HIV-I PROTEASE - CLONING, EXPRESSION, AND PURIFICATION

A new method for obtaining HIV-I protease was suggested. Fusion protei ns composed of the N-terminal fragment of human gamma-interferon and H IV-I protease connected with (Asp)(4)Lys (protein I) or Asp-Pro (prote in II) linkers were expressed in Escherichia coli cells. The fusion pr oteins were produced as insoluble inclusion bodies in the 20% yield of total cell protein. Protein I was cleaved by enterokinase. The solubi lity of protein I was increased by treating with Na-sulfite/Na-tetrath ionate under denaturing conditions. Optimal conditions for efficient a cidic hydrolysis of protein II at Asp-Pro bond were found. The hydroly sis products were separated by reversed-phase FPLC. The amount of tryp tophan and cysteine residues in the enzyme obtained was estimated. The activity of HIV-I protease was determined using the chromogenic pepti de AlaArgVal NleNphGluAlaNleNH(2) and a high-mol-wt substrate consisti ng of beta-galactosidase and a fragment of gag proteins, including p17 -p24 processing site.