Expression and Purification of Enzymatically Active Recombinant Granzyme B in a Baculovirus System

Granzyme B (GranB), a serine protease stored in the granules of cytotoxic T lymphocytes and natural killer cells, can initiate target cell apoptosis. To produce large amounts of purified active enzyme, recombinant murine granzyme B (rGranB) was expressed from baculovirus in insect cells. The express...

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Veröffentlicht in:Biochemical and biophysical research communications 1998-02, Vol.243 (2), p.384-389
Hauptverfasser: Xia, Zhinan, Kam, Chih-Min, Huang, Chifu, Powers, James C., Mandle, Robert J., Stevens, Richard L., Lieberman, Judy
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container_end_page 389
container_issue 2
container_start_page 384
container_title Biochemical and biophysical research communications
container_volume 243
creator Xia, Zhinan
Kam, Chih-Min
Huang, Chifu
Powers, James C.
Mandle, Robert J.
Stevens, Richard L.
Lieberman, Judy
description Granzyme B (GranB), a serine protease stored in the granules of cytotoxic T lymphocytes and natural killer cells, can initiate target cell apoptosis. To produce large amounts of purified active enzyme, recombinant murine granzyme B (rGranB) was expressed from baculovirus in insect cells. The expressed rGranB is secreted into the culture medium and can be readily purified to homogeneity by one-step affinity chromatography to yield 1.5 mg enzyme per liter insect cell medium. RGranB is recognized by a GranB-specific anti-peptide antibody and is active against synthetic substrate Boc-Ala-Ala-Asp-SBzl with kinetic constant (kcat/Km45,000 M−1s−1) comparable to purified human GranB. RGranB processes the caspase pro-CPP32 into its enzymatically active form and induces DNA fragmentation in isolated nuclei in the presence of cytosolic factors. The ability to express enzymatically active rGranB using the baculovirus system will help elucidate the role of this granzyme in the immune response.
doi_str_mv 10.1006/bbrc.1998.8102
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To produce large amounts of purified active enzyme, recombinant murine granzyme B (rGranB) was expressed from baculovirus in insect cells. The expressed rGranB is secreted into the culture medium and can be readily purified to homogeneity by one-step affinity chromatography to yield 1.5 mg enzyme per liter insect cell medium. RGranB is recognized by a GranB-specific anti-peptide antibody and is active against synthetic substrate Boc-Ala-Ala-Asp-SBzl with kinetic constant (kcat/Km45,000 M−1s−1) comparable to purified human GranB. RGranB processes the caspase pro-CPP32 into its enzymatically active form and induces DNA fragmentation in isolated nuclei in the presence of cytosolic factors. 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ispartof Biochemical and biophysical research communications, 1998-02, Vol.243 (2), p.384-389
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source MEDLINE; Access via ScienceDirect (Elsevier)
subjects Animals
Baculoviridae - genetics
Cysteine Endopeptidases - metabolism
DNA Fragmentation - genetics
Gene Expression - genetics
Granzymes
Killer Cells, Natural - enzymology
Kinetics
Peptides - metabolism
Protein Precursors - metabolism
Recombinant Proteins - genetics
Serine Endopeptidases - genetics
Serine Endopeptidases - isolation & purification
Spodoptera - genetics
T-Lymphocytes, Cytotoxic - enzymology
title Expression and Purification of Enzymatically Active Recombinant Granzyme B in a Baculovirus System
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