| About 10 g of the frozen cells from 2 l culture were suspended in 40 ml of the extraction buffer and passed through French Press three times at 1200 psi to disrupt them. The extraction buffer was 20 mM Tris–HCl, pH 7.5, 50 mM NaCl, 1 mM β-mercaptoethanol (β-ME), and 1 mM EDTA. The homogenate was centrifuged at 15,000 rpm in a Beckman JA-20 rotor for 30 min. fortunately, the recombinant PDE8A1 fragments mainly existed in the pellet phase and therefore refolding was necessary to obtain soluble and active PDE8A1 protein. The pellet was dissolved in 10 ml buffer of 6 M guanidine, 0.1 M Tris–HCl, pH 8.0, under slow orbital shaking at room temperature for 3 h. The dissolved mixture was centrifuged at 15,000 rpm for 20 min to remove the insoluble debris.The supernatant was loaded into a Ni–NTA column ( = 2.5 cm, 25 ml QIAGEN agarose beads). The column was washed with 100 ml buffer of 8 M urea, 0.1 M Tris–HCl, pH 8.0, and eluted with the same buffer plus 0.5 M arginine. The fractions of the PDE8A1 catalytic domain from the elution were combined. The protein concentration was estimated by the absorption of A280 (1.097 U = 1 mg/ml), as calculated by program ProtParam [38]. Fifty milligrams of the PDE8A catalytic domain at 2 mg/ml (25 ml of protein solution) was added dropwise into 1.7 l of the refolding buffer under mild stir. The refolding buffer was 0.5 M Tris–HCl, pH 7.0, 20 mM MgCl2, 20 mM MnCl2, 20 μM ZnSO4, 0.7 M arginine, 30% glycerol, 10 mM NaCl, 1 mM KCl, and 10 mM DTT. The refolding was carried out at 30 μg/ml protein concentration without shaking at 4 °C for 3 days.To concentrate the refolded PDE8A1, the dilute refolding system was mixed with 15 g of hydroxyapatite HTP GEL (Bio-Rad) that was presoaked in water. After stirred at room temperature for 15 min, the suspension was filtered with a filter paper. The beads were collected, re-suspended in 100 ml of 20 mM Tris–HCl, pH 8.0, 50 mM NaCl, 1 mM β-ME, and then packed into a column. The column was washed with 50 ml of the same buffer and eluted with 100 ml of 20 mM Tris–HCl, pH 8.0, 100 mM KH2PO4, 1 mM β-ME. The fractions were combined and dialyzed against 0.5 l of 20 mM Tris–HCl, pH 7.5, 50 mM NaCl, 1 mM β-ME twice, 1 h and overnight.To remove the His-tag, 2.5 mM CaCl2 and 1 μg/ml bovine thrombin (Haematologic Tech., Inc.) were added for digestion at 25 °C for 1 h. The digested PDE8A1 was loaded into a Q-Sepharose column (GE Healthcare) that was pre-equilibrated with a buffer of 50 mM NaCl, 20 mM Tris–HCl, pH 7.5, 1 mM β-ME, 1 mM MgCl2. The column was washed with 200 ml of 20 mM Tris–HCl, pH 7.5, 100 mM NaCl, 1 mM β-ME, 1 mM EDTA, and then PDE8A1 was eluted out with the same buffer except for 300 mM NaCl. After being concentrated to about 10 ml, the protein was loaded into a gel filtration column Sephacryl S300 (GE Healthcare) and eluted with a buffer of 20 mM Tris–HCl, pH 7.5, 50 mM NaCl, 1 mM β-ME, 1 mM MgCl2. The protein was finally concentrated by Amicon cell and ultrafiltration membrane YM30 and its purity was estimated by the SDS gel.The fragment of PDE8A1 (205–820) that contains both PAS and catalytic domains was subcloned into pET15b and expressed in E. coli under the similar procedure, and refolded in a buffer of 50 mM Tris–HCl, pH 7.0, 1.25 M urea, 1 M arginine, 40 mM MnCl2, 10 mM NaCl, 1 mM KCl, and 10 mM DTT. |