Purification and Characterization of T7 Head-Tail Connectors Expressed from the Cloned Gene
Identifieur interne : 000111 ( Istex/Corpus ); précédent : 000110; suivant : 000112Purification and Characterization of T7 Head-Tail Connectors Expressed from the Cloned Gene
Auteurs : Mario E. Cerritelli ; William F. StudierSource :
- Journal of Molecular Biology [ 0022-2836 ] ; 1996.
English descriptors
- KwdEn :
- Teeft :
- Academic press, Agarose, Atpase activity, Bacteriophage, Biol, Capsid, Carrascosa, Cerritelli, Cerritelli studier, Connector, Connector preparation, Connector preparations, Connector protein, Connector protein eluting, Connectors bind, Connectors figure, Deae column, Electron microscopy, Ethidium bromide, Hydroxylapatite, Hydroxylapatite chromatography, Hydroxylapatite column, Kocsis, Large aggregates, Linear gradient, Mass analysis, Mature form, Monomer, Monomer peak, Nacl, Owthrough, Packaging process, Peak fractions, Phage, Phosphocellulose, Phosphocellulose column, Phosphocellulose columns, Portal protein, Proteinase, Rotational symmetry analysis, Same position, Scanning transmission electron microscopy, Single peak, Small amount, Sodium dodecyl sulfate, Stable connectors, Studier, Supercoiled, Superose, Uorescence.
Abstract
Abstract: Cloned gene8, which specifies the protein of the head – tail connector of bacteriophage T7, was expressed inEscherichia coli. Extracts prepared in a low-salt buffer gave rise to free monomers, assembled connectors, and various complexes and aggregates. Connectors isolated as single peaks from DEAE-Sepharose and phosphocellulose chromatography gave separate peaks of monomers and stable connectors upon hydroxylapatite chromatography, perhaps because of dissociation of monomer – connector complexes or disassembly of unstable connectors. Electron microscopy showed that the connectors readily formed ordered arrays after hydroxylapatite chromatography, but not before. Addition of 100 mM NaCl to the buffer used to prepare extracts eliminated most complexes and aggregates and gave rise almost entirely to monomers and stable connectors that formed arrays even before hydroxylapatite chromatography. The distribution of masses determined by scanning transmission electron microscopy would be consistent with a mixed population of stable connectors containing 12 or 13 monomers, and the same preparation gave two bands upon agarose gel electrophoresis. Connectors bound linear, circular and supercoiled DNA, whereas monomers did not, as determined by a gel-shift assay. No ATPase activity was detected in either monomer or connector preparations in the absence or presence of DNA.
Url:
DOI: 10.1006/jmbi.1996.0251
Links to Exploration step
ISTEX:4A47E338F554C99963754D365DC06EAA6E53C305Le document en format XML
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Extracts prepared in a low-salt buffer gave rise to free monomers, assembled connectors, and various complexes and aggregates. Connectors isolated as single peaks from DEAE-Sepharose and phosphocellulose chromatography gave separate peaks of monomers and stable connectors upon hydroxylapatite chromatography, perhaps because of dissociation of monomer – connector complexes or disassembly of unstable connectors. Electron microscopy showed that the connectors readily formed ordered arrays after hydroxylapatite chromatography, but not before. Addition of 100 mM NaCl to the buffer used to prepare extracts eliminated most complexes and aggregates and gave rise almost entirely to monomers and stable connectors that formed arrays even before hydroxylapatite chromatography. The distribution of masses determined by scanning transmission electron microscopy would be consistent with a mixed population of stable connectors containing 12 or 13 monomers, and the same preparation gave two bands upon agarose gel electrophoresis. Connectors bound linear, circular and supercoiled DNA, whereas monomers did not, as determined by a gel-shift assay. 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Extracts prepared in a low-salt buffer gave rise to free monomers, assembled connectors, and various complexes and aggregates. Connectors isolated as single peaks from DEAE-Sepharose and phosphocellulose chromatography gave separate peaks of monomers and stable connectors upon hydroxylapatite chromatography, perhaps because of dissociation of monomer – connector complexes or disassembly of unstable connectors. Electron microscopy showed that the connectors readily formed ordered arrays after hydroxylapatite chromatography, but not before. Addition of 100 mM NaCl to the buffer used to prepare extracts eliminated most complexes and aggregates and gave rise almost entirely to monomers and stable connectors that formed arrays even before hydroxylapatite chromatography. The distribution of masses determined by scanning transmission electron microscopy would be consistent with a mixed population of stable connectors containing 12 or 13 monomers, and the same preparation gave two bands upon agarose gel electrophoresis. Connectors bound linear, circular and supercoiled DNA, whereas monomers did not, as determined by a gel-shift assay. 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E. Cerritelli, Laboratory of Structural Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, MD 20892-2755, USA.</note><affiliation>Present address: M. E. Cerritelli, Laboratory of Structural Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, MD 20892-2755, USA.</affiliation><affiliation>Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA</affiliation></author><author xml:id="author-0001"><persName><forename type="first">William F.</forename><surname>Studier</surname></persName><note type="correspondence"><p>Corresponding author</p></note><affiliation>Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA</affiliation></author><idno type="istex">4A47E338F554C99963754D365DC06EAA6E53C305</idno><idno type="ark">ark:/67375/6H6-9G4RH44R-P</idno><idno type="DOI">10.1006/jmbi.1996.0251</idno><idno type="PII">S0022-2836(96)90251-X</idno></analytic><monogr><title level="j">Journal of Molecular Biology</title><title level="j" type="abbrev">YJMBI</title><idno type="pISSN">0022-2836</idno><idno type="PII">S0022-2836(00)X0062-9</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1996"></date><biblScope unit="volume">258</biblScope><biblScope unit="issue">2</biblScope><biblScope unit="page" from="299">299</biblScope><biblScope unit="page" to="307">307</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>1996</date></creation><langUsage><language ident="en">en</language></langUsage><abstract xml:lang="en"><p>Abstract: Cloned gene8, which specifies the protein of the head – tail connector of bacteriophage T7, was expressed inEscherichia coli. Extracts prepared in a low-salt buffer gave rise to free monomers, assembled connectors, and various complexes and aggregates. Connectors isolated as single peaks from DEAE-Sepharose and phosphocellulose chromatography gave separate peaks of monomers and stable connectors upon hydroxylapatite chromatography, perhaps because of dissociation of monomer – connector complexes or disassembly of unstable connectors. Electron microscopy showed that the connectors readily formed ordered arrays after hydroxylapatite chromatography, but not before. Addition of 100 mM NaCl to the buffer used to prepare extracts eliminated most complexes and aggregates and gave rise almost entirely to monomers and stable connectors that formed arrays even before hydroxylapatite chromatography. The distribution of masses determined by scanning transmission electron microscopy would be consistent with a mixed population of stable connectors containing 12 or 13 monomers, and the same preparation gave two bands upon agarose gel electrophoresis. Connectors bound linear, circular and supercoiled DNA, whereas monomers did not, as determined by a gel-shift assay. No ATPase activity was detected in either monomer or connector preparations in the absence or presence of DNA.</p></abstract><textClass xml:lang="en"><keywords scheme="keyword"><list><head>Keywords</head><item><term>bacteriophage T7</term></item><item><term>head–tail connector</term></item><item><term>portal protein</term></item><item><term>purification</term></item><item><term>DNA binding</term></item></list></keywords></textClass></profileDesc><revisionDesc><change when="1996-01-11">Modified</change><change when="1996">Published</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-9G4RH44R-P/fulltext.txt</uri></json:item></fulltext><metadata><istex:metadataXml wicri:clean="Elsevier converted-article found"><istex:xmlDeclaration>version="1.0" encoding="utf-8"</istex:xmlDeclaration><istex:docType PUBLIC="-//ES//DTD journal article DTD version 4.5.2//EN//XML" URI="art452.dtd" name="istex:docType"></istex:docType><istex:document><converted-article version="4.5.2" docsubtype="fla" xml:lang="en"><item-info><jid>YJMBI</jid><aid>90251</aid><ce:pii>S0022-2836(96)90251-X</ce:pii><ce:doi>10.1006/jmbi.1996.0251</ce:doi><ce:copyright type="full-transfer" year="1996">Academic Press</ce:copyright></item-info><head><ce:dochead><ce:textfn>Regular article</ce:textfn></ce:dochead><ce:title>Purification and Characterization of T7 Head-Tail Connectors Expressed from the Cloned Gene</ce:title><ce:author-group><ce:author><ce:given-name>Mario E.</ce:given-name><ce:surname>Cerritelli</ce:surname><ce:cross-ref refid="A1"><ce:sup>a</ce:sup></ce:cross-ref><ce:cross-ref refid="FN1"><ce:sup>f1</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>William F.</ce:given-name><ce:surname>Studier</ce:surname><ce:cross-ref refid="A1"><ce:sup>a</ce:sup></ce:cross-ref><ce:cross-ref refid="FN2">*</ce:cross-ref></ce:author><ce:affiliation id="A1"><ce:label>a</ce:label><ce:textfn>Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA</ce:textfn></ce:affiliation><ce:footnote id="FN1"><ce:label>f1</ce:label><ce:note-para>Present address: M. E. Cerritelli, Laboratory of Structural Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, MD 20892-2755, USA.</ce:note-para></ce:footnote><ce:footnote id="FN2"><ce:label>f2</ce:label><ce:note-para>Corresponding author</ce:note-para></ce:footnote></ce:author-group><ce:date-received day="19" month="9" year="1995"></ce:date-received><ce:date-revised day="11" month="1" year="1996"></ce:date-revised><ce:date-accepted day="22" month="1" year="1996"></ce:date-accepted><ce:abstract><ce:section-title>Abstract</ce:section-title><ce:abstract-sec><ce:simple-para>Cloned gene<ce:italic>8</ce:italic>, which specifies the protein of the head – tail connector of bacteriophage T7, was expressed in<ce:italic>Escherichia coli</ce:italic>. Extracts prepared in a low-salt buffer gave rise to free monomers, assembled connectors, and various complexes and aggregates. Connectors isolated as single peaks from DEAE-Sepharose and phosphocellulose chromatography gave separate peaks of monomers and stable connectors upon hydroxylapatite chromatography, perhaps because of dissociation of monomer – connector complexes or disassembly of unstable connectors. Electron microscopy showed that the connectors readily formed ordered arrays after hydroxylapatite chromatography, but not before. Addition of 100 mM NaCl to the buffer used to prepare extracts eliminated most complexes and aggregates and gave rise almost entirely to monomers and stable connectors that formed arrays even before hydroxylapatite chromatography. The distribution of masses determined by scanning transmission electron microscopy would be consistent with a mixed population of stable connectors containing 12 or 13 monomers, and the same preparation gave two bands upon agarose gel electrophoresis. Connectors bound linear, circular and supercoiled DNA, whereas monomers did not, as determined by a gel-shift assay. No ATPase activity was detected in either monomer or connector preparations in the absence or presence of DNA.</ce:simple-para></ce:abstract-sec></ce:abstract><ce:keywords><ce:section-title>Keywords</ce:section-title><ce:keyword><ce:text>bacteriophage T7</ce:text></ce:keyword><ce:keyword><ce:text>head–tail connector</ce:text></ce:keyword><ce:keyword><ce:text>portal protein</ce:text></ce:keyword><ce:keyword><ce:text>purification</ce:text></ce:keyword><ce:keyword><ce:text>DNA binding</ce:text></ce:keyword></ce:keywords></head></converted-article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo lang="en"><title>Purification and Characterization of T7 Head-Tail Connectors Expressed from the Cloned Gene</title></titleInfo><titleInfo type="alternative" lang="en" contentType="CDATA"><title>Purification and Characterization of T7 Head-Tail Connectors Expressed from the Cloned Gene</title></titleInfo><name type="personal"><namePart type="given">Mario E.</namePart><namePart type="family">Cerritelli</namePart><affiliation>Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA</affiliation><description>Present address: M. E. Cerritelli, Laboratory of Structural Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, MD 20892-2755, USA.</description><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">William F.</namePart><namePart type="family">Studier</namePart><affiliation>Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA</affiliation><description>Corresponding author</description><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="Full-length article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">1996</dateIssued><dateModified encoding="w3cdtf">1996-01-11</dateModified><copyrightDate encoding="w3cdtf">1996</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract lang="en">Abstract: Cloned gene8, which specifies the protein of the head – tail connector of bacteriophage T7, was expressed inEscherichia coli. Extracts prepared in a low-salt buffer gave rise to free monomers, assembled connectors, and various complexes and aggregates. Connectors isolated as single peaks from DEAE-Sepharose and phosphocellulose chromatography gave separate peaks of monomers and stable connectors upon hydroxylapatite chromatography, perhaps because of dissociation of monomer – connector complexes or disassembly of unstable connectors. Electron microscopy showed that the connectors readily formed ordered arrays after hydroxylapatite chromatography, but not before. Addition of 100 mM NaCl to the buffer used to prepare extracts eliminated most complexes and aggregates and gave rise almost entirely to monomers and stable connectors that formed arrays even before hydroxylapatite chromatography. The distribution of masses determined by scanning transmission electron microscopy would be consistent with a mixed population of stable connectors containing 12 or 13 monomers, and the same preparation gave two bands upon agarose gel electrophoresis. Connectors bound linear, circular and supercoiled DNA, whereas monomers did not, as determined by a gel-shift assay. No ATPase activity was detected in either monomer or connector preparations in the absence or presence of DNA.</abstract><note type="content">Section title: Regular article</note><subject lang="en"><genre>Keywords</genre><topic>bacteriophage T7</topic><topic>head–tail connector</topic><topic>portal protein</topic><topic>purification</topic><topic>DNA binding</topic></subject><relatedItem type="host"><titleInfo><title>Journal of Molecular Biology</title></titleInfo><titleInfo type="abbreviated"><title>YJMBI</title></titleInfo><genre type="journal" authority="ISTEX" authorityURI="https://publication-type.data.istex.fr" valueURI="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">1996</dateIssued></originInfo><identifier type="ISSN">0022-2836</identifier><identifier type="PII">S0022-2836(00)X0062-9</identifier><part><date>1996</date><detail type="volume"><number>258</number><caption>vol.</caption></detail><detail type="issue"><number>2</number><caption>no.</caption></detail><extent unit="issue-pages"><start>213</start><end>410</end></extent><extent unit="pages"><start>299</start><end>307</end></extent></part></relatedItem><identifier type="istex">4A47E338F554C99963754D365DC06EAA6E53C305</identifier><identifier type="ark">ark:/67375/6H6-9G4RH44R-P</identifier><identifier type="DOI">10.1006/jmbi.1996.0251</identifier><identifier type="PII">S0022-2836(96)90251-X</identifier><accessCondition type="use and reproduction" contentType="copyright">©1996 Academic Press</accessCondition><recordInfo><recordContentSource authority="ISTEX" authorityURI="https://loaded-corpus.data.istex.fr" valueURI="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M">elsevier</recordContentSource><recordOrigin>Academic Press, ©1996</recordOrigin></recordInfo></mods><json:item><extension>json</extension><original>false</original><mimetype>application/json</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-9G4RH44R-P/record.json</uri></json:item></metadata><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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