Expression and purification of SARS coronavirus proteins using SUMO-fusions.
Identifieur interne : 004835 ( Main/Exploration ); précédent : 004834; suivant : 004836Expression and purification of SARS coronavirus proteins using SUMO-fusions.
Auteurs : Xun Zuo [États-Unis] ; Michael R. Mattern ; Robin Tan ; Shuisen Li ; John Hall ; David E. Sterner ; Joshua Shoo ; Hiep Tran ; Peter Lim ; Stefan G. Sarafianos ; Lubna Kazi ; Sonia Navas-Martin ; Susan R. Weiss ; Tauseef R. ButtSource :
- Protein expression and purification [ 1046-5928 ] ; 2005.
Descripteurs français
- KwdFr :
- Cysteine endopeptidases (biosynthèse), Cysteine endopeptidases (génétique), Cysteine endopeptidases (isolement et purification), Escherichia coli (génétique), Expression des gènes (génétique), Glycoprotéine de spicule des coronavirus, Glycoprotéines membranaires (biosynthèse), Glycoprotéines membranaires (génétique), Glycoprotéines membranaires (isolement et purification), Histidine (génétique), Peptide hydrolases (métabolisme), Petites protéines modificatrices apparentées à l'ubiquitine (génétique), Protéines de fusion recombinantes (biosynthèse), Protéines de fusion recombinantes (génétique), Protéines de fusion recombinantes (métabolisme), Protéines de l'enveloppe virale (biosynthèse), Protéines de l'enveloppe virale (génétique), Protéines de l'enveloppe virale (isolement et purification), Protéines nucléocapside (biosynthèse), Protéines nucléocapside (génétique), Protéines nucléocapside (isolement et purification), Protéines virales (génétique), Protéines virales (isolement et purification), Protéines virales (métabolisme), Vecteurs génétiques (génétique), Virus du SRAS (génétique).
- MESH :
- biosynthèse : Cysteine endopeptidases, Glycoprotéines membranaires, Protéines de fusion recombinantes, Protéines de l'enveloppe virale, Protéines nucléocapside.
- génétique : Cysteine endopeptidases, Escherichia coli, Expression des gènes, Glycoprotéines membranaires, Histidine, Petites protéines modificatrices apparentées à l'ubiquitine, Protéines de fusion recombinantes, Protéines de l'enveloppe virale, Protéines nucléocapside, Protéines virales, Vecteurs génétiques, Virus du SRAS.
- isolement et purification : Cysteine endopeptidases, Glycoprotéines membranaires, Protéines de l'enveloppe virale, Protéines nucléocapside, Protéines virales.
- métabolisme : Peptide hydrolases, Protéines de fusion recombinantes, Protéines virales.
- Glycoprotéine de spicule des coronavirus.
English descriptors
- KwdEn :
- Cysteine Endopeptidases (biosynthesis), Cysteine Endopeptidases (genetics), Cysteine Endopeptidases (isolation & purification), Escherichia coli (genetics), Gene Expression (genetics), Genetic Vectors (genetics), Histidine (genetics), Membrane Glycoproteins (biosynthesis), Membrane Glycoproteins (genetics), Membrane Glycoproteins (isolation & purification), Nucleocapsid Proteins (biosynthesis), Nucleocapsid Proteins (genetics), Nucleocapsid Proteins (isolation & purification), Peptide Hydrolases (metabolism), Recombinant Fusion Proteins (biosynthesis), Recombinant Fusion Proteins (genetics), Recombinant Fusion Proteins (metabolism), SARS Virus (genetics), Small Ubiquitin-Related Modifier Proteins (genetics), Spike Glycoprotein, Coronavirus, Viral Envelope Proteins (biosynthesis), Viral Envelope Proteins (genetics), Viral Envelope Proteins (isolation & purification), Viral Proteins (genetics), Viral Proteins (isolation & purification), Viral Proteins (metabolism).
- MESH :
- chemical , biosynthesis : Cysteine Endopeptidases, Membrane Glycoproteins, Nucleocapsid Proteins, Recombinant Fusion Proteins, Viral Envelope Proteins.
- chemical , genetics : Cysteine Endopeptidases, Histidine, Membrane Glycoproteins, Nucleocapsid Proteins, Recombinant Fusion Proteins, Small Ubiquitin-Related Modifier Proteins, Viral Envelope Proteins, Viral Proteins.
- chemical , isolation & purification : Cysteine Endopeptidases, Membrane Glycoproteins, Nucleocapsid Proteins, Viral Envelope Proteins, Viral Proteins.
- genetics : Escherichia coli, Gene Expression, Genetic Vectors, SARS Virus.
- chemical , metabolism : Peptide Hydrolases, Recombinant Fusion Proteins, Viral Proteins.
- chemical : Spike Glycoprotein, Coronavirus.
Abstract
Severe acute respiratory syndrome coronavirus (SARS-CoV) proteins belong to a large group of proteins that is difficult to express in traditional expression systems. The ability to express and purify SARS-CoV proteins in large quantities is critical for basic research and for development of pharmaceutical agents. The work reported here demonstrates: (1) fusion of SUMO (small ubiquitin-related modifier), a 100 amino acid polypeptide, to the N-termini of SARS-CoV proteins dramatically enhances expression in Escherichia coli cells and (2) 6x His-tagged SUMO-fusions facilitate rapid purification of the viral proteins on a large scale. We have exploited the natural chaperoning properties of SUMO to develop an expression system suitable for proteins that cannot be expressed by traditional methodologies. A unique feature of the system is the SUMO tag, which enhances expression, facilitates purification, and can be efficiently cleaved by a SUMO-specific protease to generate native protein with a desired N-terminus. We have purified various SARS-CoV proteins under either native or denaturing conditions. These purified proteins have been used to generate highly specific polyclonal antibodies. Our study suggests that the SUMO-fusion technology will be useful for enhancing expression and purification of the viral proteins for structural and functional studies as well as for therapeutic uses.
DOI: 10.1016/j.pep.2005.02.004
PubMed: 15939295
Affiliations:
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Le document en format XML
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Mattern</name></author><author><name sortKey="Tan, Robin" sort="Tan, Robin" uniqKey="Tan R" first="Robin" last="Tan">Robin Tan</name></author><author><name sortKey="Li, Shuisen" sort="Li, Shuisen" uniqKey="Li S" first="Shuisen" last="Li">Shuisen Li</name></author><author><name sortKey="Hall, John" sort="Hall, John" uniqKey="Hall J" first="John" last="Hall">John Hall</name></author><author><name sortKey="Sterner, David E" sort="Sterner, David E" uniqKey="Sterner D" first="David E" last="Sterner">David E. 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Mattern</name></author><author><name sortKey="Tan, Robin" sort="Tan, Robin" uniqKey="Tan R" first="Robin" last="Tan">Robin Tan</name></author><author><name sortKey="Li, Shuisen" sort="Li, Shuisen" uniqKey="Li S" first="Shuisen" last="Li">Shuisen Li</name></author><author><name sortKey="Hall, John" sort="Hall, John" uniqKey="Hall J" first="John" last="Hall">John Hall</name></author><author><name sortKey="Sterner, David E" sort="Sterner, David E" uniqKey="Sterner D" first="David E" last="Sterner">David E. Sterner</name></author><author><name sortKey="Shoo, Joshua" sort="Shoo, Joshua" uniqKey="Shoo J" first="Joshua" last="Shoo">Joshua Shoo</name></author><author><name sortKey="Tran, Hiep" sort="Tran, Hiep" uniqKey="Tran H" first="Hiep" last="Tran">Hiep Tran</name></author><author><name sortKey="Lim, Peter" sort="Lim, Peter" uniqKey="Lim P" first="Peter" last="Lim">Peter Lim</name></author><author><name sortKey="Sarafianos, Stefan G" sort="Sarafianos, Stefan G" uniqKey="Sarafianos S" first="Stefan G" last="Sarafianos">Stefan G. Sarafianos</name></author><author><name sortKey="Kazi, Lubna" sort="Kazi, Lubna" uniqKey="Kazi L" first="Lubna" last="Kazi">Lubna Kazi</name></author><author><name sortKey="Navas Martin, Sonia" sort="Navas Martin, Sonia" uniqKey="Navas Martin S" first="Sonia" last="Navas-Martin">Sonia Navas-Martin</name></author><author><name sortKey="Weiss, Susan R" sort="Weiss, Susan R" uniqKey="Weiss S" first="Susan R" last="Weiss">Susan R. Weiss</name></author><author><name sortKey="Butt, Tauseef R" sort="Butt, Tauseef R" uniqKey="Butt T" first="Tauseef R" last="Butt">Tauseef R. Butt</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>Cysteine Endopeptidases (biosynthesis)</term><term>Cysteine Endopeptidases (genetics)</term><term>Cysteine Endopeptidases (isolation & purification)</term><term>Escherichia coli (genetics)</term><term>Gene Expression (genetics)</term><term>Genetic Vectors (genetics)</term><term>Histidine (genetics)</term><term>Membrane Glycoproteins (biosynthesis)</term><term>Membrane Glycoproteins (genetics)</term><term>Membrane Glycoproteins (isolation & purification)</term><term>Nucleocapsid Proteins (biosynthesis)</term><term>Nucleocapsid Proteins (genetics)</term><term>Nucleocapsid Proteins (isolation & purification)</term><term>Peptide Hydrolases (metabolism)</term><term>Recombinant Fusion Proteins (biosynthesis)</term><term>Recombinant Fusion Proteins (genetics)</term><term>Recombinant Fusion Proteins (metabolism)</term><term>SARS Virus (genetics)</term><term>Small Ubiquitin-Related Modifier Proteins (genetics)</term><term>Spike Glycoprotein, Coronavirus</term><term>Viral Envelope Proteins (biosynthesis)</term><term>Viral Envelope Proteins (genetics)</term><term>Viral Envelope Proteins (isolation & purification)</term><term>Viral Proteins (genetics)</term><term>Viral Proteins (isolation & purification)</term><term>Viral Proteins (metabolism)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Cysteine endopeptidases (biosynthèse)</term><term>Cysteine endopeptidases (génétique)</term><term>Cysteine endopeptidases (isolement et purification)</term><term>Escherichia coli (génétique)</term><term>Expression des gènes (génétique)</term><term>Glycoprotéine de spicule des coronavirus</term><term>Glycoprotéines membranaires (biosynthèse)</term><term>Glycoprotéines membranaires (génétique)</term><term>Glycoprotéines membranaires (isolement et purification)</term><term>Histidine (génétique)</term><term>Peptide hydrolases (métabolisme)</term><term>Petites protéines modificatrices apparentées à l'ubiquitine (génétique)</term><term>Protéines de fusion recombinantes (biosynthèse)</term><term>Protéines de fusion recombinantes (génétique)</term><term>Protéines de fusion recombinantes (métabolisme)</term><term>Protéines de l'enveloppe virale (biosynthèse)</term><term>Protéines de l'enveloppe virale (génétique)</term><term>Protéines de l'enveloppe virale (isolement et purification)</term><term>Protéines nucléocapside (biosynthèse)</term><term>Protéines nucléocapside (génétique)</term><term>Protéines nucléocapside (isolement et purification)</term><term>Protéines virales (génétique)</term><term>Protéines virales (isolement et purification)</term><term>Protéines virales (métabolisme)</term><term>Vecteurs génétiques (génétique)</term><term>Virus du SRAS (génétique)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="biosynthesis" xml:lang="en"><term>Cysteine Endopeptidases</term><term>Membrane Glycoproteins</term><term>Nucleocapsid Proteins</term><term>Recombinant Fusion Proteins</term><term>Viral Envelope Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Cysteine Endopeptidases</term><term>Histidine</term><term>Membrane Glycoproteins</term><term>Nucleocapsid Proteins</term><term>Recombinant Fusion Proteins</term><term>Small Ubiquitin-Related Modifier Proteins</term><term>Viral Envelope Proteins</term><term>Viral Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="isolation & purification" xml:lang="en"><term>Cysteine Endopeptidases</term><term>Membrane Glycoproteins</term><term>Nucleocapsid Proteins</term><term>Viral Envelope Proteins</term><term>Viral Proteins</term></keywords><keywords scheme="MESH" qualifier="biosynthèse" xml:lang="fr"><term>Cysteine endopeptidases</term><term>Glycoprotéines membranaires</term><term>Protéines de fusion recombinantes</term><term>Protéines de l'enveloppe virale</term><term>Protéines nucléocapside</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Escherichia coli</term><term>Gene Expression</term><term>Genetic Vectors</term><term>SARS Virus</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Cysteine endopeptidases</term><term>Escherichia coli</term><term>Expression des gènes</term><term>Glycoprotéines membranaires</term><term>Histidine</term><term>Petites protéines modificatrices apparentées à l'ubiquitine</term><term>Protéines de fusion recombinantes</term><term>Protéines de l'enveloppe virale</term><term>Protéines nucléocapside</term><term>Protéines virales</term><term>Vecteurs génétiques</term><term>Virus du SRAS</term></keywords><keywords scheme="MESH" qualifier="isolement et purification" xml:lang="fr"><term>Cysteine endopeptidases</term><term>Glycoprotéines membranaires</term><term>Protéines de l'enveloppe virale</term><term>Protéines nucléocapside</term><term>Protéines virales</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Peptide Hydrolases</term><term>Recombinant Fusion Proteins</term><term>Viral Proteins</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Peptide hydrolases</term><term>Protéines de fusion recombinantes</term><term>Protéines virales</term></keywords><keywords scheme="MESH" type="chemical" xml:lang="en"><term>Spike Glycoprotein, Coronavirus</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Glycoprotéine de spicule des coronavirus</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Severe acute respiratory syndrome coronavirus (SARS-CoV) proteins belong to a large group of proteins that is difficult to express in traditional expression systems. The ability to express and purify SARS-CoV proteins in large quantities is critical for basic research and for development of pharmaceutical agents. The work reported here demonstrates: (1) fusion of SUMO (small ubiquitin-related modifier), a 100 amino acid polypeptide, to the N-termini of SARS-CoV proteins dramatically enhances expression in Escherichia coli cells and (2) 6x His-tagged SUMO-fusions facilitate rapid purification of the viral proteins on a large scale. We have exploited the natural chaperoning properties of SUMO to develop an expression system suitable for proteins that cannot be expressed by traditional methodologies. A unique feature of the system is the SUMO tag, which enhances expression, facilitates purification, and can be efficiently cleaved by a SUMO-specific protease to generate native protein with a desired N-terminus. We have purified various SARS-CoV proteins under either native or denaturing conditions. These purified proteins have been used to generate highly specific polyclonal antibodies. Our study suggests that the SUMO-fusion technology will be useful for enhancing expression and purification of the viral proteins for structural and functional studies as well as for therapeutic uses.</div></front></TEI><affiliations><list><country><li>États-Unis</li></country><region><li>Pennsylvanie</li></region></list><tree><noCountry><name sortKey="Butt, Tauseef R" sort="Butt, Tauseef R" uniqKey="Butt T" first="Tauseef R" last="Butt">Tauseef R. Butt</name><name sortKey="Hall, John" sort="Hall, John" uniqKey="Hall J" first="John" last="Hall">John Hall</name><name sortKey="Kazi, Lubna" sort="Kazi, Lubna" uniqKey="Kazi L" first="Lubna" last="Kazi">Lubna Kazi</name><name sortKey="Li, Shuisen" sort="Li, Shuisen" uniqKey="Li S" first="Shuisen" last="Li">Shuisen Li</name><name sortKey="Lim, Peter" sort="Lim, Peter" uniqKey="Lim P" first="Peter" last="Lim">Peter Lim</name><name sortKey="Mattern, Michael R" sort="Mattern, Michael R" uniqKey="Mattern M" first="Michael R" last="Mattern">Michael R. Mattern</name><name sortKey="Navas Martin, Sonia" sort="Navas Martin, Sonia" uniqKey="Navas Martin S" first="Sonia" last="Navas-Martin">Sonia Navas-Martin</name><name sortKey="Sarafianos, Stefan G" sort="Sarafianos, Stefan G" uniqKey="Sarafianos S" first="Stefan G" last="Sarafianos">Stefan G. Sarafianos</name><name sortKey="Shoo, Joshua" sort="Shoo, Joshua" uniqKey="Shoo J" first="Joshua" last="Shoo">Joshua Shoo</name><name sortKey="Sterner, David E" sort="Sterner, David E" uniqKey="Sterner D" first="David E" last="Sterner">David E. Sterner</name><name sortKey="Tan, Robin" sort="Tan, Robin" uniqKey="Tan R" first="Robin" last="Tan">Robin Tan</name><name sortKey="Tran, Hiep" sort="Tran, Hiep" uniqKey="Tran H" first="Hiep" last="Tran">Hiep Tran</name><name sortKey="Weiss, Susan R" sort="Weiss, Susan R" uniqKey="Weiss S" first="Susan R" last="Weiss">Susan R. Weiss</name></noCountry><country name="États-Unis"><region name="Pennsylvanie"><name sortKey="Zuo, Xun" sort="Zuo, Xun" uniqKey="Zuo X" first="Xun" last="Zuo">Xun Zuo</name></region></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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