EXPRESSION OF FUNCTIONAL RECOMBINANT HUMAN PROCATHEPSIN-B IN MAMMALIAN-CELLS

Cathepsin B has been implicated in numerous pathobiological processes. In order to study its interactions with other proteins implicated in these processes, quantities of functional recombinant cathepsin B are needed. Therefore, we expressed recombinant human procathepsin B in ma mmalian cells (BSC-1 monkey kidney cells and HeLa human cervical carci noma cells) using a vaccinia virus expression system. The recombinant human procathepsin B appeared to be authentic and expressed in its nat ive conformation as indicated by. (1) N-terminal sequencing; (2) molec ular size; (3) processing intracellularly to mature double-chain cathe psin B; (4) in vitro cleavage by pepsin to mature cathepsin B coincide nt with appearance of activity against a selective synthetic substrate ; and (5) substrate/inhibitor profiles. This is the first report of th e expression of functional recombinant human procathepsin B in mammali an cells. We also report a single-step immunoaffinity purification pro cedure for the isolation of electrophoretically pure proenzyme. By the methodologies described, human procathepsin B can now be obtained in high yield. This should facilitate studies of its interactions with pr otease inhibitors, other proteases, extracellular matrices, cell-surfa ce proteins and biological substrates that may be of relevance to the pathobiological functions of this enzyme.