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...
Gespeichert in:
| Veröffentlicht in: | Biochemical and biophysical research communications 1998-02, Vol.243 (2), p.384-389 |
|---|---|
| Hauptverfasser: | , , , , , , |
| Format: | Artikel |
| Sprache: | eng |
| Schlagworte: | |
| Online-Zugang: | Volltext |
| Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
| 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 |
| format | Article |
| fullrecord | <record><control><sourceid>proquest_cross</sourceid><recordid>TN_cdi_proquest_miscellaneous_79720867</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><els_id>S0006291X98981025</els_id><sourcerecordid>79720867</sourcerecordid><originalsourceid>FETCH-LOGICAL-c339t-47b1cf11b410765d1c83d2538bfd50cd0ee9c3faa3da3a67fcd8fe0ada0794813</originalsourceid><addsrcrecordid>eNp1kE1r3DAQhkVJSbfbXnsr6JSbtzO215aOSdimgUBLm0BvQh6NQMUfW8lesvn1tdmlt5yGmXnmhXmE-ISwQYDqS9NE2qDWaqMQ8jdihaAhyxHKC7GCmchyjb_fifcp_QFALCt9KS51qUChWolm97yPnFIYeml7J39MMfhAdlwGg5e7_uXYzR3Ztj3KaxrDgeVPpqFrQm_7Ud5FuyAsb2SYI-SNpakdDiFOSf46ppG7D-Ktt23ij-e6Fk9fd4-337KH73f3t9cPGRWFHrOybpA8YlMi1NXWIanC5dtCNd5tgRwwayq8tYWzha1qT055Buss1PM7WKzF1Sl3H4e_E6fRdCERt63teZiSqXWdg6rqGdycQIpDSpG92cfQ2Xg0CGaRahapZpFqFqnzwedz8tR07P7jZ4vzXp32PL93CBxNosA9sQuRaTRuCK9F_wO4OIf9</addsrcrecordid><sourcetype>Aggregation Database</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>79720867</pqid></control><display><type>article</type><title>Expression and Purification of Enzymatically Active Recombinant Granzyme B in a Baculovirus System</title><source>MEDLINE</source><source>Access via ScienceDirect (Elsevier)</source><creator>Xia, Zhinan ; Kam, Chih-Min ; Huang, Chifu ; Powers, James C. ; Mandle, Robert J. ; Stevens, Richard L. ; Lieberman, Judy</creator><creatorcontrib>Xia, Zhinan ; Kam, Chih-Min ; Huang, Chifu ; Powers, James C. ; Mandle, Robert J. ; Stevens, Richard L. ; Lieberman, Judy</creatorcontrib><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.</description><identifier>ISSN: 0006-291X</identifier><identifier>EISSN: 1090-2104</identifier><identifier>DOI: 10.1006/bbrc.1998.8102</identifier><identifier>PMID: 9480818</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>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</subject><ispartof>Biochemical and biophysical research communications, 1998-02, Vol.243 (2), p.384-389</ispartof><rights>1998 Academic Press</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c339t-47b1cf11b410765d1c83d2538bfd50cd0ee9c3faa3da3a67fcd8fe0ada0794813</citedby><cites>FETCH-LOGICAL-c339t-47b1cf11b410765d1c83d2538bfd50cd0ee9c3faa3da3a67fcd8fe0ada0794813</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1006/bbrc.1998.8102$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>315,782,786,3552,27931,27932,46002</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/9480818$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Xia, Zhinan</creatorcontrib><creatorcontrib>Kam, Chih-Min</creatorcontrib><creatorcontrib>Huang, Chifu</creatorcontrib><creatorcontrib>Powers, James C.</creatorcontrib><creatorcontrib>Mandle, Robert J.</creatorcontrib><creatorcontrib>Stevens, Richard L.</creatorcontrib><creatorcontrib>Lieberman, Judy</creatorcontrib><title>Expression and Purification of Enzymatically Active Recombinant Granzyme B in a Baculovirus System</title><title>Biochemical and biophysical research communications</title><addtitle>Biochem Biophys Res Commun</addtitle><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.</description><subject>Animals</subject><subject>Baculoviridae - genetics</subject><subject>Cysteine Endopeptidases - metabolism</subject><subject>DNA Fragmentation - genetics</subject><subject>Gene Expression - genetics</subject><subject>Granzymes</subject><subject>Killer Cells, Natural - enzymology</subject><subject>Kinetics</subject><subject>Peptides - metabolism</subject><subject>Protein Precursors - metabolism</subject><subject>Recombinant Proteins - genetics</subject><subject>Serine Endopeptidases - genetics</subject><subject>Serine Endopeptidases - isolation & purification</subject><subject>Spodoptera - genetics</subject><subject>T-Lymphocytes, Cytotoxic - enzymology</subject><issn>0006-291X</issn><issn>1090-2104</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1998</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp1kE1r3DAQhkVJSbfbXnsr6JSbtzO215aOSdimgUBLm0BvQh6NQMUfW8lesvn1tdmlt5yGmXnmhXmE-ISwQYDqS9NE2qDWaqMQ8jdihaAhyxHKC7GCmchyjb_fifcp_QFALCt9KS51qUChWolm97yPnFIYeml7J39MMfhAdlwGg5e7_uXYzR3Ztj3KaxrDgeVPpqFrQm_7Ud5FuyAsb2SYI-SNpakdDiFOSf46ppG7D-Ktt23ij-e6Fk9fd4-337KH73f3t9cPGRWFHrOybpA8YlMi1NXWIanC5dtCNd5tgRwwayq8tYWzha1qT055Buss1PM7WKzF1Sl3H4e_E6fRdCERt63teZiSqXWdg6rqGdycQIpDSpG92cfQ2Xg0CGaRahapZpFqFqnzwedz8tR07P7jZ4vzXp32PL93CBxNosA9sQuRaTRuCK9F_wO4OIf9</recordid><startdate>19980213</startdate><enddate>19980213</enddate><creator>Xia, Zhinan</creator><creator>Kam, Chih-Min</creator><creator>Huang, Chifu</creator><creator>Powers, James C.</creator><creator>Mandle, Robert J.</creator><creator>Stevens, Richard L.</creator><creator>Lieberman, Judy</creator><general>Elsevier Inc</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>19980213</creationdate><title>Expression and Purification of Enzymatically Active Recombinant Granzyme B in a Baculovirus System</title><author>Xia, Zhinan ; Kam, Chih-Min ; Huang, Chifu ; Powers, James C. ; Mandle, Robert J. ; Stevens, Richard L. ; Lieberman, Judy</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c339t-47b1cf11b410765d1c83d2538bfd50cd0ee9c3faa3da3a67fcd8fe0ada0794813</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1998</creationdate><topic>Animals</topic><topic>Baculoviridae - genetics</topic><topic>Cysteine Endopeptidases - metabolism</topic><topic>DNA Fragmentation - genetics</topic><topic>Gene Expression - genetics</topic><topic>Granzymes</topic><topic>Killer Cells, Natural - enzymology</topic><topic>Kinetics</topic><topic>Peptides - metabolism</topic><topic>Protein Precursors - metabolism</topic><topic>Recombinant Proteins - genetics</topic><topic>Serine Endopeptidases - genetics</topic><topic>Serine Endopeptidases - isolation & purification</topic><topic>Spodoptera - genetics</topic><topic>T-Lymphocytes, Cytotoxic - enzymology</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Xia, Zhinan</creatorcontrib><creatorcontrib>Kam, Chih-Min</creatorcontrib><creatorcontrib>Huang, Chifu</creatorcontrib><creatorcontrib>Powers, James C.</creatorcontrib><creatorcontrib>Mandle, Robert J.</creatorcontrib><creatorcontrib>Stevens, Richard L.</creatorcontrib><creatorcontrib>Lieberman, Judy</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Biochemical and biophysical research communications</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Xia, Zhinan</au><au>Kam, Chih-Min</au><au>Huang, Chifu</au><au>Powers, James C.</au><au>Mandle, Robert J.</au><au>Stevens, Richard L.</au><au>Lieberman, Judy</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Expression and Purification of Enzymatically Active Recombinant Granzyme B in a Baculovirus System</atitle><jtitle>Biochemical and biophysical research communications</jtitle><addtitle>Biochem Biophys Res Commun</addtitle><date>1998-02-13</date><risdate>1998</risdate><volume>243</volume><issue>2</issue><spage>384</spage><epage>389</epage><pages>384-389</pages><issn>0006-291X</issn><eissn>1090-2104</eissn><abstract>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.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>9480818</pmid><doi>10.1006/bbrc.1998.8102</doi><tpages>6</tpages></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 0006-291X |
| ispartof | Biochemical and biophysical research communications, 1998-02, Vol.243 (2), p.384-389 |
| issn | 0006-291X 1090-2104 |
| language | eng |
| recordid | cdi_proquest_miscellaneous_79720867 |
| 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 |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2024-12-07T16%3A19%3A42IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-proquest_cross&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Expression%20and%20Purification%20of%20Enzymatically%20Active%20Recombinant%20Granzyme%20B%20in%20a%20Baculovirus%20System&rft.jtitle=Biochemical%20and%20biophysical%20research%20communications&rft.au=Xia,%20Zhinan&rft.date=1998-02-13&rft.volume=243&rft.issue=2&rft.spage=384&rft.epage=389&rft.pages=384-389&rft.issn=0006-291X&rft.eissn=1090-2104&rft_id=info:doi/10.1006/bbrc.1998.8102&rft_dat=%3Cproquest_cross%3E79720867%3C/proquest_cross%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=79720867&rft_id=info:pmid/9480818&rft_els_id=S0006291X98981025&rfr_iscdi=true |