Ni nta agarose column

Constructs encoding a human PRDX6 protein were obtained and expressed in E. coli cells, strain BL21(DE3), as described earlier.^{18 (link)} The obtained recombinant proteins, harboring His-tag, were purified by affinity chromatography on the Ni-NTA-agarose column (Thermo Fisher Scientific, USA), according to the column manufacturer’s instructions. Protein isolation was performed as described earlier.^{18 (link)} The purity of the obtained proteins was at least 98%, based on SDS-PAGE method. PRDX6 was diluted at a concentration of 10 mg/ml in phosphate buffer (1.7 mM KH₂PO₄, 5.2 mM Na₂HPO₄, 150 mM NaCl, pH 7.4) and stored in the freezer at –20°C. After 2 month of storage, no reduction of enzymatic activity was observed. To evaluate functional activity of PRDX6, its ability to reduce hydrogen peroxide (H₂O₂) and tert-butyl-hydroperoxide (t-BOOH) was determined by a slightly modified Kang’s method.¹⁹ The results showed that activity of recombinant PRDX6 was 230 nmol/min/mg of protein (measured with H₂O₂) or 100 nmol/min/mg of protein (measured with t-BOOH).

Expression of recombinant UGT1 and UGT2 were referred to the method described by Kim et al.^{29 (link)} Briefly, the glycosyltransferase genes from Oryza sativa (UGT1, GenBank accession no. AK121682) and S. rebaudiana (UGT2, GenBank accession no. AY345974) were codon-optimized and synthesized by GenScript (Nanjing, China). Respectively, the resulting UGT1 or UGT2 was cloned in the pET28a(+) vector and transformed into the E. coli BL21(DE3) competent cells (TransGen Biotech, Beijing, China), resulting in the recombinant E. coli BL21 (pet28a-SrUGT76G1) strain or E. coli BL21 (pet28a-OsEUGT11) strain. The recombinant strain was cultivated in 400 mL Luria–Bertani (LB) medium containing kanamycin (50 mg mL⁻¹) in 1 L flask until the OD₆₀₀ reached approximately 0.6. The protein expression was induced by the addition of IPTG 0.1 mM IPTG and cells were incubated ∼14 hours at 18 °C. The cells were collected from the medium and resuspended in the Tris–HCl buffer at pH 7.0. Subsequently, the cells were lysed by ultra-sonication in an ice-water bath. After centrifugation, the His-tagged protein in the supernatant was purified through the affinity adsorption on Ni-NTA agarose column (Invitrogen). Protein mass concentration was measured by TaKaRa Bradford Protein Assay Kit with bovine serum albumin as a reference protein.

To express recombinant proteins, a culture of E. coli strain Rosetta 2 (DE3) grown in LB media at 37°C to a density of 0.6 (595 nm) was induced overnight with 0.5 mM isopropyl β-d-thiogalactoside (IPTG) at 16°C. The harvested cells were re-suspended in lysis buffer (20 mM Tris–HCl, pH 8, 1 M NaCl, 0.5 M NaBr, 20 mM imidazole and 2 mM Tris(2-carboxyethyl)phosphine (TCEP)), lysed by sonication, and centrifuged at 18 000 rpm (46 285 g) for 30 min. The supernatant was loaded onto a Ni-NTA agarose column (Invitrogen), washed with lysis buffer, and eluted in the same buffer containing 0.5 M imidazole. The eluted protein was then cleaved with His-tagged TEV protease to remove the histidine tag while dialyzing against 20 mM Tris–HCl, pH 8, 1 M NaCl and 2 mM TCEP overnight to remove the imidazole. Cleaved and dialyzed protein was then again loaded onto the Ni-NTA agarose column, and the flow-through containing the histidine tag-free protein was collected.