Purification and Renaturation of Japanese Encephalitis Virus Nonstructural Glycoprotein NS1 Overproduced by Insect Cells
Identifieur interne : 002123 ( Istex/Corpus ); précédent : 002122; suivant : 002124Purification and Renaturation of Japanese Encephalitis Virus Nonstructural Glycoprotein NS1 Overproduced by Insect Cells
Auteurs : M. Flamand ; M. Chevalier ; E. Henchal ; M. Girard ; V. DeubelSource :
- Protein Expression and Purification [ 1046-5928 ] ; 1995.
Abstract
Abstract: The nonstructural protein NS1 of Japanese encephalitis virus is a major immunogen produced during flavivirus infection. However, the function of this protein has not been identified. To analyze its biochemical properties and evaluate its potential activity in the virus life cycle, the protein was produced in Spodoptera frugiperda insect cells (Sf9), using a recombinant baculovirus, and purified. As described previously by M. Flamand, V. Deubel, and M. Girard (1992, Virology 191, 826-836), a small fraction of the synthesized recombinant protein could mature into a dimer, whereas the major part was retained in intracellular aggregates. This insolubility was used to recover the protein in a purified form using a two-step procedure. Isolated inclusion bodies, in which NS1 constituted over 60% of the protein, were solubilized in 8 M urea. NS1 was further purified by reverse-phase HPLC and recovered at over 90% purity with an overall yield of over 60%. Conditions promoting reoxidation-renaturation of the purified protein were then investigated at a concentration of 100 μg/ml at pH 8. The presence of 8 M urea during reoxidation of NS1 with oxidized glutathione was essential prior to renaturation by dialysis to avoid reaggregation, the main side pathway of refolding in vitro. Three major species, all monomeric, were resolved by nonreducing SDS-PAGE. The form showing the lowest apparent molecular weight comigrated with native unreduced NS1 and was recognized by a monoclonal antibody directed against a conformational epitope strictly dependent on the native structure of the protein. Thus, this form, representing over 30% of the renatured products, may have reached the native conformation of the protein. This simple renaturation-reoxidation procedure may be applicable to other disulfide bond-containing proteins to be recovered in the native state from inclusion bodies.
Url:
DOI: 10.1006/prep.1995.1069
Links to Exploration step
ISTEX:1144D979B71F7B6DD58FE9EBEDAB25D90DB38DDBLe document en format XML
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Henchal</name><affiliation><mods:affiliation>Inst Pasteur, Unite Arbovirus & Virus Fievres Hemorrag, 25 Rue Dr Roux, F 75015 Paris, France; Pasteur Merieux Serums & Vaccins, F 69280 Marcy Letoile, France; USA, Med Res Inst Infect Dis, Div Virol, Dept Molec Virol, Frederick, MD 21702, USA and Inst Pasteur, Unite Virol Molec, F 75015 Paris, France</mods:affiliation></affiliation></author><author><name sortKey="Girard, M" sort="Girard, M" uniqKey="Girard M" first="M." last="Girard">M. Girard</name><affiliation><mods:affiliation>Inst Pasteur, Unite Arbovirus & Virus Fievres Hemorrag, 25 Rue Dr Roux, F 75015 Paris, France; Pasteur Merieux Serums & Vaccins, F 69280 Marcy Letoile, France; USA, Med Res Inst Infect Dis, Div Virol, Dept Molec Virol, Frederick, MD 21702, USA and Inst Pasteur, Unite Virol Molec, F 75015 Paris, France</mods:affiliation></affiliation></author><author><name sortKey="Deubel, V" sort="Deubel, V" uniqKey="Deubel V" first="V." last="Deubel">V. Deubel</name><affiliation><mods:affiliation>Inst Pasteur, Unite Arbovirus & Virus Fievres Hemorrag, 25 Rue Dr Roux, F 75015 Paris, France; Pasteur Merieux Serums & Vaccins, F 69280 Marcy Letoile, France; USA, Med Res Inst Infect Dis, Div Virol, Dept Molec Virol, Frederick, MD 21702, USA and Inst Pasteur, Unite Virol Molec, F 75015 Paris, France</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j">Protein Expression and Purification</title><title level="j" type="abbrev">YPREP</title><idno type="ISSN">1046-5928</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1995">1995</date><biblScope unit="volume">6</biblScope><biblScope unit="issue">4</biblScope><biblScope unit="page" from="519">519</biblScope><biblScope unit="page" to="527">527</biblScope></imprint><idno type="ISSN">1046-5928</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">1046-5928</idno></seriesStmt></fileDesc><profileDesc><textClass></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Abstract: The nonstructural protein NS1 of Japanese encephalitis virus is a major immunogen produced during flavivirus infection. However, the function of this protein has not been identified. To analyze its biochemical properties and evaluate its potential activity in the virus life cycle, the protein was produced in Spodoptera frugiperda insect cells (Sf9), using a recombinant baculovirus, and purified. As described previously by M. Flamand, V. Deubel, and M. Girard (1992, Virology 191, 826-836), a small fraction of the synthesized recombinant protein could mature into a dimer, whereas the major part was retained in intracellular aggregates. This insolubility was used to recover the protein in a purified form using a two-step procedure. Isolated inclusion bodies, in which NS1 constituted over 60% of the protein, were solubilized in 8 M urea. NS1 was further purified by reverse-phase HPLC and recovered at over 90% purity with an overall yield of over 60%. Conditions promoting reoxidation-renaturation of the purified protein were then investigated at a concentration of 100 μg/ml at pH 8. The presence of 8 M urea during reoxidation of NS1 with oxidized glutathione was essential prior to renaturation by dialysis to avoid reaggregation, the main side pathway of refolding in vitro. Three major species, all monomeric, were resolved by nonreducing SDS-PAGE. The form showing the lowest apparent molecular weight comigrated with native unreduced NS1 and was recognized by a monoclonal antibody directed against a conformational epitope strictly dependent on the native structure of the protein. Thus, this form, representing over 30% of the renatured products, may have reached the native conformation of the protein. This simple renaturation-reoxidation procedure may be applicable to other disulfide bond-containing proteins to be recovered in the native state from inclusion bodies.</div></front></TEI><istex><corpusName>elsevier</corpusName><author><json:item><name>M. 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Deubel</name><affiliations><json:string>Inst Pasteur, Unite Arbovirus & Virus Fievres Hemorrag, 25 Rue Dr Roux, F 75015 Paris, France; Pasteur Merieux Serums & Vaccins, F 69280 Marcy Letoile, France; USA, Med Res Inst Infect Dis, Div Virol, Dept Molec Virol, Frederick, MD 21702, USA and Inst Pasteur, Unite Virol Molec, F 75015 Paris, France</json:string></affiliations></json:item></author><arkIstex>ark:/67375/6H6-F5L7QX2M-Q</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>Full-length article</json:string></originalGenre><abstract>Abstract: The nonstructural protein NS1 of Japanese encephalitis virus is a major immunogen produced during flavivirus infection. However, the function of this protein has not been identified. To analyze its biochemical properties and evaluate its potential activity in the virus life cycle, the protein was produced in Spodoptera frugiperda insect cells (Sf9), using a recombinant baculovirus, and purified. As described previously by M. Flamand, V. Deubel, and M. Girard (1992, Virology 191, 826-836), a small fraction of the synthesized recombinant protein could mature into a dimer, whereas the major part was retained in intracellular aggregates. This insolubility was used to recover the protein in a purified form using a two-step procedure. Isolated inclusion bodies, in which NS1 constituted over 60% of the protein, were solubilized in 8 M urea. NS1 was further purified by reverse-phase HPLC and recovered at over 90% purity with an overall yield of over 60%. Conditions promoting reoxidation-renaturation of the purified protein were then investigated at a concentration of 100 μg/ml at pH 8. The presence of 8 M urea during reoxidation of NS1 with oxidized glutathione was essential prior to renaturation by dialysis to avoid reaggregation, the main side pathway of refolding in vitro. Three major species, all monomeric, were resolved by nonreducing SDS-PAGE. The form showing the lowest apparent molecular weight comigrated with native unreduced NS1 and was recognized by a monoclonal antibody directed against a conformational epitope strictly dependent on the native structure of the protein. Thus, this form, representing over 30% of the renatured products, may have reached the native conformation of the protein. 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Pasteur Merieux Serums & Vaccins, F 69280 Marcy Letoile, France; USA, Med Res Inst Infect Dis, Div Virol, Dept Molec Virol, Frederick, MD 21702, USA and Inst Pasteur, Unite Virol Molec, F 75015 Paris, France</affiliation></author><author xml:id="author-0001"><persName><forename type="first">M.</forename><surname>Chevalier</surname></persName><affiliation>Inst Pasteur, Unite Arbovirus & Virus Fievres Hemorrag, 25 Rue Dr Roux, F 75015 Paris, France; Pasteur Merieux Serums & Vaccins, F 69280 Marcy Letoile, France; USA, Med Res Inst Infect Dis, Div Virol, Dept Molec Virol, Frederick, MD 21702, USA and Inst Pasteur, Unite Virol Molec, F 75015 Paris, France</affiliation></author><author xml:id="author-0002"><persName><forename type="first">E.</forename><surname>Henchal</surname></persName><affiliation>Inst Pasteur, Unite Arbovirus & Virus Fievres Hemorrag, 25 Rue Dr Roux, F 75015 Paris, France; Pasteur Merieux Serums & Vaccins, F 69280 Marcy Letoile, France; USA, Med Res Inst Infect Dis, Div Virol, Dept Molec Virol, Frederick, MD 21702, USA and Inst Pasteur, Unite Virol Molec, F 75015 Paris, France</affiliation></author><author xml:id="author-0003"><persName><forename type="first">M.</forename><surname>Girard</surname></persName><affiliation>Inst Pasteur, Unite Arbovirus & Virus Fievres Hemorrag, 25 Rue Dr Roux, F 75015 Paris, France; Pasteur Merieux Serums & Vaccins, F 69280 Marcy Letoile, France; USA, Med Res Inst Infect Dis, Div Virol, Dept Molec Virol, Frederick, MD 21702, USA and Inst Pasteur, Unite Virol Molec, F 75015 Paris, France</affiliation></author><author xml:id="author-0004"><persName><forename type="first">V.</forename><surname>Deubel</surname></persName><affiliation>Inst Pasteur, Unite Arbovirus & Virus Fievres Hemorrag, 25 Rue Dr Roux, F 75015 Paris, France; Pasteur Merieux Serums & Vaccins, F 69280 Marcy Letoile, France; USA, Med Res Inst Infect Dis, Div Virol, Dept Molec Virol, Frederick, MD 21702, USA and Inst Pasteur, Unite Virol Molec, F 75015 Paris, France</affiliation></author><idno type="istex">1144D979B71F7B6DD58FE9EBEDAB25D90DB38DDB</idno><idno type="ark">ark:/67375/6H6-F5L7QX2M-Q</idno><idno type="DOI">10.1006/prep.1995.1069</idno><idno type="PII">S1046-5928(85)71069-8</idno></analytic><monogr><title level="j">Protein Expression and Purification</title><title level="j" type="abbrev">YPREP</title><idno type="pISSN">1046-5928</idno><idno type="PII">S1046-5928(00)X0067-8</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1995"></date><biblScope unit="volume">6</biblScope><biblScope unit="issue">4</biblScope><biblScope unit="page" from="519">519</biblScope><biblScope unit="page" to="527">527</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>1995</date></creation><langUsage><language ident="en">en</language></langUsage><abstract xml:lang="en"><p>Abstract: The nonstructural protein NS1 of Japanese encephalitis virus is a major immunogen produced during flavivirus infection. However, the function of this protein has not been identified. To analyze its biochemical properties and evaluate its potential activity in the virus life cycle, the protein was produced in Spodoptera frugiperda insect cells (Sf9), using a recombinant baculovirus, and purified. As described previously by M. Flamand, V. Deubel, and M. Girard (1992, Virology 191, 826-836), a small fraction of the synthesized recombinant protein could mature into a dimer, whereas the major part was retained in intracellular aggregates. This insolubility was used to recover the protein in a purified form using a two-step procedure. Isolated inclusion bodies, in which NS1 constituted over 60% of the protein, were solubilized in 8 M urea. NS1 was further purified by reverse-phase HPLC and recovered at over 90% purity with an overall yield of over 60%. Conditions promoting reoxidation-renaturation of the purified protein were then investigated at a concentration of 100 μg/ml at pH 8. The presence of 8 M urea during reoxidation of NS1 with oxidized glutathione was essential prior to renaturation by dialysis to avoid reaggregation, the main side pathway of refolding in vitro. Three major species, all monomeric, were resolved by nonreducing SDS-PAGE. The form showing the lowest apparent molecular weight comigrated with native unreduced NS1 and was recognized by a monoclonal antibody directed against a conformational epitope strictly dependent on the native structure of the protein. Thus, this form, representing over 30% of the renatured products, may have reached the native conformation of the protein. This simple renaturation-reoxidation procedure may be applicable to other disulfide bond-containing proteins to be recovered in the native state from inclusion bodies.</p></abstract></profileDesc><revisionDesc><change when="1995">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-F5L7QX2M-Q/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>YPREP</jid><aid>71069</aid><ce:pii>S1046-5928(85)71069-8</ce:pii><ce:doi>10.1006/prep.1995.1069</ce:doi><ce:copyright type="full-transfer" year="1995">Academic Press</ce:copyright></item-info><head><ce:dochead><ce:textfn>Regular Article</ce:textfn></ce:dochead><ce:title>Purification and Renaturation of Japanese Encephalitis Virus Nonstructural Glycoprotein NS1 Overproduced by Insect Cells</ce:title><ce:author-group><ce:author><ce:surname>Flamand</ce:surname><ce:given-name>M.</ce:given-name></ce:author><ce:author><ce:surname>Chevalier</ce:surname><ce:given-name>M.</ce:given-name></ce:author><ce:author><ce:surname>Henchal</ce:surname><ce:given-name>E.</ce:given-name></ce:author><ce:author><ce:surname>Girard</ce:surname><ce:given-name>M.</ce:given-name></ce:author><ce:author><ce:surname>Deubel</ce:surname><ce:given-name>V.</ce:given-name></ce:author><ce:affiliation><ce:textfn>Inst Pasteur, Unite Arbovirus & Virus Fievres Hemorrag, 25 Rue Dr Roux, F 75015 Paris, France; Pasteur Merieux Serums & Vaccins, F 69280 Marcy Letoile, France; USA, Med Res Inst Infect Dis, Div Virol, Dept Molec Virol, Frederick, MD 21702, USA and Inst Pasteur, Unite Virol Molec, F 75015 Paris, France</ce:textfn></ce:affiliation></ce:author-group><ce:abstract><ce:section-title>Abstract</ce:section-title><ce:abstract-sec><ce:simple-para>The nonstructural protein NS1 of Japanese encephalitis virus is a major immunogen produced during flavivirus infection. However, the function of this protein has not been identified. To analyze its biochemical properties and evaluate its potential activity in the virus life cycle, the protein was produced in <ce:italic>Spodoptera frugiperda</ce:italic> insect cells (Sf9), using a recombinant baculovirus, and purified. As described previously by M. Flamand, V. Deubel, and M. Girard (1992, <ce:italic>Virology</ce:italic> 191, 826-836), a small fraction of the synthesized recombinant protein could mature into a dimer, whereas the major part was retained in intracellular aggregates. This insolubility was used to recover the protein in a purified form using a two-step procedure. Isolated inclusion bodies, in which NS1 constituted over 60% of the protein, were solubilized in 8 M urea. NS1 was further purified by reverse-phase HPLC and recovered at over 90% purity with an overall yield of over 60%. Conditions promoting reoxidation-renaturation of the purified protein were then investigated at a concentration of 100 μg/ml at pH 8. The presence of 8 M urea during reoxidation of NS1 with oxidized glutathione was essential prior to renaturation by dialysis to avoid reaggregation, the main side pathway of refolding <ce:italic>in vitro</ce:italic>. Three major species, all monomeric, were resolved by nonreducing SDS-PAGE. The form showing the lowest apparent molecular weight comigrated with native unreduced NS1 and was recognized by a monoclonal antibody directed against a conformational epitope strictly dependent on the native structure of the protein. Thus, this form, representing over 30% of the renatured products, may have reached the native conformation of the protein. This simple renaturation-reoxidation procedure may be applicable to other disulfide bond-containing proteins to be recovered in the native state from inclusion bodies.</ce:simple-para></ce:abstract-sec></ce:abstract></head></converted-article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo lang="en"><title>Purification and Renaturation of Japanese Encephalitis Virus Nonstructural Glycoprotein NS1 Overproduced by Insect Cells</title></titleInfo><titleInfo type="alternative" lang="en" contentType="CDATA"><title>Purification and Renaturation of Japanese Encephalitis Virus Nonstructural Glycoprotein NS1 Overproduced by Insect Cells</title></titleInfo><name type="personal"><namePart type="given">M.</namePart><namePart type="family">Flamand</namePart><affiliation>Inst Pasteur, Unite Arbovirus & Virus Fievres Hemorrag, 25 Rue Dr Roux, F 75015 Paris, France; Pasteur Merieux Serums & Vaccins, F 69280 Marcy Letoile, France; USA, Med Res Inst Infect Dis, Div Virol, Dept Molec Virol, Frederick, MD 21702, USA and Inst Pasteur, Unite Virol Molec, F 75015 Paris, France</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">M.</namePart><namePart type="family">Chevalier</namePart><affiliation>Inst Pasteur, Unite Arbovirus & Virus Fievres Hemorrag, 25 Rue Dr Roux, F 75015 Paris, France; Pasteur Merieux Serums & Vaccins, F 69280 Marcy Letoile, France; USA, Med Res Inst Infect Dis, Div Virol, Dept Molec Virol, Frederick, MD 21702, USA and Inst Pasteur, Unite Virol Molec, F 75015 Paris, France</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">E.</namePart><namePart type="family">Henchal</namePart><affiliation>Inst Pasteur, Unite Arbovirus & Virus Fievres Hemorrag, 25 Rue Dr Roux, F 75015 Paris, France; Pasteur Merieux Serums & Vaccins, F 69280 Marcy Letoile, France; USA, Med Res Inst Infect Dis, Div Virol, Dept Molec Virol, Frederick, MD 21702, USA and Inst Pasteur, Unite Virol Molec, F 75015 Paris, France</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">M.</namePart><namePart type="family">Girard</namePart><affiliation>Inst Pasteur, Unite Arbovirus & Virus Fievres Hemorrag, 25 Rue Dr Roux, F 75015 Paris, France; Pasteur Merieux Serums & Vaccins, F 69280 Marcy Letoile, France; USA, Med Res Inst Infect Dis, Div Virol, Dept Molec Virol, Frederick, MD 21702, USA and Inst Pasteur, Unite Virol Molec, F 75015 Paris, France</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">V.</namePart><namePart type="family">Deubel</namePart><affiliation>Inst Pasteur, Unite Arbovirus & Virus Fievres Hemorrag, 25 Rue Dr Roux, F 75015 Paris, France; Pasteur Merieux Serums & Vaccins, F 69280 Marcy Letoile, France; USA, Med Res Inst Infect Dis, Div Virol, Dept Molec Virol, Frederick, MD 21702, USA and Inst Pasteur, Unite Virol Molec, F 75015 Paris, France</affiliation><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">1995</dateIssued><copyrightDate encoding="w3cdtf">1995</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract lang="en">Abstract: The nonstructural protein NS1 of Japanese encephalitis virus is a major immunogen produced during flavivirus infection. However, the function of this protein has not been identified. To analyze its biochemical properties and evaluate its potential activity in the virus life cycle, the protein was produced in Spodoptera frugiperda insect cells (Sf9), using a recombinant baculovirus, and purified. As described previously by M. Flamand, V. Deubel, and M. Girard (1992, Virology 191, 826-836), a small fraction of the synthesized recombinant protein could mature into a dimer, whereas the major part was retained in intracellular aggregates. This insolubility was used to recover the protein in a purified form using a two-step procedure. Isolated inclusion bodies, in which NS1 constituted over 60% of the protein, were solubilized in 8 M urea. NS1 was further purified by reverse-phase HPLC and recovered at over 90% purity with an overall yield of over 60%. Conditions promoting reoxidation-renaturation of the purified protein were then investigated at a concentration of 100 μg/ml at pH 8. The presence of 8 M urea during reoxidation of NS1 with oxidized glutathione was essential prior to renaturation by dialysis to avoid reaggregation, the main side pathway of refolding in vitro. Three major species, all monomeric, were resolved by nonreducing SDS-PAGE. The form showing the lowest apparent molecular weight comigrated with native unreduced NS1 and was recognized by a monoclonal antibody directed against a conformational epitope strictly dependent on the native structure of the protein. Thus, this form, representing over 30% of the renatured products, may have reached the native conformation of the protein. This simple renaturation-reoxidation procedure may be applicable to other disulfide bond-containing proteins to be recovered in the native state from inclusion bodies.</abstract><note type="content">Section title: Regular Article</note><relatedItem type="host"><titleInfo><title>Protein Expression and Purification</title></titleInfo><titleInfo type="abbreviated"><title>YPREP</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">1995</dateIssued></originInfo><identifier type="ISSN">1046-5928</identifier><identifier type="PII">S1046-5928(00)X0067-8</identifier><part><date>1995</date><detail type="volume"><number>6</number><caption>vol.</caption></detail><detail type="issue"><number>4</number><caption>no.</caption></detail><extent unit="issue-pages"><start>389</start><end>558</end></extent><extent unit="pages"><start>519</start><end>527</end></extent></part></relatedItem><identifier type="istex">1144D979B71F7B6DD58FE9EBEDAB25D90DB38DDB</identifier><identifier type="ark">ark:/67375/6H6-F5L7QX2M-Q</identifier><identifier type="DOI">10.1006/prep.1995.1069</identifier><identifier type="PII">S1046-5928(85)71069-8</identifier><accessCondition type="use and reproduction" contentType="copyright">©1995 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, ©1995</recordOrigin></recordInfo></mods><json:item><extension>json</extension><original>false</original><mimetype>application/json</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-F5L7QX2M-Q/record.json</uri></json:item></metadata><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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