Confirming Glycosyltransferase Activity of Gly

To confirm the glycosyltransferase activity of Gly, two different in vitro assays were performed. Recombinant Gly enzyme was expressed and purified as described previously (15 (link)). For gel shift assay, 15 μl of purified His-Gly (20 μm) and 10 μl of Gtf123-dGT1-GalT2 modified rFap1 (50 μm) were added into 50 μl of reaction buffer (20 mm Tris, pH 8.0, 100 mm NaCl, and 10 mm Mg²⁺). 10 mm EDTA was used as metal ion chelator. 10 mm UDP-Glc or UDP-Gal was used as sugar donor. The mixture was incubated in test tube at 37 °C water bath for 15 h. Then sample loading buffer was added to the samples for SDS-PAGE.
For the UDP-Glo Glycosyltransferase Assay (Promega) (31 ), 5 μl of the glycosyltransferase reaction mixtures that contain 20 μm recombinant Gly enzyme, 50 μm rFap1, or rFap1-Gtf123-dGT1, or rFap1-Gtf123-dGT1-GalT2 as a substrate and 25 μm UDP-Glc, or UDP-Gal were incubated in a solid white 384-well plate. After a 1-h incubation at room temperature, 5 μl of UDP detection reagent was added to each well. After another 1-h incubation at room temperature, luminescence was recorded using a BioTek Microplate reader. The values represent the mean of three experimental replicates.