High level expression of Ricinus communis casbene synthase in Escherichia coli and characterization of the recombinant enzyme
Casbene synthase catalyzes the cyclization of geranylgeranyl diphosphate (2) to casbene (1), a diterpene phytoalexin with antibacterial and antifungal activity that is produced by seedlings of castor bean (Ricinus communis L.) in response to fungal attack. We report the high-level expression of casb...
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| Veröffentlicht in: | Archives of biochemistry and biophysics 1996-12, Vol.336 (2), p.283 |
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| creator | Hill, A M Cane, D E Mau, C J West, C A |
| description | Casbene synthase catalyzes the cyclization of geranylgeranyl diphosphate (2) to casbene (1), a diterpene phytoalexin with antibacterial and antifungal activity that is produced by seedlings of castor bean (Ricinus communis L.) in response to fungal attack. We report the high-level expression of casbene synthase cDNA in Escherichia coli as insoluble inclusion bodies, the solubilization and refolding of active casbene synthase, and the kinetic and product analysis of the recombinant enzyme. To overcome problems apparently associated with the presence in the casbene synthase gene of rare Arg codons, as well as the intrinsic antibacterial activity of casbene itself, the casbene synthase gene was expressed in an E. coli host harboring the pSM102 vector that encodes the dnaY gene for tArg(AGA/G), using an expression vector, pET-21d(+), carrying the tightly controlled T7lac promoter. |
| doi_str_mv | 10.1006/abbi.1996.0559 |
| format | Article |
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We report the high-level expression of casbene synthase cDNA in Escherichia coli as insoluble inclusion bodies, the solubilization and refolding of active casbene synthase, and the kinetic and product analysis of the recombinant enzyme. To overcome problems apparently associated with the presence in the casbene synthase gene of rare Arg codons, as well as the intrinsic antibacterial activity of casbene itself, the casbene synthase gene was expressed in an E. coli host harboring the pSM102 vector that encodes the dnaY gene for tArg(AGA/G), using an expression vector, pET-21d(+), carrying the tightly controlled T7lac promoter.</description><identifier>ISSN: 0003-9861</identifier><identifier>EISSN: 1096-0384</identifier><identifier>DOI: 10.1006/abbi.1996.0559</identifier><identifier>PMID: 8954576</identifier><language>eng</language><publisher>United States</publisher><subject>Escherichia coli ; Gas Chromatography-Mass Spectrometry ; Inclusion Bodies - enzymology ; liasas ; lyase ; lyases ; Lyases - genetics ; Lyases - isolation & purification ; Lyases - metabolism ; Phosphorus-Oxygen Lyases ; Plants, Toxic ; Recombinant Proteins - genetics ; Recombinant Proteins - isolation & purification ; Recombinant Proteins - metabolism ; ricinus communis ; Ricinus communis - enzymology ; Solubility</subject><ispartof>Archives of biochemistry and biophysics, 1996-12, Vol.336 (2), p.283</ispartof><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/8954576$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Hill, A M</creatorcontrib><creatorcontrib>Cane, D E</creatorcontrib><creatorcontrib>Mau, C J</creatorcontrib><creatorcontrib>West, C A</creatorcontrib><creatorcontrib>Brown University, Providence, RI</creatorcontrib><title>High level expression of Ricinus communis casbene synthase in Escherichia coli and characterization of the recombinant enzyme</title><title>Archives of biochemistry and biophysics</title><addtitle>Arch Biochem Biophys</addtitle><description>Casbene synthase catalyzes the cyclization of geranylgeranyl diphosphate (2) to casbene (1), a diterpene phytoalexin with antibacterial and antifungal activity that is produced by seedlings of castor bean (Ricinus communis L.) in response to fungal attack. We report the high-level expression of casbene synthase cDNA in Escherichia coli as insoluble inclusion bodies, the solubilization and refolding of active casbene synthase, and the kinetic and product analysis of the recombinant enzyme. To overcome problems apparently associated with the presence in the casbene synthase gene of rare Arg codons, as well as the intrinsic antibacterial activity of casbene itself, the casbene synthase gene was expressed in an E. coli host harboring the pSM102 vector that encodes the dnaY gene for tArg(AGA/G), using an expression vector, pET-21d(+), carrying the tightly controlled T7lac promoter.</description><subject>Escherichia coli</subject><subject>Gas Chromatography-Mass Spectrometry</subject><subject>Inclusion Bodies - enzymology</subject><subject>liasas</subject><subject>lyase</subject><subject>lyases</subject><subject>Lyases - genetics</subject><subject>Lyases - isolation & purification</subject><subject>Lyases - metabolism</subject><subject>Phosphorus-Oxygen Lyases</subject><subject>Plants, Toxic</subject><subject>Recombinant Proteins - genetics</subject><subject>Recombinant Proteins - isolation & purification</subject><subject>Recombinant Proteins - metabolism</subject><subject>ricinus communis</subject><subject>Ricinus communis - enzymology</subject><subject>Solubility</subject><issn>0003-9861</issn><issn>1096-0384</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1996</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNotkEFLwzAcxYMoc06v3pR8gc5_0iRNjjKmEwaCunNJ0mSNtOloOnEDv7uF7fQe7z1-h4fQPYE5ARBP2pgwJ0qJOXCuLtCUgBIZ5JJdoikA5JmSglyjm5S-AQhhgk7QRCrOeCGm6G8VtjVu3I9rsPvd9S6l0EXcefwRbIj7hG3XtvsYRqOTcdHhdIhDrZPDIeJlsrXrg62DHodNwDpW2Na613YY86MezrShdrh3I8uEqOOAXTweWneLrrxukrs76wxtXpZfi1W2fn99WzyvM0-pGrI8r4iX1INWWuZGcl55Jh2TxAtLbMGJYkQSC4JbziV1xlAqhJCUVQyYzWfo4cTd7U3rqnLXh1b3h_J8w9g_nnqvu1Jv-5DKzed4agFQSEpU_g8_FmpI</recordid><startdate>19961215</startdate><enddate>19961215</enddate><creator>Hill, A M</creator><creator>Cane, D E</creator><creator>Mau, C J</creator><creator>West, C A</creator><scope>FBQ</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope></search><sort><creationdate>19961215</creationdate><title>High level expression of Ricinus communis casbene synthase in Escherichia coli and characterization of the recombinant enzyme</title><author>Hill, A M ; Cane, D E ; Mau, C J ; West, C A</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-f229t-33d1f82f0a9a83b855df48e481f6c1c75194181c065c5582ebb22666824d404c3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1996</creationdate><topic>Escherichia coli</topic><topic>Gas Chromatography-Mass Spectrometry</topic><topic>Inclusion Bodies - enzymology</topic><topic>liasas</topic><topic>lyase</topic><topic>lyases</topic><topic>Lyases - genetics</topic><topic>Lyases - isolation & purification</topic><topic>Lyases - metabolism</topic><topic>Phosphorus-Oxygen Lyases</topic><topic>Plants, Toxic</topic><topic>Recombinant Proteins - genetics</topic><topic>Recombinant Proteins - isolation & purification</topic><topic>Recombinant Proteins - metabolism</topic><topic>ricinus communis</topic><topic>Ricinus communis - enzymology</topic><topic>Solubility</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Hill, A M</creatorcontrib><creatorcontrib>Cane, D E</creatorcontrib><creatorcontrib>Mau, C J</creatorcontrib><creatorcontrib>West, C A</creatorcontrib><creatorcontrib>Brown University, Providence, RI</creatorcontrib><collection>AGRIS</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><jtitle>Archives of biochemistry and biophysics</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Hill, A M</au><au>Cane, D E</au><au>Mau, C J</au><au>West, C A</au><aucorp>Brown University, Providence, RI</aucorp><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>High level expression of Ricinus communis casbene synthase in Escherichia coli and characterization of the recombinant enzyme</atitle><jtitle>Archives of biochemistry and biophysics</jtitle><addtitle>Arch Biochem Biophys</addtitle><date>1996-12-15</date><risdate>1996</risdate><volume>336</volume><issue>2</issue><spage>283</spage><pages>283-</pages><issn>0003-9861</issn><eissn>1096-0384</eissn><abstract>Casbene synthase catalyzes the cyclization of geranylgeranyl diphosphate (2) to casbene (1), a diterpene phytoalexin with antibacterial and antifungal activity that is produced by seedlings of castor bean (Ricinus communis L.) in response to fungal attack. We report the high-level expression of casbene synthase cDNA in Escherichia coli as insoluble inclusion bodies, the solubilization and refolding of active casbene synthase, and the kinetic and product analysis of the recombinant enzyme. To overcome problems apparently associated with the presence in the casbene synthase gene of rare Arg codons, as well as the intrinsic antibacterial activity of casbene itself, the casbene synthase gene was expressed in an E. coli host harboring the pSM102 vector that encodes the dnaY gene for tArg(AGA/G), using an expression vector, pET-21d(+), carrying the tightly controlled T7lac promoter.</abstract><cop>United States</cop><pmid>8954576</pmid><doi>10.1006/abbi.1996.0559</doi></addata></record> |
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| ispartof | Archives of biochemistry and biophysics, 1996-12, Vol.336 (2), p.283 |
| issn | 0003-9861 1096-0384 |
| language | eng |
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| source | MEDLINE; ScienceDirect Journals (5 years ago - present) |
| subjects | Escherichia coli Gas Chromatography-Mass Spectrometry Inclusion Bodies - enzymology liasas lyase lyases Lyases - genetics Lyases - isolation & purification Lyases - metabolism Phosphorus-Oxygen Lyases Plants, Toxic Recombinant Proteins - genetics Recombinant Proteins - isolation & purification Recombinant Proteins - metabolism ricinus communis Ricinus communis - enzymology Solubility |
| title | High level expression of Ricinus communis casbene synthase in Escherichia coli and characterization of the recombinant enzyme |
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