| Products/Services Used | Details | Operation |
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| Peptide Synthesis | The recombinant E. coli cells were cultured in a Luria–Bertani medium containing 100 mg L−1 ampicillin at 37 °C and 200 rpm. After optical density (OD600) reached 0.6–0.8, the recombinant E. coli cells were induced with 0.1 mM isopropyl-β-D-thiogalactopyranoside for 20 h at 16 °C and 200 rpm. Subsequently, induced cells were harvested by centrifugation at 5000 g for 10 min at 4 °C and resuspended in lysis buffer (50 mM Tris-HCl, 25 mM imidazole, and 150 mM NaCl, pH 7.5). After disruption by high-press homogenization, the resultant cell debris was removed by centrifugation at 17,000 g for 60 min at 4 °C. The supernatant was applied to an AKTA Purifier system equipped with a nickel nitrilotriacetic acid agarose affinity column (GE Healthcare, Piscataway, NJ, USA). The purity of the purified proteins was analyzed by 4%–12% SurePAGE Bis-Tris gels (GenScript, Nanjing, China). Protein concentration was determined by bicinchoninic acid assay using bovine serum albumin as a reference according to the manufacturer’s instructions (Solarbio, Beijing, China). Purified UGT88E18 and SuSy were kept in 50 mM Tis-HCl (pH 7.5) at −80 °C. | Get A Quote |
Calycosin-7-O-β-D-glucoside (Cy7G) is one of the principal components of Radix astragali. This isoflavonoid glucoside is regarded as an indicator to assess the quality of R. astragali and exhibits diverse pharmacological activities. In this study, uridine diphosphate-dependent glucosyltransferase (UGT) UGT88E18 was isolated from Glycine max and expressed in Escherichia coli. Recombinant UGT88E18 could selectively and effectively glucosylate the C7 hydroxyl group of calycosin to synthesize Cy7G. A one-pot reaction by coupling UGT88E18 to sucrose synthase (SuSy) from G. max was developed. The UGT88E18–SuSy cascade reaction could recycle the costly uridine diphosphate glucose (UDPG) from cheap sucrose a... More
