Expression of SARS-coronavirus spike glycoprotein in Pichia pastoris.
Identifieur interne : 001A41 ( PubMed/Corpus ); précédent : 001A40; suivant : 001A42Expression of SARS-coronavirus spike glycoprotein in Pichia pastoris.
Auteurs : Chi-Pang Chuck ; Chi-Hang Wong ; Larry Ming-Cheung Chow ; Kwok-Pui Fung ; Mary Miu-Yee Waye ; Stephen Kwok-Wing TsuiSource :
- Virus genes [ 0920-8569 ] ; 2009.
English descriptors
- KwdEn :
- Chromatography, Affinity, Escherichia coli (genetics), Escherichia coli (metabolism), Membrane Glycoproteins (biosynthesis), Membrane Glycoproteins (chemistry), Membrane Glycoproteins (genetics), Membrane Glycoproteins (isolation & purification), Peptidyl-Dipeptidase A (metabolism), Pichia (genetics), Pichia (metabolism), Protein Binding, Recombinant Proteins (chemistry), Recombinant Proteins (genetics), Recombinant Proteins (isolation & purification), Recombinant Proteins (metabolism), SARS Virus (genetics), Solubility, Spike Glycoprotein, Coronavirus, Viral Envelope Proteins (biosynthesis), Viral Envelope Proteins (chemistry), Viral Envelope Proteins (genetics), Viral Envelope Proteins (isolation & purification).
- MESH :
- chemical , biosynthesis : Membrane Glycoproteins, Viral Envelope Proteins.
- chemical , chemistry : Membrane Glycoproteins, Recombinant Proteins, Viral Envelope Proteins.
- genetics : Escherichia coli, Membrane Glycoproteins, Pichia, Recombinant Proteins, SARS Virus, Viral Envelope Proteins.
- chemical , isolation & purification : Membrane Glycoproteins, Recombinant Proteins, Viral Envelope Proteins.
- metabolism : Escherichia coli, Peptidyl-Dipeptidase A, Pichia, Recombinant Proteins.
- Chromatography, Affinity, Protein Binding, Solubility, Spike Glycoprotein, Coronavirus.
Abstract
To establish a rapid and economical method for the expression of viral proteins in high yield and purity by Pichia pastoris, the S protein of the SARS-CoV was selected in this study. Six S glycoprotein fragments were expressed in Escherichia coli BL21 and yeast KM71H strains. After purification by affinity chromatography, the protein identities were confirmed by western blot analysis, N-terminal sequencing and mass spectrometry. The proteins expressed in E. coli were low in solubility and bound by GroEL. They still formed soluble aggregates even when the GroEL was removed by urea. The proteins expressed in P. pastoris were relatively soluble. The maximal yield of the RBD reached 46 mg/l with purity greater than 95%. Pull-down assay revealed that ACE2 was specifically captured from cell lysate, indicating that the RBD was biologically active. The glycosylated and deglycosylated RBD was then subjected to SEC and results showed that deglycosylated RBD formed soluble aggregates again. Taken together, pure and biological active RBD of the S protein could be expressed in P. pastoris, and the P. pastoris expression platform will be a good alternative for the expression of viral proteins, in particular, the highly glycosylated surface proteins that mediate the tissue tropism and viral entry.
DOI: 10.1007/s11262-008-0292-3
PubMed: 18958613
Links to Exploration step
pubmed:18958613Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Expression of SARS-coronavirus spike glycoprotein in Pichia pastoris.</title><author><name sortKey="Chuck, Chi Pang" sort="Chuck, Chi Pang" uniqKey="Chuck C" first="Chi-Pang" last="Chuck">Chi-Pang Chuck</name><affiliation><nlm:affiliation>Department of Biochemistry, The Chinese University of Hong Kong, Shatin, Hong Kong.</nlm:affiliation></affiliation></author><author><name sortKey="Wong, Chi Hang" sort="Wong, Chi Hang" uniqKey="Wong C" first="Chi-Hang" last="Wong">Chi-Hang Wong</name></author><author><name sortKey="Chow, Larry Ming Cheung" sort="Chow, Larry Ming Cheung" uniqKey="Chow L" first="Larry Ming-Cheung" last="Chow">Larry Ming-Cheung Chow</name></author><author><name sortKey="Fung, Kwok Pui" sort="Fung, Kwok Pui" uniqKey="Fung K" first="Kwok-Pui" last="Fung">Kwok-Pui Fung</name></author><author><name sortKey="Waye, Mary Miu Yee" sort="Waye, Mary Miu Yee" uniqKey="Waye M" first="Mary Miu-Yee" last="Waye">Mary Miu-Yee Waye</name></author><author><name sortKey="Tsui, Stephen Kwok Wing" sort="Tsui, Stephen Kwok Wing" uniqKey="Tsui S" first="Stephen Kwok-Wing" last="Tsui">Stephen Kwok-Wing Tsui</name></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2009">2009</date><idno type="RBID">pubmed:18958613</idno><idno type="pmid">18958613</idno><idno type="doi">10.1007/s11262-008-0292-3</idno><idno type="wicri:Area/PubMed/Corpus">001A41</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Corpus" wicri:corpus="PubMed">001A41</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">Expression of SARS-coronavirus spike glycoprotein in Pichia pastoris.</title><author><name sortKey="Chuck, Chi Pang" sort="Chuck, Chi Pang" uniqKey="Chuck C" first="Chi-Pang" last="Chuck">Chi-Pang Chuck</name><affiliation><nlm:affiliation>Department of Biochemistry, The Chinese University of Hong Kong, Shatin, Hong Kong.</nlm:affiliation></affiliation></author><author><name sortKey="Wong, Chi Hang" sort="Wong, Chi Hang" uniqKey="Wong C" first="Chi-Hang" last="Wong">Chi-Hang Wong</name></author><author><name sortKey="Chow, Larry Ming Cheung" sort="Chow, Larry Ming Cheung" uniqKey="Chow L" first="Larry Ming-Cheung" last="Chow">Larry Ming-Cheung Chow</name></author><author><name sortKey="Fung, Kwok Pui" sort="Fung, Kwok Pui" uniqKey="Fung K" first="Kwok-Pui" last="Fung">Kwok-Pui Fung</name></author><author><name sortKey="Waye, Mary Miu Yee" sort="Waye, Mary Miu Yee" uniqKey="Waye M" first="Mary Miu-Yee" last="Waye">Mary Miu-Yee Waye</name></author><author><name sortKey="Tsui, Stephen Kwok Wing" sort="Tsui, Stephen Kwok Wing" uniqKey="Tsui S" first="Stephen Kwok-Wing" last="Tsui">Stephen Kwok-Wing Tsui</name></author></analytic><series><title level="j">Virus genes</title><idno type="ISSN">0920-8569</idno><imprint><date when="2009" type="published">2009</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Chromatography, Affinity</term><term>Escherichia coli (genetics)</term><term>Escherichia coli (metabolism)</term><term>Membrane Glycoproteins (biosynthesis)</term><term>Membrane Glycoproteins (chemistry)</term><term>Membrane Glycoproteins (genetics)</term><term>Membrane Glycoproteins (isolation & purification)</term><term>Peptidyl-Dipeptidase A (metabolism)</term><term>Pichia (genetics)</term><term>Pichia (metabolism)</term><term>Protein Binding</term><term>Recombinant Proteins (chemistry)</term><term>Recombinant Proteins (genetics)</term><term>Recombinant Proteins (isolation & purification)</term><term>Recombinant Proteins (metabolism)</term><term>SARS Virus (genetics)</term><term>Solubility</term><term>Spike Glycoprotein, Coronavirus</term><term>Viral Envelope Proteins (biosynthesis)</term><term>Viral Envelope Proteins (chemistry)</term><term>Viral Envelope Proteins (genetics)</term><term>Viral Envelope Proteins (isolation & purification)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="biosynthesis" xml:lang="en"><term>Membrane Glycoproteins</term><term>Viral Envelope Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Membrane Glycoproteins</term><term>Recombinant Proteins</term><term>Viral Envelope Proteins</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Escherichia coli</term><term>Membrane Glycoproteins</term><term>Pichia</term><term>Recombinant Proteins</term><term>SARS Virus</term><term>Viral Envelope Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="isolation & purification" xml:lang="en"><term>Membrane Glycoproteins</term><term>Recombinant Proteins</term><term>Viral Envelope Proteins</term></keywords><keywords scheme="MESH" qualifier="metabolism" xml:lang="en"><term>Escherichia coli</term><term>Peptidyl-Dipeptidase A</term><term>Pichia</term><term>Recombinant Proteins</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Chromatography, Affinity</term><term>Protein Binding</term><term>Solubility</term><term>Spike Glycoprotein, Coronavirus</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">To establish a rapid and economical method for the expression of viral proteins in high yield and purity by Pichia pastoris, the S protein of the SARS-CoV was selected in this study. Six S glycoprotein fragments were expressed in Escherichia coli BL21 and yeast KM71H strains. After purification by affinity chromatography, the protein identities were confirmed by western blot analysis, N-terminal sequencing and mass spectrometry. The proteins expressed in E. coli were low in solubility and bound by GroEL. They still formed soluble aggregates even when the GroEL was removed by urea. The proteins expressed in P. pastoris were relatively soluble. The maximal yield of the RBD reached 46 mg/l with purity greater than 95%. Pull-down assay revealed that ACE2 was specifically captured from cell lysate, indicating that the RBD was biologically active. The glycosylated and deglycosylated RBD was then subjected to SEC and results showed that deglycosylated RBD formed soluble aggregates again. Taken together, pure and biological active RBD of the S protein could be expressed in P. pastoris, and the P. pastoris expression platform will be a good alternative for the expression of viral proteins, in particular, the highly glycosylated surface proteins that mediate the tissue tropism and viral entry.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">18958613</PMID><DateCompleted><Year>2009</Year><Month>04</Month><Day>16</Day></DateCompleted><DateRevised><Year>2020</Year><Month>04</Month><Day>15</Day></DateRevised><Article PubModel="Print-Electronic"><Journal><ISSN IssnType="Print">0920-8569</ISSN><JournalIssue CitedMedium="Print"><Volume>38</Volume><Issue>1</Issue><PubDate><Year>2009</Year><Month>Feb</Month></PubDate></JournalIssue><Title>Virus genes</Title><ISOAbbreviation>Virus Genes</ISOAbbreviation></Journal><ArticleTitle>Expression of SARS-coronavirus spike glycoprotein in Pichia pastoris.</ArticleTitle><Pagination><MedlinePgn>1-9</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1007/s11262-008-0292-3</ELocationID><Abstract><AbstractText>To establish a rapid and economical method for the expression of viral proteins in high yield and purity by Pichia pastoris, the S protein of the SARS-CoV was selected in this study. Six S glycoprotein fragments were expressed in Escherichia coli BL21 and yeast KM71H strains. After purification by affinity chromatography, the protein identities were confirmed by western blot analysis, N-terminal sequencing and mass spectrometry. The proteins expressed in E. coli were low in solubility and bound by GroEL. They still formed soluble aggregates even when the GroEL was removed by urea. The proteins expressed in P. pastoris were relatively soluble. The maximal yield of the RBD reached 46 mg/l with purity greater than 95%. Pull-down assay revealed that ACE2 was specifically captured from cell lysate, indicating that the RBD was biologically active. The glycosylated and deglycosylated RBD was then subjected to SEC and results showed that deglycosylated RBD formed soluble aggregates again. Taken together, pure and biological active RBD of the S protein could be expressed in P. pastoris, and the P. pastoris expression platform will be a good alternative for the expression of viral proteins, in particular, the highly glycosylated surface proteins that mediate the tissue tropism and viral entry.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Chuck</LastName><ForeName>Chi-Pang</ForeName><Initials>CP</Initials><AffiliationInfo><Affiliation>Department of Biochemistry, The Chinese University of Hong Kong, Shatin, Hong Kong.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Wong</LastName><ForeName>Chi-Hang</ForeName><Initials>CH</Initials></Author><Author ValidYN="Y"><LastName>Chow</LastName><ForeName>Larry Ming-Cheung</ForeName><Initials>LM</Initials></Author><Author ValidYN="Y"><LastName>Fung</LastName><ForeName>Kwok-Pui</ForeName><Initials>KP</Initials></Author><Author ValidYN="Y"><LastName>Waye</LastName><ForeName>Mary Miu-Yee</ForeName><Initials>MM</Initials></Author><Author ValidYN="Y"><LastName>Tsui</LastName><ForeName>Stephen Kwok-Wing</ForeName><Initials>SK</Initials></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013485">Research Support, Non-U.S. Gov't</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2008</Year><Month>10</Month><Day>29</Day></ArticleDate></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>Virus Genes</MedlineTA><NlmUniqueID>8803967</NlmUniqueID><ISSNLinking>0920-8569</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="C578553">MHV surface projection glycoprotein</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D008562">Membrane 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