The candidates were expressed in N-terminally His-tagged form using soluBL21 (DE3) (Genlantis) cells transformed with pOPINF plasmids carrying the desired construct and purified similar to a procedure published previously (25). A flask containing 1 liter of yeast extract and Tryptone medium and 50 μg/ml carbenicillin was inoculated with 1 ml of overnight culture of the expression strain and incubated at 37 °C until the A600 reached 0.6–0.8. The temperature was then reduced to 18 °C, protein production was induced by adding 0.25 mm isopropyl 1-thio-β-d-galactopyranoside, and incubation was continued for 16–20 h. Cells were harvested by centrifugation at 4000 × g for 20 min at 4 °C and resuspended in 50 ml of buffer A (50 mm Tris-HCl, pH 8.0, 50 mm glycine, 5% v/v glycerol, 500 mm NaCl, 20 mm imidazole, and 1 mm β-mercaptoethanol) containing 0.5 mg/ml lysozyme and one tablet of Complete EDTA free protease inhibitor (Roche). Cells were disrupted by sonication on ice for 7 min (2-s sonication, 3-s break). Cell debris was removed from the lysate by centrifugation at 35,000 × g for 20 min at 4 °C. The supernatant was injected on a His-Trap 5-ml nickel affinity column attached to an Äkta purifier (GE Healthcare). Protein was eluted with buffer A containing 500 mm imidazole. Iridoid synthase–containing fractions were pooled, concentrated, and washed with size exclusion buffer B (20 mm HEPES, pH 7.5, 150 mm NaCl, and 1 mm β-mercaptoethanol) in an Amicon centrifugal filter (Millipore) with 30-kDa molecular mass cutoff. For further purification, the protein was loaded onto a HiLoad 16/600 Superdex 200 pg (GE Healthcare) size exclusion column and eluted with buffer B. Protein concentration was determined in triplicate on a Nanodrop spectrophotometer (Thermo Fisher Scientific) using absorbance at 280 nm and calculated extinction coefficients (ExPASy ProtParam; Am18679, 99,350 m−1 cm−1; Am18685, 93,390 m−1 cm−1; Am26155, 101,870 m−1 cm−1; Am29566, 81,360 m−1 cm−1). Protein was flash-frozen in liquid nitrogen and stored at −20 °C until further assays were performed.