Recombinant Expression of Glycosyltransferases

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.