Properties of Drosophila melanogaster prophenoloxidases expressed in Escherichia coli

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 Escherichia 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. Additional Cu²⁺, either added into the culture medium during PPO induction or directly mixed with the purified rPPO preparations, was necessary to produce Cu²⁺ associated proenzymes. Cu²⁺ associated PPOs showed obvious enzyme activities after activation by either ethanol or cetylpyridinium chloride, or by AMM1 (a pupal protein fraction containing native serine proteases for PPO activation). Dose responses for association of individual purified Drosophila rPPOs with Cu²⁺ showed that Drosophila rPPO1 and rPPO3 had relatively higher affinity for Cu²⁺ than rPPO2 did. Surprisingly, however, high concentration of Cu²⁺ (2mM) completely inhibited PPO activity. Each rPPO had similar activity when dopamine or l-DOPA was the substrate. However, rPPO1 alone had very high activity if l-tyrosine was used as a substrate. After activation by ethanol or 2-propanol, Km and Vmax of the three rPPOs changed as shown in the following: rPPO2<rPPO3<rPPO1. If activated by ethanol, the Km and Vmax of each rPPO were lower than by 2-propanol. Due to the difficulty in obtaining functional PPOs via traditional purification methods, the method established in this study will be helpful to produce active insect recombinant PPOs for the study of PPO properties and functions in the future.