Recombinant Expression And Enzyme Activity Analysis Of The Prophenoloxidases Of Drosophila Melanogaster

Insect prophenoloxidases (PPOs) are a group of important innate immunity proteins. Although there have been numerous studies dealing with the PPO activation cascade, the detailed biochemical behaviors of the PPO family proteins remain to be clearly established. This is due primarily to the difficulty in obtaining adequate amounts of PPO proteins for comprehensive characterization. In this study, we expressed three Drosophila melanogaster PPO genes in E. coli, and extensively evaluated expression conditions for obtaining soluble proteins. Through the manipulation of expression conditions, particularly the culture temperature of PPO-transformed E. coli cells, we were able to obtain large quantities of soluble recombinant PPO proteins. The results are as follows:1. All Drosophila melanogaster PPO1/2/3genes could be expressed in E. coli. At37℃almost all PPO proteins were expressed in conclusion body， which were more than proteins expressed at16℃. At16℃, about half of expressed proteins were in conclusion body and all the left were soluble protein. PPO1and PPO3expressed in E. coli cells have the same molecular weight as PPO1and PPO3expressed in Drosophila melanogaster S2cell according to Western blot assay. The reason might be that PPO1and PPO3didn’t need posttranslational modification or too much modification. However, the reason why PPO2expressed in Drosophila melanogaster S2cell was little bigger than that expressed in E. coli cells might be due to PPO2posttranslational modification in S2cells.2. Activity assay of PPO expressed in E. coli cell at16℃. Both transformational E. coli cells and their crude protein could be activated by30%ethanol or0.02%CPC. PPO1had the strongest activity among three PPOs, and PPO3had stronger activity than PPO2.30%ethanol could activate much more PPOs than0.02%CPC for PPO1and PPO3, but0.02%CPC was better than30%ethanol for PPO2.3. Expression amounts of PPO1. The amouts of PPO1expressed in E. coli cell at16℃gradually increased in12h. It reached at submit when PPO1was induced at the12t h h.4. The influence30%ethanol and Cu2+on the PPO1activity. We could dectect the obvious enzyme activity no matter which one, Cu2+or30%ethanol, was added firstly. However, we observed that the activated PPOs had higher activitiy when Cu2+was added before30%ethanol. 5. Dose responses of each purified PPO to Cu2+. For all three Drosophila PPOs expressed in E. coli, they had no enzyme activity when there was no Cu2+in medium or buffer. The dose responses of each PPO to Cu2+showed that2-10μM Cu2+was enough to turn PPO1and PPO3active but at least100μM Cu2+was necessary for PPO2. When Cu2+was higher than500μM, each PPO activity decreased. Less or no enzyme activities were observed when there was2000μM Cu2+in solution.The method established in this study will be helpful to produce insect specific recombinant PPOs, which may not be able to be obtained via traditional purification methods in order to compare PPO properties and functions in the future.