Expression of functional recombinant human procathepsin B in mammalian cells.

Abstract

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.

• Authors: W P Ren, R Fridman, J R Zabrecky, L D Morris, N A Day, B F Sloane
• Journal: The Biochemical journal (Biochem J) Vol. 319 ( Pt 3) Pg. 793-800 (Nov 01 1996) ISSN: 0264-6021 [Print] England
• PMID: 8920982 (Publication Type: Journal Article, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Carbohydrates
• DNA Primers
• Enzyme Precursors
• Recombinant Proteins
• procathepsin B
• Cathepsin B

Topics

• Adenocarcinoma
• Animals
• Carbohydrates (analysis)
• Cathepsin B (biosynthesis, isolation & purification, metabolism)
• Cell Line
• Chlorocebus aethiops
• Chromatography, Affinity
• DNA Primers
• Enzyme Precursors (biosynthesis, isolation & purification, metabolism)
• Genetic Vectors
• Glycosylation
• HeLa Cells
• Humans
• Isoelectric Focusing
• Kinetics
• Polymerase Chain Reaction
• Recombinant Proteins (biosynthesis, isolation & purification, metabolism)
• Stomach Neoplasms
• Substrate Specificity
• Transfection
• Tumor Cells, Cultured
• Vaccinia virus