CHARACTERIZATION OF RECOMBINANT HUMAN CATHEPSIN-B EXPRESSED AT HIGH-LEVELS IN BACULOVIRUS

The lysosomal cysteine protease cathepsin B has been studied intensely for many years because of its unique characteristics and its potentia l involvement in disease states. A reproducible, high yield expression system for active recombinant protein is key to biochemical and bioph ysical studies as well as rational drug design. Although several micro bial and mammalian expression systems for recombinant human cathepsin B have been described, these have been limited by low or variable yiel ds. Further, in some of these systems hyper-glycosylation of the enzym e near the active site affects its activity. We describe a baculovirus expression system and purification scheme that solve all of these pro blems. Yields of active, protected enzyme were reproducibly in excess of 25 mg/L. Since this protein was not hyper-glycosylated, it had grea ter activity than cathepsin B produced in yeast systems as indicated b y a threefold increase in K-cat. In addition, the biophysical properti es of the baculovirus-expressed cathepsin B, as measured by dynamic li ght scattering, were more amenable to crystallographic study since the data indicated proteins of more uniform size. Therefore, this system for the production of recombinant human cathepsin B constitutes a majo r improvement in both quantity and quality over those previously repor ted. Further, we demonstrate that the manner of expression and purific ation of this enzyme has profound effects on its kinetic and physical parameters.