Prokaryotic expression, refolding, and purification of fragment 450-650 of the spike protein of SARS-coronavirus.
Identifieur interne : 002950 ( PubMed/Corpus ); précédent : 002949; suivant : 002951Prokaryotic expression, refolding, and purification of fragment 450-650 of the spike protein of SARS-coronavirus.
Auteurs : Jin-Cun Zhao ; Zhen-Dong Zhao ; Wei Wang ; Xiao-Ming GaoSource :
- Protein expression and purification [ 1046-5928 ] ; 2005.
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
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pubmed:15642467Le document en format XML
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Wei" sort="Wang, Wei" 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></analytic><series><title level="j">Protein expression and purification</title><idno type="ISSN">1046-5928</idno><imprint><date when="2005" type="published">2005</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" 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>Escherichia coli (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 (isolation & purification)</term><term>Recombinant Proteins (metabolism)</term><term>SARS Virus (chemistry)</term><term>Transformation, Genetic</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Histidine</term><term>Membrane Glycoproteins</term><term>Peptide Fragments</term><term>Recombinant Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Membrane Glycoproteins</term><term>Peptide Fragments</term></keywords><keywords scheme="MESH" type="chemical" qualifier="immunology" xml:lang="en"><term>Membrane Glycoproteins</term><term>Peptide Fragments</term><term>Recombinant Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="isolation & purification" xml:lang="en"><term>Peptide Fragments</term><term>Recombinant Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Membrane Glycoproteins</term><term>Peptide Fragments</term><term>Recombinant Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="pharmacology" xml:lang="en"><term>Freund's Adjuvant</term></keywords><keywords scheme="MESH" qualifier="chemistry" xml:lang="en"><term>SARS Virus</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Escherichia coli</term></keywords><keywords scheme="MESH" qualifier="growth & development" xml:lang="en"><term>Escherichia coli</term></keywords><keywords scheme="MESH" qualifier="metabolism" xml:lang="en"><term>Escherichia coli</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></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><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">15642467</PMID><DateCompleted><Year>2005</Year><Month>05</Month><Day>26</Day></DateCompleted><DateRevised><Year>2020</Year><Month>04</Month><Day>09</Day></DateRevised><Article PubModel="Print"><Journal><ISSN IssnType="Print">1046-5928</ISSN><JournalIssue CitedMedium="Print"><Volume>39</Volume><Issue>2</Issue><PubDate><Year>2005</Year><Month>Feb</Month></PubDate></JournalIssue><Title>Protein expression and purification</Title><ISOAbbreviation>Protein Expr. Purif.</ISOAbbreviation></Journal><ArticleTitle>Prokaryotic expression, refolding, and purification of fragment 450-650 of the spike protein of SARS-coronavirus.</ArticleTitle><Pagination><MedlinePgn>169-74</MedlinePgn></Pagination><Abstract><AbstractText>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.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Zhao</LastName><ForeName>Jin-Cun</ForeName><Initials>JC</Initials><AffiliationInfo><Affiliation>Department of Immunology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China. zhaojincun@hotmail.com</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Zhao</LastName><ForeName>Zhen-Dong</ForeName><Initials>ZD</Initials></Author><Author ValidYN="Y"><LastName>Wang</LastName><ForeName>Wei</ForeName><Initials>W</Initials></Author><Author ValidYN="Y"><LastName>Gao</LastName><ForeName>Xiao-Ming</ForeName><Initials>XM</Initials></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013485">Research Support, Non-U.S. 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