Fig. 2.

Purification of recombinant OsCOMT protein and enzyme activity. (A) Purification of N-terminal 6× histidine-tagged OsCOMT. ASMT activity of purified OsCOMT according to (B) enzyme concentration and (C) temperature. E. coli harbouring pET300-OsCOMT was incubated with 1mM IPTG (isopropyl-β-d-thiogalactopyranoside) at 28 °C for 5h. Protein samples were separated by SDS-PAGE and stained with Coomassie blue. M, molecular mass standards; lane 1, total proteins in 15-µl aliquots of bacterial culture without IPTG; lane 2, total proteins in 15-µl aliquots of bacterial culture with IPTG; lane 3, 20 µg soluble protein; lane 4, 10 µg OsCOMT purified by affinity chromatography. In vitro melatonin production was measured at 37 °C or varying temperatures using purified N-terminal 6× histidine-tagged OsCOMT. These data represent the mean ± standard deviation of triplicate experiments.