High-Level Expression of Functional Rat Neuronal Nitric Oxide Synthase in Escherichia coli
The neuronal nitric oxide synthase (nNOS) has been successfully overexpressed in Escherichia coli, with average yields of 125-150 nmol (20-24 mg) of enzyme per liter of cells. The cDNA for nNOS was subcloned into the pCW vector under the control of the tac promotor and was coexpressed with the chape...
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| Veröffentlicht in: | Proceedings of the National Academy of Sciences - PNAS 1995-08, Vol.92 (18), p.8428-8432 |
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| container_title | Proceedings of the National Academy of Sciences - PNAS |
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| creator | Roman, Linda J. Sheta, Essam A. Martasek, Pavel Gross, Steven S. Liu, Qing Bettie Sue Siler Masters |
| description | The neuronal nitric oxide synthase (nNOS) has been successfully overexpressed in Escherichia coli, with average yields of 125-150 nmol (20-24 mg) of enzyme per liter of cells. The cDNA for nNOS was subcloned into the pCW vector under the control of the tac promotor and was coexpressed with the chaperonins groEL and groES in the protease-deficient BL21 strain of E. coli. The enzyme produced is replete with heme and flavins and, after overnight incubation with tetrahydrobiopterin, contains 0.7 pmol of tetrahydrobiopterin per pmol of nNOS. nNOS is isolated as a predominantly high-spin heme protein and demonstrates spectral properties that are identical to those of nNOS isolated from stably transfected human kidney 293 cells. It binds Nω-nitroarginine dependent on the presence of bound tetrahy-drobiopterin and exhibits a Kdof 45 nM. The enzyme is completely functional; the specific activity is 450 nmol/min per mg. This overexpression system will be extremely useful for rapid, inexpensive preparation of large amounts of active nNOS for use in mechanistic and structure/function studies, as well as for drug design and development. |
| doi_str_mv | 10.1073/pnas.92.18.8428 |
| format | Article |
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The cDNA for nNOS was subcloned into the pCW vector under the control of the tac promotor and was coexpressed with the chaperonins groEL and groES in the protease-deficient BL21 strain of E. coli. The enzyme produced is replete with heme and flavins and, after overnight incubation with tetrahydrobiopterin, contains 0.7 pmol of tetrahydrobiopterin per pmol of nNOS. nNOS is isolated as a predominantly high-spin heme protein and demonstrates spectral properties that are identical to those of nNOS isolated from stably transfected human kidney 293 cells. It binds Nω-nitroarginine dependent on the presence of bound tetrahy-drobiopterin and exhibits a Kdof 45 nM. The enzyme is completely functional; the specific activity is 450 nmol/min per mg. This overexpression system will be extremely useful for rapid, inexpensive preparation of large amounts of active nNOS for use in mechanistic and structure/function studies, as well as for drug design and development.</description><identifier>ISSN: 0027-8424</identifier><identifier>EISSN: 1091-6490</identifier><identifier>DOI: 10.1073/pnas.92.18.8428</identifier><identifier>PMID: 7545302</identifier><language>eng</language><publisher>United States: National Academy of Sciences of the United States of America</publisher><subject>Amino Acid Oxidoreductases - genetics ; Animals ; Arginine - analogs & derivatives ; Arginine - metabolism ; Bacteria ; Base Sequence ; Biochemistry ; Cell Line ; Chaperonin 10 - genetics ; Chaperonin 60 - genetics ; Chaperonins ; Cloning, Molecular ; Cytochrome P-450 Enzyme System - genetics ; Cytochromes ; Deoxyribonucleic acid ; DNA ; DNA Primers ; DNA, Complementary ; Enzymes ; Escherichia coli ; Escherichia coli - genetics ; Hydrolysis ; Imidazoles ; Kidneys ; Male ; Molecular Sequence Data ; Neurons ; Neurons - enzymology ; Nitric Oxide Synthase ; Nitroarginine ; Oxides ; Plasmids ; Promoter Regions, Genetic ; Protein Binding ; Protein isoforms ; Proteins ; Pterins ; Pterins - metabolism ; Rats ; Recombinant Proteins - genetics ; Rodents ; Spectrum Analysis</subject><ispartof>Proceedings of the National Academy of Sciences - PNAS, 1995-08, Vol.92 (18), p.8428-8432</ispartof><rights>Copyright 1995 The National Academy of Sciences of the United States of America</rights><rights>Copyright National Academy of Sciences Aug 29, 1995</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c586t-f6225bd0e6284cb71034a50b9e4688ca7ffdfce9bb16091a045bdbcfb8232b0c3</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Uhttp://www.pnas.org/content/92/18.cover.gif</thumbnail><linktopdf>$$Uhttps://www.jstor.org/stable/pdf/2368071$$EPDF$$P50$$Gjstor$$H</linktopdf><linktohtml>$$Uhttps://www.jstor.org/stable/2368071$$EHTML$$P50$$Gjstor$$H</linktohtml><link.rule.ids>230,315,728,781,785,804,886,27929,27930,53796,53798,58022,58255</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/7545302$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Roman, Linda J.</creatorcontrib><creatorcontrib>Sheta, Essam A.</creatorcontrib><creatorcontrib>Martasek, Pavel</creatorcontrib><creatorcontrib>Gross, Steven S.</creatorcontrib><creatorcontrib>Liu, Qing</creatorcontrib><creatorcontrib>Bettie Sue Siler Masters</creatorcontrib><title>High-Level Expression of Functional Rat Neuronal Nitric Oxide Synthase in Escherichia coli</title><title>Proceedings of the National Academy of Sciences - PNAS</title><addtitle>Proc Natl Acad Sci U S A</addtitle><description>The neuronal nitric oxide synthase (nNOS) has been successfully overexpressed in Escherichia coli, with average yields of 125-150 nmol (20-24 mg) of enzyme per liter of cells. The cDNA for nNOS was subcloned into the pCW vector under the control of the tac promotor and was coexpressed with the chaperonins groEL and groES in the protease-deficient BL21 strain of E. coli. The enzyme produced is replete with heme and flavins and, after overnight incubation with tetrahydrobiopterin, contains 0.7 pmol of tetrahydrobiopterin per pmol of nNOS. nNOS is isolated as a predominantly high-spin heme protein and demonstrates spectral properties that are identical to those of nNOS isolated from stably transfected human kidney 293 cells. It binds Nω-nitroarginine dependent on the presence of bound tetrahy-drobiopterin and exhibits a Kdof 45 nM. The enzyme is completely functional; the specific activity is 450 nmol/min per mg. This overexpression system will be extremely useful for rapid, inexpensive preparation of large amounts of active nNOS for use in mechanistic and structure/function studies, as well as for drug design and development.</description><subject>Amino Acid Oxidoreductases - genetics</subject><subject>Animals</subject><subject>Arginine - analogs & derivatives</subject><subject>Arginine - metabolism</subject><subject>Bacteria</subject><subject>Base Sequence</subject><subject>Biochemistry</subject><subject>Cell Line</subject><subject>Chaperonin 10 - genetics</subject><subject>Chaperonin 60 - genetics</subject><subject>Chaperonins</subject><subject>Cloning, Molecular</subject><subject>Cytochrome P-450 Enzyme System - genetics</subject><subject>Cytochromes</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>DNA Primers</subject><subject>DNA, Complementary</subject><subject>Enzymes</subject><subject>Escherichia coli</subject><subject>Escherichia coli - genetics</subject><subject>Hydrolysis</subject><subject>Imidazoles</subject><subject>Kidneys</subject><subject>Male</subject><subject>Molecular Sequence Data</subject><subject>Neurons</subject><subject>Neurons - enzymology</subject><subject>Nitric Oxide Synthase</subject><subject>Nitroarginine</subject><subject>Oxides</subject><subject>Plasmids</subject><subject>Promoter Regions, Genetic</subject><subject>Protein Binding</subject><subject>Protein isoforms</subject><subject>Proteins</subject><subject>Pterins</subject><subject>Pterins - metabolism</subject><subject>Rats</subject><subject>Recombinant Proteins - genetics</subject><subject>Rodents</subject><subject>Spectrum Analysis</subject><issn>0027-8424</issn><issn>1091-6490</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1995</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkc9vFCEUx4nR1LV69qKGeNDTbB8MM0DixTRba7JpE39cvBCGZTpsZoctMM32v5dx1431oFzg8f18X3h8EXpJYE6Al2fbQce5pHMi5oJR8QjNCEhS1EzCYzQDoLzI9-wpehbjGgBkJeAEnfCKVSXQGfpx6W66YmnvbI8Xu22wMTo_YN_ii3EwKZ91j7_ohK_sGH4VVy4FZ_D1zq0s_no_pE5Hi92AF9F0Nkud09j43j1HT1rdR_visJ-i7xeLb-eXxfL60-fzj8vCVKJORVtTWjUrsDUVzDScQMl0BY20rBbCaN62q9ZY2TSkzqNpYJluTNsIWtIGTHmKPuz7bsdmY1fGDinoXm2D2-hwr7x26qEyuE7d-DvFCOGQ7e8O9uBvRxuT2rhobN_rwfoxKs6ZlGVe_wNJLTgDOnV8-xe49mPIfxcVBUJlDZxm6GwPmeBjDLY9PpiAmqJVU7RKUkWEmqLNjtd_znnkD1lm_c1Bn4y_1QcN3v8TUO3Y98nuUiZf7cl1TD4cUVrWAjgpfwLd2cI3</recordid><startdate>19950829</startdate><enddate>19950829</enddate><creator>Roman, Linda J.</creator><creator>Sheta, Essam A.</creator><creator>Martasek, Pavel</creator><creator>Gross, Steven S.</creator><creator>Liu, Qing</creator><creator>Bettie Sue Siler Masters</creator><general>National Academy of Sciences of the United States of America</general><general>National Acad Sciences</general><general>National Academy of Sciences</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>7QG</scope><scope>7QL</scope><scope>7QP</scope><scope>7QR</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TK</scope><scope>7TM</scope><scope>7TO</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>19950829</creationdate><title>High-Level Expression of Functional Rat Neuronal Nitric Oxide Synthase in Escherichia coli</title><author>Roman, Linda J. ; 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The cDNA for nNOS was subcloned into the pCW vector under the control of the tac promotor and was coexpressed with the chaperonins groEL and groES in the protease-deficient BL21 strain of E. coli. The enzyme produced is replete with heme and flavins and, after overnight incubation with tetrahydrobiopterin, contains 0.7 pmol of tetrahydrobiopterin per pmol of nNOS. nNOS is isolated as a predominantly high-spin heme protein and demonstrates spectral properties that are identical to those of nNOS isolated from stably transfected human kidney 293 cells. It binds Nω-nitroarginine dependent on the presence of bound tetrahy-drobiopterin and exhibits a Kdof 45 nM. The enzyme is completely functional; the specific activity is 450 nmol/min per mg. This overexpression system will be extremely useful for rapid, inexpensive preparation of large amounts of active nNOS for use in mechanistic and structure/function studies, as well as for drug design and development.</abstract><cop>United States</cop><pub>National Academy of Sciences of the United States of America</pub><pmid>7545302</pmid><doi>10.1073/pnas.92.18.8428</doi><tpages>5</tpages><oa>free_for_read</oa></addata></record> |
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| source | MEDLINE; JSTOR Archive Collection A-Z Listing; PubMed Central; Alma/SFX Local Collection; Free Full-Text Journals in Chemistry |
| subjects | Amino Acid Oxidoreductases - genetics Animals Arginine - analogs & derivatives Arginine - metabolism Bacteria Base Sequence Biochemistry Cell Line Chaperonin 10 - genetics Chaperonin 60 - genetics Chaperonins Cloning, Molecular Cytochrome P-450 Enzyme System - genetics Cytochromes Deoxyribonucleic acid DNA DNA Primers DNA, Complementary Enzymes Escherichia coli Escherichia coli - genetics Hydrolysis Imidazoles Kidneys Male Molecular Sequence Data Neurons Neurons - enzymology Nitric Oxide Synthase Nitroarginine Oxides Plasmids Promoter Regions, Genetic Protein Binding Protein isoforms Proteins Pterins Pterins - metabolism Rats Recombinant Proteins - genetics Rodents Spectrum Analysis |
| title | High-Level Expression of Functional Rat Neuronal Nitric Oxide Synthase in Escherichia coli |
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