Enzymatic Assay of Recombinant Sb06g029550

To obtain N-terminally His-tagged Sb06g029550 for enzymatic assays, cDNA was subcloned into the NheI–XhoI sites of a Gateway Nova pET-53-DEST vector (Merck KGaA). The plasmid was transformed into E. coli strain BL21 Star (DE3) pLysS (Life Technologies). Expression was induced by incubating the culture at 18° for 48 hr and then at 30° for 5 hr. Then, 2 mg of protein was purified from the cell pellet (5 g) with an IMAC system (Takara). The reaction mixtures (500 μl) for assay contained 0.1 M potassium phosphate buffer (pH 7.0), 1 mg of NADPH, and 200 μg of a substrate [naringenin, eriodictyol, dihydroquercetin (ChromaDex, Inc.), or (±)-taxifolin (dihydrokaempferol) (ChromaDex, Inc.)]. The recombinant protein (250 μg) was added, and the mixture was incubated for 12 hr at 30°. The reaction was terminated by extraction with ethyl acetate (500 μl). Following evaporation of 200-μl extracts, residues were dissolved in 2 N HCl in 50% methanol, incubated for 1 hr at 80°, dried, and dissolved in 200 μl of 0.1% formic acid in water. The reaction mixtures were acid-treated to convert the direct reaction products, flavan-3, 4-diol, and flavan-4-ol, to their respective anthocyanidin or 3-deoxyanthocyanidins, and analyzed by LC-MS/MS (Sawada et al. 2009 (link)).