Prokaryotic expression, refolding, and purification of fragment 450-650 of the spike protein of SARS-coronavirus.
Identifieur interne : 004709 ( Main/Exploration ); précédent : 004708; suivant : 004710Prokaryotic expression, refolding, and purification of fragment 450-650 of the spike protein of SARS-coronavirus.
Auteurs : Jin-Cun Zhao [République populaire de Chine] ; Zhen-Dong Zhao ; Wei Wang ; Xiao-Ming GaoSource :
- Protein expression and purification [ 1046-5928 ] ; 2005.
Descripteurs français
- KwdFr :
- Adjuvant Freund (pharmacologie), Animaux, Clonage moléculaire, Escherichia coli (croissance et développement), Escherichia coli (génétique), Escherichia coli (métabolisme), Femelle, Fragments peptidiques (), Fragments peptidiques (génétique), Fragments peptidiques (immunologie), Fragments peptidiques (isolement et purification), Fragments peptidiques (métabolisme), Glycoprotéines membranaires (), Glycoprotéines membranaires (génétique), Glycoprotéines membranaires (immunologie), Glycoprotéines membranaires (métabolisme), Histidine (), Humains, Immunisation, Injections sous-cutanées, Masse moléculaire, Pliage des protéines, Protéines recombinantes (), Protéines recombinantes (immunologie), Protéines recombinantes (isolement et purification), Protéines recombinantes (métabolisme), Rappel de vaccin, Souris, Souris de lignée BALB C, Séquence d'acides aminés, Technique de Western, Test ELISA, Transformation génétique, Vecteurs génétiques, Virus du SRAS ().
- MESH :
- croissance et développement : Escherichia coli.
- génétique : Escherichia coli, Fragments peptidiques, Glycoprotéines membranaires.
- immunologie : Fragments peptidiques, Glycoprotéines membranaires, Protéines recombinantes.
- isolement et purification : Fragments peptidiques, Protéines recombinantes.
- métabolisme : Escherichia coli, Fragments peptidiques, Glycoprotéines membranaires, Protéines recombinantes.
- pharmacologie : Adjuvant Freund.
- Animaux, Clonage moléculaire, Femelle, Fragments peptidiques, Glycoprotéines membranaires, Histidine, Humains, Immunisation, Injections sous-cutanées, Masse moléculaire, Pliage des protéines, Protéines recombinantes, Rappel de vaccin, Souris, Souris de lignée BALB C, Séquence d'acides aminés, Technique de Western, Test ELISA, Transformation génétique, Vecteurs génétiques, Virus du SRAS.
English descriptors
- KwdEn :
- Amino Acid Sequence, Animals, Blotting, Western, Cloning, Molecular, Enzyme-Linked Immunosorbent Assay, Escherichia coli (genetics), Escherichia coli (growth & development), Escherichia coli (metabolism), Female, Freund's Adjuvant (pharmacology), Genetic Vectors, Histidine (chemistry), Humans, Immunization, Immunization, Secondary, Injections, Subcutaneous, Membrane Glycoproteins (chemistry), Membrane Glycoproteins (genetics), Membrane Glycoproteins (immunology), Membrane Glycoproteins (metabolism), Mice, Mice, Inbred BALB C, Molecular Weight, Peptide Fragments (chemistry), Peptide Fragments (genetics), Peptide Fragments (immunology), Peptide Fragments (isolation & purification), Peptide Fragments (metabolism), Protein Folding, Recombinant Proteins (chemistry), Recombinant Proteins (immunology), Recombinant Proteins (isolation & purification), Recombinant Proteins (metabolism), SARS Virus (chemistry), Transformation, Genetic.
- MESH :
- chemical , chemistry : Histidine, Membrane Glycoproteins, Peptide Fragments, Recombinant Proteins.
- chemical , genetics : Membrane Glycoproteins, Peptide Fragments.
- chemical , immunology : Membrane Glycoproteins, Peptide Fragments, Recombinant Proteins.
- chemical , isolation & purification : Peptide Fragments, Recombinant Proteins.
- chemical , metabolism : Membrane Glycoproteins, Peptide Fragments, Recombinant Proteins.
- chemical , pharmacology : Freund's Adjuvant.
- chemistry : SARS Virus.
- genetics : Escherichia coli.
- growth & development : Escherichia coli.
- metabolism : Escherichia coli.
- Amino Acid Sequence, Animals, Blotting, Western, Cloning, Molecular, Enzyme-Linked Immunosorbent Assay, Female, Genetic Vectors, Humans, Immunization, Immunization, Secondary, Injections, Subcutaneous, Mice, Mice, Inbred BALB C, Molecular Weight, Protein Folding, Transformation, Genetic.
Abstract
The spike (S) glycoprotein is one of the major structure proteins of SARS-associated coronavirus (CoV). Fragment 450-650 (S450-650) of the S protein contains receptor-binding domain and neutralizing epitopes. In this study, S450-650 was expressed with a histidine tag in Escherichia coli BL21. Bacterial inclusion bodies containing the recombinant S450-650 were solubilized with 8 M urea and then applied onto a Ni-nitrilotriacetic acid column. On-column refolding and purification was performed. Reduced glutathione and oxidized glutathione were included in the refolding buffer. In the wash and elution buffers, glycerol and glucose were necessary additives to prevent protein aggregation during purification. This refolding and purification procedure allowed production of S450-650 at up to 500 microg/ml in soluble form, which maintained appropriate antigenicity and immunogenicity. It was able to induce strong IgG responses in BALB/c mice. In Western blot assays, the recombinant S450-650 was recognized by monoclonal Ab against the His-tag and also sera from a convalescent SARS patient. S450-650-based ELISA system was able to detect anti-SARS-CoV IgG Abs in patient sera.
DOI: 10.1016/j.pep.2004.10.004
PubMed: 15642467
Affiliations:
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Le document en format XML
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uniqKey="Wang W" first="Wei" last="Wang">Wei Wang</name></author><author><name sortKey="Gao, Xiao Ming" sort="Gao, Xiao Ming" uniqKey="Gao X" first="Xiao-Ming" last="Gao">Xiao-Ming Gao</name></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2005">2005</date><idno type="RBID">pubmed:15642467</idno><idno type="pmid">15642467</idno><idno type="doi">10.1016/j.pep.2004.10.004</idno><idno type="wicri:Area/PubMed/Corpus">002950</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Corpus" wicri:corpus="PubMed">002950</idno><idno type="wicri:Area/PubMed/Curation">002950</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Curation">002950</idno><idno type="wicri:Area/PubMed/Checkpoint">002571</idno><idno type="wicri:explorRef" wicri:stream="Checkpoint" wicri:step="PubMed">002571</idno><idno type="wicri:Area/Ncbi/Merge">000D15</idno><idno type="wicri:Area/Ncbi/Curation">000D15</idno><idno 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(genetics)</term><term>Escherichia coli (growth & development)</term><term>Escherichia coli (metabolism)</term><term>Female</term><term>Freund's Adjuvant (pharmacology)</term><term>Genetic Vectors</term><term>Histidine (chemistry)</term><term>Humans</term><term>Immunization</term><term>Immunization, Secondary</term><term>Injections, Subcutaneous</term><term>Membrane Glycoproteins (chemistry)</term><term>Membrane Glycoproteins (genetics)</term><term>Membrane Glycoproteins (immunology)</term><term>Membrane Glycoproteins (metabolism)</term><term>Mice</term><term>Mice, Inbred BALB C</term><term>Molecular Weight</term><term>Peptide Fragments (chemistry)</term><term>Peptide Fragments (genetics)</term><term>Peptide Fragments (immunology)</term><term>Peptide Fragments (isolation & purification)</term><term>Peptide Fragments (metabolism)</term><term>Protein Folding</term><term>Recombinant Proteins (chemistry)</term><term>Recombinant Proteins (immunology)</term><term>Recombinant Proteins 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coli</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Escherichia coli</term><term>Fragments peptidiques</term><term>Glycoprotéines membranaires</term></keywords><keywords scheme="MESH" qualifier="immunologie" xml:lang="fr"><term>Fragments peptidiques</term><term>Glycoprotéines membranaires</term><term>Protéines recombinantes</term></keywords><keywords scheme="MESH" qualifier="isolement et purification" xml:lang="fr"><term>Fragments peptidiques</term><term>Protéines recombinantes</term></keywords><keywords scheme="MESH" qualifier="metabolism" xml:lang="en"><term>Escherichia coli</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Escherichia coli</term><term>Fragments peptidiques</term><term>Glycoprotéines membranaires</term><term>Protéines recombinantes</term></keywords><keywords scheme="MESH" qualifier="pharmacologie" xml:lang="fr"><term>Adjuvant Freund</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Amino Acid Sequence</term><term>Animals</term><term>Blotting, Western</term><term>Cloning, Molecular</term><term>Enzyme-Linked Immunosorbent Assay</term><term>Female</term><term>Genetic Vectors</term><term>Humans</term><term>Immunization</term><term>Immunization, Secondary</term><term>Injections, Subcutaneous</term><term>Mice</term><term>Mice, Inbred BALB C</term><term>Molecular Weight</term><term>Protein Folding</term><term>Transformation, Genetic</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Animaux</term><term>Clonage moléculaire</term><term>Femelle</term><term>Fragments peptidiques</term><term>Glycoprotéines membranaires</term><term>Histidine</term><term>Humains</term><term>Immunisation</term><term>Injections sous-cutanées</term><term>Masse moléculaire</term><term>Pliage des protéines</term><term>Protéines recombinantes</term><term>Rappel de vaccin</term><term>Souris</term><term>Souris de lignée BALB C</term><term>Séquence d'acides aminés</term><term>Technique de Western</term><term>Test ELISA</term><term>Transformation génétique</term><term>Vecteurs génétiques</term><term>Virus du SRAS</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The spike (S) glycoprotein is one of the major structure proteins of SARS-associated coronavirus (CoV). Fragment 450-650 (S450-650) of the S protein contains receptor-binding domain and neutralizing epitopes. In this study, S450-650 was expressed with a histidine tag in Escherichia coli BL21. Bacterial inclusion bodies containing the recombinant S450-650 were solubilized with 8 M urea and then applied onto a Ni-nitrilotriacetic acid column. On-column refolding and purification was performed. Reduced glutathione and oxidized glutathione were included in the refolding buffer. In the wash and elution buffers, glycerol and glucose were necessary additives to prevent protein aggregation during purification. This refolding and purification procedure allowed production of S450-650 at up to 500 microg/ml in soluble form, which maintained appropriate antigenicity and immunogenicity. It was able to induce strong IgG responses in BALB/c mice. In Western blot assays, the recombinant S450-650 was recognized by monoclonal Ab against the His-tag and also sera from a convalescent SARS patient. S450-650-based ELISA system was able to detect anti-SARS-CoV IgG Abs in patient sera.</div></front></TEI><affiliations><list><country><li>République populaire de Chine</li></country><settlement><li>Pékin</li></settlement></list><tree><noCountry><name sortKey="Gao, Xiao Ming" sort="Gao, Xiao Ming" uniqKey="Gao X" first="Xiao-Ming" last="Gao">Xiao-Ming Gao</name><name sortKey="Wang, Wei" sort="Wang, Wei" uniqKey="Wang W" first="Wei" last="Wang">Wei Wang</name><name sortKey="Zhao, Zhen Dong" sort="Zhao, Zhen Dong" uniqKey="Zhao Z" first="Zhen-Dong" last="Zhao">Zhen-Dong Zhao</name></noCountry><country name="République populaire de Chine"><noRegion><name sortKey="Zhao, Jin Cun" sort="Zhao, Jin Cun" uniqKey="Zhao J" first="Jin-Cun" last="Zhao">Jin-Cun Zhao</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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