FIGURE 2.
Purification and biochemical characterization of E22O. A-C, E22O was purified from the N. rileyi-conditioned medium by sequential use of phenyl-Sepharose HP hydrophobic interaction chromatography (A), Superdex 200 pg gel-filtration chromatography (B), and HiTrap Q column chromatography (C). Fractions indicated using the hatched bars in A and B were pooled and purified further in B and C, respectively. Fractions indicated using the closed bar in C contained pure E22O; they were pooled and then used for further biochemical analyses. D and E, molecular mass of E22O was determined by SDS-PAGE (D) and Superdex 200 pg gel-filtration chromatography (E). F, kinetic analysis. Various concentrations of ecdysone or 20-hydroxyecdysone were incubated with 2.5 ng/50 μl of purified E22O in phosphate buffer (pH 7.0) and amounts of 22-dehydroecdysteroids formed were measured after 10 min. Kcat values were calculated using the molecular mass of E22O as 76 kDa.