Extended Data Fig. 6: VC1 expression, purification, and assays.
From: VC1 catalyses a key step in the biosynthesis of vicine in faba bean
(a) Polyacrylamide gel electrophoresis (PAGE) showing the affinity chromatography of His-tagged VC1 on a Ni-NTA matrix. Protein was visualized on a Stain-FreeTM pre-cast gel using the ChemiDocTM gel imaging system (BioRad). L, lysate; P, pellet; FT, flow-through; W1-3, three consecutive wash fractions; E1-4, elutions with increasing concentration of imidazole (20, 50, 100, and 250 mM, respectively); M, molecular weight marker (given in kDa). The expected molecular weight of His-tagged VC1 was 51.3 kDa. After buffer exchange to remove the imidazole, fraction E4 was used for the subsequent assays. The expression and purification of VC1 was carried out several times with similar results. (b) Representative result of the GTP cyclohydrolase II assays measuring the appearance of DARPP, which presents an absorption maximum at 310 nm. The graph shows the increase in absorbance at 310 nm (∆A310) against time for a control (no enzyme) and for an assay with purified VC1.
