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 mammalian cells (BSC-1 monkey kidney cells and HeLa human cervical carcinoma cells) using a vaccinia virus expression system. The recombinant human procathepsin B appeared to be authentic and expressed in its native conformation as indicated by: (1) N-terminal sequencing; (2) molecular size; (3) processing intracellularly to mature double-chain cathepsin B; (4) in vitro cleavage by pepsin to mature cathepsin B coincident with appearance of activity against a selective synthetic substrate; and (5) substrate/inhibitor profiles. This is the first report of the expression of functional recombinant human procathepsin B in mammalian cells. We also report a single-step immunoaffinity purification procedure 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 protease inhibitors, other proteases, extracellular matrices, cell-surface proteins and biological substrates that may be of relevance to the pathobiological functions of this enzyme.
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November 1996
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Research Article|
November 01 1996
Expression of functional recombinant human procathepsin B in mammalian cells
Wei-Ping REN;
Wei-Ping REN
*Department of Pharmacology, Wayne State University, Detroit, MI 48201, U.S.A.
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Rafael FRIDMAN;
Rafael FRIDMAN
†Department of Pathology, Wayne State University, Detroit, MI 48201, U.S.A.
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James R. ZABRECKY;
James R. ZABRECKY
‡Department of Oncogene Science, Cambridge, MA 02412, U.S.A.
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Leticia D. MORRIS;
Leticia D. MORRIS
‡Department of Oncogene Science, Cambridge, MA 02412, U.S.A.
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Nancy A DAY;
Nancy A DAY
*Department of Pharmacology, Wayne State University, Detroit, MI 48201, U.S.A.
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Bonnie F. SLOANE
Bonnie F. SLOANE
§
*Department of Pharmacology, Wayne State University, Detroit, MI 48201, U.S.A.
§To whom correspondence and reprint requests should be addressed.
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Publisher: Portland Press Ltd
Received:
February 26 1996
Revision Received:
June 17 1996
Accepted:
July 02 1996
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1996
1996
Biochem J (1996) 319 (3): 793–800.
Article history
Received:
February 26 1996
Revision Received:
June 17 1996
Accepted:
July 02 1996
Citation
Wei-Ping REN, Rafael FRIDMAN, James R. ZABRECKY, Leticia D. MORRIS, Nancy A DAY, Bonnie F. SLOANE; Expression of functional recombinant human procathepsin B in mammalian cells. Biochem J 1 November 1996; 319 (3): 793–800. doi: https://doi.org/10.1042/bj3190793
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