*3.5. The Recombinant CamPhoD Isolation and Purification*

For isolation of CamPhoD, CaCl2 and MgCl2 were added to the recombinant cell extract to a final concentration of 10 mM, DNase was (SkyGen, Moscow, Russia) to a final concentration of 5 μg/mL, these were incubated at 37 ◦C for 1 h, and then centrifuged at 11000 rpm for 20 min. The resulting supernatant was introduced into a 25 × 3.2 cm Ni-IMAC-Sepharose column (GE Healthcare Life Sciences, Buckinghamshire, UK) equilibrated with 25 mM Tris-HCl, pH 9.0 (buffer A), and washed with five volumes of the same buffer. The recombinant protein was eluted with a linear gradient of 0–0.5 M imidazole in 25 mM Tris-HCl buffer, pH 9.0, and 0.5 M NaCl (6 column volume), at a rate of 1.3 mL/min. The CamPhoD-containing fraction was purified on a 10 × 1.4 cm Source 15 Q column (GE Healthcare Life Sciences) equilibrated with buffer A, then the protein was eluted with a linear gradient of 0–0.5 M NaCl in the 25 mM Tris-HCl buffer at pH 9.0. Ion exchange chromatography was performed at a rate of 1 mL min; the volume of the fractions was 1 mL. The CamPhoD-containing fractions were collected and treated with enterokinase at a final concentration of 1 U per 1 mg of protein for 22 h at 25 ◦C. Then, the protein solution was applied to a Superdex 200 PG column (105 × 2 cm) (GE Healthcare Life Sciences), previously equilibrated with buffer A with 0.15 M NaCl at a rate of 0.5 mL/min, with 1 mL fractions. The CamPhoD-containing fractions were collected and subjected to chromatography using a mono-Q HR column (4 × 0,8 cm) (GE Healthcare Life Sciences, Buckinghamshire, UK) equilibrated with buffer A, washed with 10 volumes of buffer A, and then the target protein was eluted with a linear gradient of 0–0.5 M NaCl in buffer A at a rate of 0.5 mL/min and with fractions of 1 mL. The purified preparation of CamPhoD was used to study the physicochemical properties and substrate specificity.
