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. 2021 Jun 25;38(2):205–218. doi: 10.5511/plantbiotechnology.21.0222a

Figure 1. UGD activity of purified UGD recombinant proteins. (A) Electrophoresis of purified recombinant proteins. Purified His-tagged recombinant proteins were detected in a UV-stained SDS-PAGE gel (left panel) and by anti-His detection in a western blotting membrane transferred from an identical gel (right panel). UGDs were detected as ∼50 kDa. Lanes 1–5: purified recombinant GuUGD1–5 proteins (10, 10, 7.5, 5, and 15 µl of eluted GuUGD1, GuUGD2, GuUGD3, GuUGD4, and GuUGD5 solution were loaded, respectively); E: protein solution removed non-interactive protein for the TALON resin, extracted from IPTG-induced E. coli transformed with empty vector (10 µl of protein solution was loaded); A: purified recombinant AtUGD2 protein (7.5 µl of protein solution was loaded). (B) UPLC–MS chromatograms at m/z 579.1 for the in vitro reaction products. All reaction products were diluted 10 times after 1 day of incubation using 1 µg of each purified UGD (protein solution extracted from E. coli transformed with empty vector was used at the same volume used for UGD-containing protein solutions). 100% corresponds to the intensity indicated in products catalyzed by AtUGD2, which was used as a positive control. UDP-glucuronic acid was used as an authentic standard.

Figure 1. UGD activity of purified UGD recombinant proteins. (A) Electrophoresis of purified recombinant proteins. Purified His-tagged recombinant proteins were detected in a UV-stained SDS-PAGE gel (left panel) and by anti-His detection in a western blotting membrane transferred from an identical gel (right panel). UGDs were detected as ∼50 kDa. Lanes 1–5: purified recombinant GuUGD1–5 proteins (10, 10, 7.5, 5, and 15 µl of eluted GuUGD1, GuUGD2, GuUGD3, GuUGD4, and GuUGD5 solution were loaded, respectively); E: protein solution removed non-interactive protein for the TALON resin, extracted from IPTG-induced E. coli transformed with empty vector (10 µl of protein solution was loaded); A: purified recombinant AtUGD2 protein (7.5 µl of protein solution was loaded). (B) UPLC–MS chromatograms at m/z 579.1 for the in vitro reaction products. All reaction products were diluted 10 times after 1 day of incubation using 1 µg of each purified UGD (protein solution extracted from E. coli transformed with empty vector was used at the same volume used for UGD-containing protein solutions). 100% corresponds to the intensity indicated in products catalyzed by AtUGD2, which was used as a positive control. UDP-glucuronic acid was used as an authentic standard.