorous stirring on dry ice. After 1 hour, the precipitant was pelleted (10,000 × g, 20 min) and re-dissolved for 1 hour on ice into 150 mL 20 mM BisTris buffer, 150 mM NaCl, pH 6.5, supplemented with 1.5 mM dithiodipyridine and 0.02% [w:v] NaN3. The sample was dialysed at 4°C against ion exchange buffer (50 mM BisTris buffer, 5 mM CaCl2, pH 6.5 with 0.02% NaN3), then clarified by ultracentrifugation at 4°C for 1 hour at 100,000 × g. The resulting product was loaded onto 50 g of pre-equilibrated DE-52 Whatman anion-exchange resin (GE Healthcare, Pittsburgh, PA, USA) at a flow rate of 1 mL/min. The column was washed with 200 mL of the same buffer and a gradient of NaCl (0 to 500 mM in 400 mL) was applied to elute bound proteins. Fractions were assayed for the RgpB activity using L-BAPNA as substrate. The fractions of flow through containing the majority of RgpB were pooled and dialysed against Ni-Sepharose binding buffer (20 mM sodium phosphate buffer, 500 mM NaCl, 20 mM imidazole, pH 7.4 with 0.02% NaN3). The retentate obtained was applied to a 10 mL pre-equilibrated Ni2+-Sepharose 6 Fast Flow matrix (GE Healthcare, Pittsburgh, PA) and bound proteins were eluted with binding buffer supplemented with 500 mM imidazole. Two peaks containing protease activity were separated in this manner corresponding to the affinity-bound and unbound proteins. Fractions of those two peaks were combined separately and dialysed at 4 °C against 20 mM BisTris 150 mM NaCl, 5 mM CaCl2, pH 6.8 with 0.02% NaN3. Protein concentration of the final samples was determined by BCA Assay (Sigma) and the active fraction of RgpB-6His was determined by titration against D-Phe-Phe-Arg-chloromethylketone (FFR-CMK) as published previously (Potempa and Nguyen, 2007).
Amidase activity using L-BAPNA as substrate; 1 unit = A405nm of 1.00/ml/min at 37°C