Cloning, expression, and characterization of DNA polymerase I from the hyperthermophilic archaea Thermococcus fumicolans
The DNA polymerase I gene of a newly described deep-sea hydrothermal vent Archaea species, Thermococcus fumicolans, from IFREMERS's collection of hyperthermophiles has been cloned in Escherichia coli. As in Thermococcus litoralis, the gene is split by two intervening sequences (IVS) encoding in...
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| Veröffentlicht in: | Extremophiles : life under extreme conditions 2000-08, Vol.4 (4), p.215-225 |
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| creator | CAMBON-BONAVITA, M.-A SCHMITT, P BARBIER, G QUERELLOU, J ZIEGER, M FLAMAN, J.-M LESONGEUR, F RAGUENES, G BINDEL, D FRISCH, N LAKKIS, Z DUPRET, D |
| description | The DNA polymerase I gene of a newly described deep-sea hydrothermal vent Archaea species, Thermococcus fumicolans, from IFREMERS's collection of hyperthermophiles has been cloned in Escherichia coli. As in Thermococcus litoralis, the gene is split by two intervening sequences (IVS) encoding inteins inserted in sites A and C of family B DNA polymerases. The entire DNA polymerase gene, containing both inteins, was expressed at 30 degrees C in E. coli strain BL21(DE3)pLysS using the pARHS2 expression vector. The native polypeptide precursor of 170kDa was obtained, and intein splicing as well as ligation of the three exteins was observed in vitro after heat exposure. The recombinant enzyme was purified and some of its activities were characterized: polymerization, thermostability, exonuclease activities, and fidelity. |
| doi_str_mv | 10.1007/PL00010714 |
| format | Article |
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The recombinant enzyme was purified and some of its activities were characterized: polymerization, thermostability, exonuclease activities, and fidelity.</description><identifier>ISSN: 1431-0651</identifier><identifier>EISSN: 1433-4909</identifier><identifier>DOI: 10.1007/PL00010714</identifier><identifier>PMID: 10972190</identifier><language>eng</language><publisher>Heidelberg: Springer</publisher><subject>Bacteriology ; Biological and medical sciences ; Cloning, Molecular ; DNA Polymerase I - genetics ; DNA Polymerase I - isolation & purification ; DNA Polymerase I - metabolism ; Enzyme Stability ; Escherichia coli ; Exonucleases - genetics ; Exonucleases - isolation & purification ; Exonucleases - metabolism ; Fundamental and applied biological sciences. Psychology ; Genetics ; Magnesium - pharmacology ; Microbiology ; Polymerase Chain Reaction ; Protein Splicing ; Recombinant Proteins - isolation & purification ; Recombinant Proteins - metabolism ; Sequence Analysis, DNA ; Space life sciences ; Temperature ; Thermococcus - enzymology ; Thermococcus - genetics</subject><ispartof>Extremophiles : life under extreme conditions, 2000-08, Vol.4 (4), p.215-225</ispartof><rights>2000 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c314t-91853ed5825d5c1fc988b8fec0c1d7b3b6b7554042cb642db055f9c1f27852c83</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780,27903,27904</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=1540677$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/10972190$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>CAMBON-BONAVITA, M.-A</creatorcontrib><creatorcontrib>SCHMITT, P</creatorcontrib><creatorcontrib>BARBIER, G</creatorcontrib><creatorcontrib>QUERELLOU, J</creatorcontrib><creatorcontrib>ZIEGER, M</creatorcontrib><creatorcontrib>FLAMAN, J.-M</creatorcontrib><creatorcontrib>LESONGEUR, F</creatorcontrib><creatorcontrib>RAGUENES, G</creatorcontrib><creatorcontrib>BINDEL, D</creatorcontrib><creatorcontrib>FRISCH, N</creatorcontrib><creatorcontrib>LAKKIS, Z</creatorcontrib><creatorcontrib>DUPRET, D</creatorcontrib><title>Cloning, expression, and characterization of DNA polymerase I from the hyperthermophilic archaea Thermococcus fumicolans</title><title>Extremophiles : life under extreme conditions</title><addtitle>Extremophiles</addtitle><description>The DNA polymerase I gene of a newly described deep-sea hydrothermal vent Archaea species, Thermococcus fumicolans, from IFREMERS's collection of hyperthermophiles has been cloned in Escherichia coli. As in Thermococcus litoralis, the gene is split by two intervening sequences (IVS) encoding inteins inserted in sites A and C of family B DNA polymerases. The entire DNA polymerase gene, containing both inteins, was expressed at 30 degrees C in E. coli strain BL21(DE3)pLysS using the pARHS2 expression vector. The native polypeptide precursor of 170kDa was obtained, and intein splicing as well as ligation of the three exteins was observed in vitro after heat exposure. The recombinant enzyme was purified and some of its activities were characterized: polymerization, thermostability, exonuclease activities, and fidelity.</description><subject>Bacteriology</subject><subject>Biological and medical sciences</subject><subject>Cloning, Molecular</subject><subject>DNA Polymerase I - genetics</subject><subject>DNA Polymerase I - isolation & purification</subject><subject>DNA Polymerase I - metabolism</subject><subject>Enzyme Stability</subject><subject>Escherichia coli</subject><subject>Exonucleases - genetics</subject><subject>Exonucleases - isolation & purification</subject><subject>Exonucleases - metabolism</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Genetics</subject><subject>Magnesium - pharmacology</subject><subject>Microbiology</subject><subject>Polymerase Chain Reaction</subject><subject>Protein Splicing</subject><subject>Recombinant Proteins - isolation & purification</subject><subject>Recombinant Proteins - metabolism</subject><subject>Sequence Analysis, DNA</subject><subject>Space life sciences</subject><subject>Temperature</subject><subject>Thermococcus - enzymology</subject><subject>Thermococcus - genetics</subject><issn>1431-0651</issn><issn>1433-4909</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2000</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpN0E9LwzAYBvAgipvTix9AchAPsmqSNkt7HPPfYKiHeS7p28RF2qYmLWx-euM2mKe8vPnx8PIgdEnJHSVE3L8vCCGUCJocoSFN4jhKMpIdb2cakQmnA3Tm_VdAPHycogElmWA0I0O0nlW2Mc3nGKt165T3xjZjLJsSw0o6CZ1y5kd2YYutxg-vU9zaalMrJ73Cc6ydrXG3Uni1aZULg6ttuzKVASxdSFASL7dLsAC9x7qvDdhKNv4cnWhZeXWxf0fo4-lxOXuJFm_P89l0EUFMky7KaMpjVfKU8ZID1ZClaZFqBQRoKYq4mBSC84QkDIpJwsqCcK6zAJlIOYM0HqGbXW7r7HevfJfXxoOqwg3K9j4XjLE0plmAtzsIznrvlM5bZ2rpNjkl-V_P-aHngK_2qX1Rq_If3RUbwPUeSA-y0k42YPzBhZMnQsS_wVyF9A</recordid><startdate>20000801</startdate><enddate>20000801</enddate><creator>CAMBON-BONAVITA, M.-A</creator><creator>SCHMITT, P</creator><creator>BARBIER, G</creator><creator>QUERELLOU, J</creator><creator>ZIEGER, M</creator><creator>FLAMAN, J.-M</creator><creator>LESONGEUR, F</creator><creator>RAGUENES, G</creator><creator>BINDEL, D</creator><creator>FRISCH, N</creator><creator>LAKKIS, Z</creator><creator>DUPRET, D</creator><general>Springer</general><scope>IQODW</scope><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>20000801</creationdate><title>Cloning, expression, and characterization of DNA polymerase I from the hyperthermophilic archaea Thermococcus fumicolans</title><author>CAMBON-BONAVITA, M.-A ; SCHMITT, P ; BARBIER, G ; QUERELLOU, J ; ZIEGER, M ; FLAMAN, J.-M ; LESONGEUR, F ; RAGUENES, G ; BINDEL, D ; FRISCH, N ; LAKKIS, Z ; DUPRET, D</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c314t-91853ed5825d5c1fc988b8fec0c1d7b3b6b7554042cb642db055f9c1f27852c83</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2000</creationdate><topic>Bacteriology</topic><topic>Biological and medical sciences</topic><topic>Cloning, Molecular</topic><topic>DNA Polymerase I - genetics</topic><topic>DNA Polymerase I - isolation & purification</topic><topic>DNA Polymerase I - metabolism</topic><topic>Enzyme Stability</topic><topic>Escherichia coli</topic><topic>Exonucleases - genetics</topic><topic>Exonucleases - isolation & purification</topic><topic>Exonucleases - metabolism</topic><topic>Fundamental and applied biological sciences. 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As in Thermococcus litoralis, the gene is split by two intervening sequences (IVS) encoding inteins inserted in sites A and C of family B DNA polymerases. The entire DNA polymerase gene, containing both inteins, was expressed at 30 degrees C in E. coli strain BL21(DE3)pLysS using the pARHS2 expression vector. The native polypeptide precursor of 170kDa was obtained, and intein splicing as well as ligation of the three exteins was observed in vitro after heat exposure. The recombinant enzyme was purified and some of its activities were characterized: polymerization, thermostability, exonuclease activities, and fidelity.</abstract><cop>Heidelberg</cop><cop>Tokyo</cop><pub>Springer</pub><pmid>10972190</pmid><doi>10.1007/PL00010714</doi><tpages>11</tpages></addata></record> |
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| subjects | Bacteriology Biological and medical sciences Cloning, Molecular DNA Polymerase I - genetics DNA Polymerase I - isolation & purification DNA Polymerase I - metabolism Enzyme Stability Escherichia coli Exonucleases - genetics Exonucleases - isolation & purification Exonucleases - metabolism Fundamental and applied biological sciences. Psychology Genetics Magnesium - pharmacology Microbiology Polymerase Chain Reaction Protein Splicing Recombinant Proteins - isolation & purification Recombinant Proteins - metabolism Sequence Analysis, DNA Space life sciences Temperature Thermococcus - enzymology Thermococcus - genetics |
| title | Cloning, expression, and characterization of DNA polymerase I from the hyperthermophilic archaea Thermococcus fumicolans |
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