1. An initial goal of this proposal is to develop a new expression system for dengue protease NS2B-NS3pro in E. coli. The idea is to search for a tag of single-domain peptide which would facilitate the expression level of NS2B-NS3pro, and help membrane sorting during expression in E. coli cell. Experiments will also be focused on the optimization of the protein expression temperature, protein induction level, and the E. coli host.
2. The next attempt will be made to extract the expressed protease from the E. coli membrane using detergents. Screening of the appropriate detergents will be performed prior to purification step by column chromatography. The protease activity of the purified protein complex will be examined in a membrane-like situation resembling the environment in vivo, and then will be intensively compared with the values from assays in the solution.
3. For the future structural study, purified NS2B-NS3 will be subjected to the crystallization screening together with detergents to obtain crystals of good quality for the subsequent preliminary crystallographic characterization. If a good x-ray diffraction data is once available, structure determination could be carried out by molecular replacement method using the NS3pro structure as a template-model.
