The immediate aim of this project is to engineer and produce active HIV protease in quantities sufficient to support the other components in this program. Purified protease will be subjected to amino acid sequence analysis to determine the amino-terminus of the mutant enzyme. This information will be used to design synthetic gene for the HIV protease which will be constructed and expressed in E. coli. Large quantities of active protease will be purified from bacterial cultures. Recombinant protease will be for structural studies and to C for screening of microbial beers for protease inhibitors. The protease will be isotopically labeled by growing the cells in media containing isotopically labeled amino acids. Following purification, this material will be provided to for NMR studies. Site-directed mutagenesis will be used to engineer specific changes in the amino acid sequence of the HIV protease. These mutant enzymes will be biologically and enzymatically characterized. The site-directed mutugenesis studies will also be used to test structural models of the protease, and, combined with the X-ray crystallographic and NMR investigations, will aid in the interpretation of structure-activity relationships for synthetic inhibitors.
