VvROMT purification was carried out through immobilized metal affinity chromatography (IMAC), using a HisTrap FF column (GE Healthcare Biosciences, Pittsburgh, PA, USA) according to the manufacturer's instructions. The clarified extract was loaded onto the column, which was preconditioned with buffer A, and after 10-bed column washes, the fusion protein was eluted with elution buffer (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 10% glycerol) using a four-step imidazole gradient (50, 100, 180, and 250 mM) or a linear gradient. The selected fractions were pooled according to visualization by SDS-PAGE or by absorbance at 280 nm. Buffer exchange using Amicon Ultra-15 Centrifugal Filter Units (NMWL 30 kDa; Merck Millipore) was performed twice with buffer A, without imidazole. GB1-ROMT fusion protein was subjected to proteolytic removal of the N-terminal GB1 protein using a His-tagged TEV protease followed by one-step final purification using IMAC, as mentioned above.