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 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.
Ren
W P
WP
Department of Pharmacology, Wayne State University, Detroit, MI 48201, USA.
Fridman
R
R
Zabrecky
J R
JR
Morris
L D
LD
Day
N A
NA
Sloane
B F
BF
D000230:Adenocarcinoma; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002401:Cathepsin B / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D002522:Chlorocebus aethiops; D002846:Chromatography, Affinity; D017931:DNA Primers; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D005822:Genetic Vectors; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D007525:Isoelectric Focusing; D007700:Kinetics; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013274:Stomach Neoplasms; D013379:Substrate Specificity; D014162:Transfection; D014407:Tumor Cells, Cultured; D014616:Vaccinia virus
D002241:Carbohydrates; D017931:DNA Primers; D004792:Enzyme Precursors; D011994:Recombinant Proteins; C057832:procathepsin B; D002401:Cathepsin B