Specific and efficient cleavage of fusion proteins by recombinant plum pox virus NIa protease.
Identifieur interne : 001B15 ( Ncbi/Checkpoint ); précédent : 001B14; suivant : 001B16Specific and efficient cleavage of fusion proteins by recombinant plum pox virus NIa protease.
Auteurs : Nuoyan Zheng [République populaire de Chine] ; José De Jesús Pérez ; Zhonghui Zhang ; Elvira Domínguez ; Juan Antonio Garcia ; Qi XieSource :
- Protein expression and purification [ 1046-5928 ] ; 2008.
Descripteurs français
- KwdFr :
- Alignement de séquences, Chlorure de sodium (pharmacologie), Concentration osmolaire, Données de séquences moléculaires, Endopeptidases (), Endopeptidases (isolement et purification), Endopeptidases (métabolisme), Escherichia coli (métabolisme), Inhibiteurs de protéases (pharmacologie), Maturation post-traductionnelle des protéines (), Protéines de capside (isolement et purification), Protéines de capside (métabolisme), Protéines de fusion recombinantes (isolement et purification), Protéines de fusion recombinantes (métabolisme), Protéines virales (), Protéines virales (isolement et purification), Protéines virales (métabolisme), Protéines à fluorescence verte (isolement et purification), Protéines à fluorescence verte (métabolisme), Spécificité du substrat (), Stabilité enzymatique (), Séquence d'acides aminés, Température, Virus de la variole du prunier (enzymologie).
- MESH :
- enzymologie : Virus de la variole du prunier.
- isolement et purification : Endopeptidases, Protéines de capside, Protéines de fusion recombinantes, Protéines virales, Protéines à fluorescence verte.
- métabolisme : Endopeptidases, Escherichia coli, Protéines de capside, Protéines de fusion recombinantes, Protéines virales, Protéines à fluorescence verte.
- pharmacologie : Chlorure de sodium, Inhibiteurs de protéases.
- Alignement de séquences, Concentration osmolaire, Données de séquences moléculaires, Endopeptidases, Maturation post-traductionnelle des protéines, Protéines virales, Spécificité du substrat, Stabilité enzymatique, Séquence d'acides aminés, Température.
English descriptors
- KwdEn :
- Amino Acid Sequence, Capsid Proteins (isolation & purification), Capsid Proteins (metabolism), Endopeptidases (chemistry), Endopeptidases (isolation & purification), Endopeptidases (metabolism), Enzyme Stability (drug effects), Escherichia coli (metabolism), Green Fluorescent Proteins (isolation & purification), Green Fluorescent Proteins (metabolism), Molecular Sequence Data, Osmolar Concentration, Plum Pox Virus (enzymology), Protease Inhibitors (pharmacology), Protein Processing, Post-Translational (drug effects), Recombinant Fusion Proteins (isolation & purification), Recombinant Fusion Proteins (metabolism), Sequence Alignment, Sodium Chloride (pharmacology), Substrate Specificity (drug effects), Temperature, Viral Proteins (chemistry), Viral Proteins (isolation & purification), Viral Proteins (metabolism).
- MESH :
- chemical , chemistry : Endopeptidases, Viral Proteins.
- chemical , isolation & purification : Capsid Proteins, Endopeptidases, Green Fluorescent Proteins, Recombinant Fusion Proteins, Viral Proteins.
- chemical , metabolism : Capsid Proteins, Endopeptidases, Green Fluorescent Proteins, Recombinant Fusion Proteins, Viral Proteins.
- drug effects : Enzyme Stability, Protein Processing, Post-Translational, Substrate Specificity.
- enzymology : Plum Pox Virus.
- metabolism : Escherichia coli.
- chemical , pharmacology : Protease Inhibitors, Sodium Chloride.
- Amino Acid Sequence, Molecular Sequence Data, Osmolar Concentration, Sequence Alignment, Temperature.
Abstract
Site-specific proteases are the most popular kind of enzymes for removing the fusion tags from fused target proteins. Nuclear inclusion protein a (NIa) proteases obtained from the family Potyviridae have become promising due to their high activities and stringencies of sequences recognition. NIa proteases from tobacco etch virus (TEV) and tomato vein mottling virus (TVMV) have been shown to process recombinant proteins successfully in vitro. In this report, recombinant PPV (plum pox virus) NIa protease was employed to process fusion proteins with artificial cleavage site in vitro. Characteristics such as catalytic ability and affecting factors (salt, temperature, protease inhibitors, detergents, and denaturing reagents) were investigated. Recombinant PPV NIa protease expressed and purified from Escherichia coli demonstrated efficient and specific processing of recombinant GFP and SARS-CoV nucleocapsid protein, with site F (N V V V H Q black triangle down A) for PPV NIa protease artificially inserted between the fusion tags and the target proteins. Its catalytic capability is similar to those of TVMV and TEV NIa protease. Recombinant PPV NIa protease reached its maximal proteolytic activity at approximately 30 degrees C. Salt concentration and only one of the tested protease inhibitors had minor influences on the proteolytic activity of PPV NIa protease. Recombinant PPV NIa protease was resistant to self-lysis for at least five days.
DOI: 10.1016/j.pep.2007.10.008
PubMed: 18024078
Affiliations:
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pubmed:18024078Le document en format XML
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sortKey="Zhang, Zhonghui" sort="Zhang, Zhonghui" uniqKey="Zhang Z" first="Zhonghui" last="Zhang">Zhonghui Zhang</name></author><author><name sortKey="Dominguez, Elvira" sort="Dominguez, Elvira" uniqKey="Dominguez E" first="Elvira" last="Domínguez">Elvira Domínguez</name></author><author><name sortKey="Garcia, Juan Antonio" sort="Garcia, Juan Antonio" uniqKey="Garcia J" first="Juan Antonio" last="Garcia">Juan Antonio Garcia</name></author><author><name sortKey="Xie, Qi" sort="Xie, Qi" uniqKey="Xie Q" first="Qi" last="Xie">Qi Xie</name></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2008">2008</date><idno type="RBID">pubmed:18024078</idno><idno type="pmid">18024078</idno><idno type="doi">10.1016/j.pep.2007.10.008</idno><idno type="wicri:Area/PubMed/Corpus">001C77</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Corpus" wicri:corpus="PubMed">001C77</idno><idno type="wicri:Area/PubMed/Curation">001C77</idno><idno 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Fusion Proteins (metabolism)</term><term>Sequence Alignment</term><term>Sodium Chloride (pharmacology)</term><term>Substrate Specificity (drug effects)</term><term>Temperature</term><term>Viral Proteins (chemistry)</term><term>Viral Proteins (isolation & purification)</term><term>Viral Proteins (metabolism)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Alignement de séquences</term><term>Chlorure de sodium (pharmacologie)</term><term>Concentration osmolaire</term><term>Données de séquences moléculaires</term><term>Endopeptidases ()</term><term>Endopeptidases (isolement et purification)</term><term>Endopeptidases (métabolisme)</term><term>Escherichia coli (métabolisme)</term><term>Inhibiteurs de protéases (pharmacologie)</term><term>Maturation post-traductionnelle des protéines ()</term><term>Protéines de capside (isolement et purification)</term><term>Protéines de capside (métabolisme)</term><term>Protéines de fusion recombinantes (isolement et purification)</term><term>Protéines de fusion recombinantes (métabolisme)</term><term>Protéines virales ()</term><term>Protéines virales (isolement et purification)</term><term>Protéines virales (métabolisme)</term><term>Protéines à fluorescence verte (isolement et purification)</term><term>Protéines à fluorescence verte (métabolisme)</term><term>Spécificité du substrat ()</term><term>Stabilité enzymatique ()</term><term>Séquence d'acides aminés</term><term>Température</term><term>Virus de la variole du prunier (enzymologie)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Endopeptidases</term><term>Viral Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="isolation & purification" xml:lang="en"><term>Capsid Proteins</term><term>Endopeptidases</term><term>Green Fluorescent Proteins</term><term>Recombinant Fusion Proteins</term><term>Viral Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Capsid Proteins</term><term>Endopeptidases</term><term>Green Fluorescent Proteins</term><term>Recombinant Fusion Proteins</term><term>Viral Proteins</term></keywords><keywords scheme="MESH" qualifier="drug effects" xml:lang="en"><term>Enzyme Stability</term><term>Protein Processing, Post-Translational</term><term>Substrate Specificity</term></keywords><keywords scheme="MESH" qualifier="enzymologie" xml:lang="fr"><term>Virus de la variole du prunier</term></keywords><keywords scheme="MESH" qualifier="enzymology" xml:lang="en"><term>Plum Pox Virus</term></keywords><keywords scheme="MESH" qualifier="isolement et purification" xml:lang="fr"><term>Endopeptidases</term><term>Protéines de capside</term><term>Protéines de fusion recombinantes</term><term>Protéines virales</term><term>Protéines à fluorescence verte</term></keywords><keywords scheme="MESH" qualifier="metabolism" xml:lang="en"><term>Escherichia coli</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Endopeptidases</term><term>Escherichia coli</term><term>Protéines de capside</term><term>Protéines de fusion recombinantes</term><term>Protéines virales</term><term>Protéines à fluorescence verte</term></keywords><keywords scheme="MESH" qualifier="pharmacologie" xml:lang="fr"><term>Chlorure de sodium</term><term>Inhibiteurs de protéases</term></keywords><keywords scheme="MESH" type="chemical" qualifier="pharmacology" xml:lang="en"><term>Protease Inhibitors</term><term>Sodium Chloride</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Amino Acid Sequence</term><term>Molecular Sequence Data</term><term>Osmolar Concentration</term><term>Sequence Alignment</term><term>Temperature</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Alignement de séquences</term><term>Concentration osmolaire</term><term>Données de séquences moléculaires</term><term>Endopeptidases</term><term>Maturation post-traductionnelle des protéines</term><term>Protéines virales</term><term>Spécificité du substrat</term><term>Stabilité enzymatique</term><term>Séquence d'acides aminés</term><term>Température</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Site-specific proteases are the most popular kind of enzymes for removing the fusion tags from fused target proteins. Nuclear inclusion protein a (NIa) proteases obtained from the family Potyviridae have become promising due to their high activities and stringencies of sequences recognition. NIa proteases from tobacco etch virus (TEV) and tomato vein mottling virus (TVMV) have been shown to process recombinant proteins successfully in vitro. In this report, recombinant PPV (plum pox virus) NIa protease was employed to process fusion proteins with artificial cleavage site in vitro. Characteristics such as catalytic ability and affecting factors (salt, temperature, protease inhibitors, detergents, and denaturing reagents) were investigated. Recombinant PPV NIa protease expressed and purified from Escherichia coli demonstrated efficient and specific processing of recombinant GFP and SARS-CoV nucleocapsid protein, with site F (N V V V H Q black triangle down A) for PPV NIa protease artificially inserted between the fusion tags and the target proteins. Its catalytic capability is similar to those of TVMV and TEV NIa protease. Recombinant PPV NIa protease reached its maximal proteolytic activity at approximately 30 degrees C. Salt concentration and only one of the tested protease inhibitors had minor influences on the proteolytic activity of PPV NIa protease. Recombinant PPV NIa protease was resistant to self-lysis for at least five days.</div></front></TEI><affiliations><list><country><li>République populaire de Chine</li></country><region><li>Guangdong</li></region><settlement><li>Jiangmen</li></settlement></list><tree><noCountry><name sortKey="Dominguez, Elvira" sort="Dominguez, Elvira" uniqKey="Dominguez E" first="Elvira" last="Domínguez">Elvira Domínguez</name><name sortKey="Garcia, Juan Antonio" sort="Garcia, Juan Antonio" uniqKey="Garcia J" first="Juan Antonio" last="Garcia">Juan Antonio Garcia</name><name sortKey="Perez, Jose De Jesus" sort="Perez, Jose De Jesus" uniqKey="Perez J" first="José De Jesús" last="Pérez">José De Jesús Pérez</name><name sortKey="Xie, Qi" sort="Xie, Qi" uniqKey="Xie Q" first="Qi" last="Xie">Qi Xie</name><name sortKey="Zhang, Zhonghui" sort="Zhang, Zhonghui" uniqKey="Zhang Z" first="Zhonghui" last="Zhang">Zhonghui Zhang</name></noCountry><country name="République populaire de Chine"><region name="Guangdong"><name sortKey="Zheng, Nuoyan" sort="Zheng, Nuoyan" uniqKey="Zheng N" first="Nuoyan" last="Zheng">Nuoyan Zheng</name></region></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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