HIV-1 Aspartic Proteinase: High-Level Production and Automated Fluorometric Screening Assay of Inhibitors
The 99-amino-acid HIV-1 aspartic proteinase was expressed to high levels in Escherichia coli using a T7 expression system. About 50% of the insoluble material after sonication of the bacteria was composed of aggregated proteinase. Subsequent renaturation and purification yielded large quantities of...
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| Veröffentlicht in: | Antiviral chemistry & chemotherapy 1990-02, Vol.1 (1), p.9-15 |
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| creator | Hirel, Ph.-H. Parker, F. Boiziau, J. Jung, G. Outerovitch, D. Dugué, A. Peltiers, C. Giuliacci, C. Boulay, R. Lelièvre, Y. Cambou, B. Mayaux, J.-F. Cartwright, T. |
| description | The 99-amino-acid HIV-1 aspartic proteinase was expressed to high levels in Escherichia coli using a T7 expression system. About 50% of the insoluble material after sonication of the bacteria was composed of aggregated proteinase. Subsequent renaturation and purification yielded large quantities of a homogeneous enzyme able to cleave various heptapeptidic substrates in vitro with a Km around 2.5 mM. A fluorometric assay has been devised to allow automated screening of HIV proteinase inhibitors based on an analogous renin assay. We used the synthetic intramolecularly quenched fluorogenic substrate Suc-TLNFPIS-4MCA based on the heptapeptide TLNFPIS, which encompasses the proteinase/reverse transcriptase junction, coupled to the fluorophore 7-amino-4-methylcoumarin and blocked at the amino-terminus by a succinyl group. The enzyme cleaves the substrate between phenylalanine and proline, and conditions were optimized for liberation of 7AMC from the generated PIS-4MCA with aminopeptidase M as secondary enzyme. 7AMC was monitored with a microplate fluorescence scanner. The known aspartic proteinase inhibitor pepstatin A consistently gave Ki = 2 × 10−6M. Other synthetic and natural compounds are currently being tested. |
| doi_str_mv | 10.1177/095632029000100103 |
| format | Article |
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About 50% of the insoluble material after sonication of the bacteria was composed of aggregated proteinase. Subsequent renaturation and purification yielded large quantities of a homogeneous enzyme able to cleave various heptapeptidic substrates in vitro with a Km around 2.5 mM. A fluorometric assay has been devised to allow automated screening of HIV proteinase inhibitors based on an analogous renin assay. We used the synthetic intramolecularly quenched fluorogenic substrate Suc-TLNFPIS-4MCA based on the heptapeptide TLNFPIS, which encompasses the proteinase/reverse transcriptase junction, coupled to the fluorophore 7-amino-4-methylcoumarin and blocked at the amino-terminus by a succinyl group. The enzyme cleaves the substrate between phenylalanine and proline, and conditions were optimized for liberation of 7AMC from the generated PIS-4MCA with aminopeptidase M as secondary enzyme. 7AMC was monitored with a microplate fluorescence scanner. The known aspartic proteinase inhibitor pepstatin A consistently gave Ki = 2 × 10−6M. Other synthetic and natural compounds are currently being tested.</description><identifier>ISSN: 2040-2066</identifier><identifier>ISSN: 0956-3202</identifier><identifier>EISSN: 2040-2066</identifier><identifier>DOI: 10.1177/095632029000100103</identifier><language>eng</language><publisher>London, England: SAGE Publications</publisher><subject>Aminopeptidase ; Antibiotics. Antiinfectious agents. Antiparasitic agents ; Antiviral agents ; Aspartic proteinase ; Automation ; Biological and medical sciences ; Enzymes ; HIV ; Human immunodeficiency virus ; Medical sciences ; Pharmacology. Drug treatments ; Phenylalanine ; Proline ; Protein purification ; Proteinase ; Proteinase inhibitors ; Renaturation ; Renin ; RNA-directed DNA polymerase ; Sonication</subject><ispartof>Antiviral chemistry & chemotherapy, 1990-02, Vol.1 (1), p.9-15</ispartof><rights>1990 SAGE Publications</rights><rights>1991 INIST-CNRS</rights><rights>1990 SAGE Publications. 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About 50% of the insoluble material after sonication of the bacteria was composed of aggregated proteinase. Subsequent renaturation and purification yielded large quantities of a homogeneous enzyme able to cleave various heptapeptidic substrates in vitro with a Km around 2.5 mM. A fluorometric assay has been devised to allow automated screening of HIV proteinase inhibitors based on an analogous renin assay. We used the synthetic intramolecularly quenched fluorogenic substrate Suc-TLNFPIS-4MCA based on the heptapeptide TLNFPIS, which encompasses the proteinase/reverse transcriptase junction, coupled to the fluorophore 7-amino-4-methylcoumarin and blocked at the amino-terminus by a succinyl group. The enzyme cleaves the substrate between phenylalanine and proline, and conditions were optimized for liberation of 7AMC from the generated PIS-4MCA with aminopeptidase M as secondary enzyme. 7AMC was monitored with a microplate fluorescence scanner. The known aspartic proteinase inhibitor pepstatin A consistently gave Ki = 2 × 10−6M. Other synthetic and natural compounds are currently being tested.</description><subject>Aminopeptidase</subject><subject>Antibiotics. Antiinfectious agents. Antiparasitic agents</subject><subject>Antiviral agents</subject><subject>Aspartic proteinase</subject><subject>Automation</subject><subject>Biological and medical sciences</subject><subject>Enzymes</subject><subject>HIV</subject><subject>Human immunodeficiency virus</subject><subject>Medical sciences</subject><subject>Pharmacology. Drug treatments</subject><subject>Phenylalanine</subject><subject>Proline</subject><subject>Protein purification</subject><subject>Proteinase</subject><subject>Proteinase inhibitors</subject><subject>Renaturation</subject><subject>Renin</subject><subject>RNA-directed DNA polymerase</subject><subject>Sonication</subject><issn>2040-2066</issn><issn>0956-3202</issn><issn>2040-2066</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1990</creationdate><recordtype>article</recordtype><sourceid>BENPR</sourceid><recordid>eNp9UNtKAzEQXUTBWv0BnwLi49pJsrvZ-FaKtYWCgpfXJU2ybUqb1CQr9O9NaaGCIAzMcOZcmMmyWwwPGDM2AF5WlADhAID3Rc-yHoECcgJVdf5rvsyuQlgB0LqkvJeZyfQzx2gYtsJHI9Grd1EbK4J-RBOzWOYz_a3Xe1h1MhpnkbAKDbvoNiJqhcbrznm30dEn8Zv0WltjF8kviB1yLZrapZmb6Hy4zi5asQ765tj72cf46X00yWcvz9PRcJZLinnMOSWsrCqpC9wywet6Xs5BcSCYEyZVrUAJgluoSVHjdC6uiKIJkTWoKi1pP7s7-G69--p0iM3Kdd6myIbQglCGocSJRQ4s6V0IXrfN1puN8LsGQ7N_afP3pUl0f7QWQYp164WVJpyUnFWs5JB4gwMviIU-xf_j_APckoFx</recordid><startdate>19900201</startdate><enddate>19900201</enddate><creator>Hirel, Ph.-H.</creator><creator>Parker, F.</creator><creator>Boiziau, J.</creator><creator>Jung, G.</creator><creator>Outerovitch, D.</creator><creator>Dugué, A.</creator><creator>Peltiers, C.</creator><creator>Giuliacci, C.</creator><creator>Boulay, R.</creator><creator>Lelièvre, Y.</creator><creator>Cambou, B.</creator><creator>Mayaux, J.-F.</creator><creator>Cartwright, T.</creator><general>SAGE Publications</general><general>International Medical Press</general><general>Sage Publications Ltd</general><scope>IQODW</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7QL</scope><scope>7QR</scope><scope>7T5</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8FD</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M7P</scope><scope>P64</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope></search><sort><creationdate>19900201</creationdate><title>HIV-1 Aspartic Proteinase: High-Level Production and Automated Fluorometric Screening Assay of Inhibitors</title><author>Hirel, Ph.-H. ; Parker, F. ; Boiziau, J. ; Jung, G. ; Outerovitch, D. ; Dugué, A. ; Peltiers, C. ; Giuliacci, C. ; Boulay, R. ; Lelièvre, Y. ; Cambou, B. ; Mayaux, J.-F. ; Cartwright, T.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c319t-9327566ce41f7a988b5b0d9021927cd8d0da21f082481900162d3a21c80d6d0d3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1990</creationdate><topic>Aminopeptidase</topic><topic>Antibiotics. Antiinfectious agents. Antiparasitic agents</topic><topic>Antiviral agents</topic><topic>Aspartic proteinase</topic><topic>Automation</topic><topic>Biological and medical sciences</topic><topic>Enzymes</topic><topic>HIV</topic><topic>Human immunodeficiency virus</topic><topic>Medical sciences</topic><topic>Pharmacology. Drug treatments</topic><topic>Phenylalanine</topic><topic>Proline</topic><topic>Protein purification</topic><topic>Proteinase</topic><topic>Proteinase inhibitors</topic><topic>Renaturation</topic><topic>Renin</topic><topic>RNA-directed DNA polymerase</topic><topic>Sonication</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Hirel, Ph.-H.</creatorcontrib><creatorcontrib>Parker, F.</creatorcontrib><creatorcontrib>Boiziau, J.</creatorcontrib><creatorcontrib>Jung, G.</creatorcontrib><creatorcontrib>Outerovitch, D.</creatorcontrib><creatorcontrib>Dugué, A.</creatorcontrib><creatorcontrib>Peltiers, C.</creatorcontrib><creatorcontrib>Giuliacci, C.</creatorcontrib><creatorcontrib>Boulay, R.</creatorcontrib><creatorcontrib>Lelièvre, Y.</creatorcontrib><creatorcontrib>Cambou, B.</creatorcontrib><creatorcontrib>Mayaux, J.-F.</creatorcontrib><creatorcontrib>Cartwright, T.</creatorcontrib><collection>Pascal-Francis</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Chemoreception Abstracts</collection><collection>Immunology Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest One Sustainability</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Natural Science Collection</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Biological Science Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Publicly Available Content Database</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><jtitle>Antiviral chemistry & chemotherapy</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Hirel, Ph.-H.</au><au>Parker, F.</au><au>Boiziau, J.</au><au>Jung, G.</au><au>Outerovitch, D.</au><au>Dugué, A.</au><au>Peltiers, C.</au><au>Giuliacci, C.</au><au>Boulay, R.</au><au>Lelièvre, Y.</au><au>Cambou, B.</au><au>Mayaux, J.-F.</au><au>Cartwright, T.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>HIV-1 Aspartic Proteinase: High-Level Production and Automated Fluorometric Screening Assay of Inhibitors</atitle><jtitle>Antiviral chemistry & chemotherapy</jtitle><date>1990-02-01</date><risdate>1990</risdate><volume>1</volume><issue>1</issue><spage>9</spage><epage>15</epage><pages>9-15</pages><issn>2040-2066</issn><issn>0956-3202</issn><eissn>2040-2066</eissn><abstract>The 99-amino-acid HIV-1 aspartic proteinase was expressed to high levels in Escherichia coli using a T7 expression system. About 50% of the insoluble material after sonication of the bacteria was composed of aggregated proteinase. Subsequent renaturation and purification yielded large quantities of a homogeneous enzyme able to cleave various heptapeptidic substrates in vitro with a Km around 2.5 mM. A fluorometric assay has been devised to allow automated screening of HIV proteinase inhibitors based on an analogous renin assay. We used the synthetic intramolecularly quenched fluorogenic substrate Suc-TLNFPIS-4MCA based on the heptapeptide TLNFPIS, which encompasses the proteinase/reverse transcriptase junction, coupled to the fluorophore 7-amino-4-methylcoumarin and blocked at the amino-terminus by a succinyl group. The enzyme cleaves the substrate between phenylalanine and proline, and conditions were optimized for liberation of 7AMC from the generated PIS-4MCA with aminopeptidase M as secondary enzyme. 7AMC was monitored with a microplate fluorescence scanner. The known aspartic proteinase inhibitor pepstatin A consistently gave Ki = 2 × 10−6M. Other synthetic and natural compounds are currently being tested.</abstract><cop>London, England</cop><pub>SAGE Publications</pub><doi>10.1177/095632029000100103</doi><tpages>7</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | Antiviral chemistry & chemotherapy, 1990-02, Vol.1 (1), p.9-15 |
| issn | 2040-2066 0956-3202 2040-2066 |
| language | eng |
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| source | Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Alma/SFX Local Collection |
| subjects | Aminopeptidase Antibiotics. Antiinfectious agents. Antiparasitic agents Antiviral agents Aspartic proteinase Automation Biological and medical sciences Enzymes HIV Human immunodeficiency virus Medical sciences Pharmacology. Drug treatments Phenylalanine Proline Protein purification Proteinase Proteinase inhibitors Renaturation Renin RNA-directed DNA polymerase Sonication |
| title | HIV-1 Aspartic Proteinase: High-Level Production and Automated Fluorometric Screening Assay of Inhibitors |
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