Expression and purification of coronavirus envelope proteins using a modified β-barrel construct.
Identifieur interne : 002534 ( Ncbi/Curation ); précédent : 002533; suivant : 002535Expression and purification of coronavirus envelope proteins using a modified β-barrel construct.
Auteurs : Krupakar Parthasarathy [Singapour] ; Huang Lu ; Wahyu Surya ; Ardcharaporn Vararattanavech ; Konstantin Pervushin ; Jaume TorresSource :
- Protein expression and purification [ 1096-0279 ] ; 2012.
Descripteurs français
- KwdFr :
- Clonage moléculaire, Dichroïsme circulaire, Données de séquences moléculaires, Double couche lipidique (), Escherichia coli (génétique), Multimérisation de protéines, Mutation, Protéines de fusion recombinantes (), Protéines de fusion recombinantes (génétique), Protéines de fusion recombinantes (isolement et purification), Protéines de l'enveloppe virale (), Protéines de l'enveloppe virale (génétique), Protéines de l'enveloppe virale (isolement et purification), Régulation positive, Structure secondaire des protéines, Syndrome respiratoire aigu sévère (virologie), Séquence d'acides aminés, Ultracentrifugation, Virus du SRAS (), Virus du SRAS (génétique), Électrophorèse sur gel de polyacrylamide.
- MESH :
- génétique : Escherichia coli, Protéines de fusion recombinantes, Protéines de l'enveloppe virale, Virus du SRAS.
- isolement et purification : Protéines de fusion recombinantes, Protéines de l'enveloppe virale.
- virologie : Syndrome respiratoire aigu sévère.
- Clonage moléculaire, Dichroïsme circulaire, Données de séquences moléculaires, Double couche lipidique, Multimérisation de protéines, Mutation, Protéines de fusion recombinantes, Protéines de l'enveloppe virale, Régulation positive, Structure secondaire des protéines, Séquence d'acides aminés, Ultracentrifugation, Virus du SRAS, Électrophorèse sur gel de polyacrylamide.
English descriptors
- KwdEn :
- Amino Acid Sequence, Circular Dichroism, Cloning, Molecular, Electrophoresis, Polyacrylamide Gel, Escherichia coli (genetics), Lipid Bilayers (chemistry), Molecular Sequence Data, Mutation, Protein Multimerization, Protein Structure, Secondary, Recombinant Fusion Proteins (chemistry), Recombinant Fusion Proteins (genetics), Recombinant Fusion Proteins (isolation & purification), SARS Virus (chemistry), SARS Virus (genetics), Severe Acute Respiratory Syndrome (virology), Ultracentrifugation, Up-Regulation, Viral Envelope Proteins (chemistry), Viral Envelope Proteins (genetics), Viral Envelope Proteins (isolation & purification).
- MESH :
- chemical , chemistry : Lipid Bilayers, Recombinant Fusion Proteins, Viral Envelope Proteins.
- chemistry : SARS Virus.
- genetics : Escherichia coli, Recombinant Fusion Proteins, SARS Virus, Viral Envelope Proteins.
- chemical , isolation & purification : Recombinant Fusion Proteins, Viral Envelope Proteins.
- virology : Severe Acute Respiratory Syndrome.
- Amino Acid Sequence, Circular Dichroism, Cloning, Molecular, Electrophoresis, Polyacrylamide Gel, Molecular Sequence Data, Mutation, Protein Multimerization, Protein Structure, Secondary, Ultracentrifugation, Up-Regulation.
Abstract
Coronavirus envelope (E) proteins are short (~100 residues) polypeptides that contain at least one transmembrane (TM) domain and a cluster of 2-3 juxtamembrane cysteines. These proteins are involved in viral morphogenesis and tropism, and their absence leads in some cases to aberrant virions, or to viral attenuation. In common to other viroporins, coronavirus envelope proteins increase membrane permeability to ions. Although an NMR-based model for the TM domain of the E protein in the severe acute respiratory syndrome virus (SARS-CoV E) has been reported, structural data and biophysical studies of full length E proteins are not available because efficient expression and purification methods for these proteins are lacking. Herein we have used a novel fusion protein consisting of a modified β-barrel to purify both wild type and cysteine-less mutants of two representatives of coronavirus E proteins: the shortest (76 residues), from SARS-CoV E, and one of the longest (109 residues), from the infectious bronchitis virus (IBV E). The fusion construct was subsequently cleaved with cyanogen bromide and all polypeptides were obtained with high purity. This is an approach that can be used in other difficult hydrophobic peptides.
DOI: 10.1016/j.pep.2012.07.005
PubMed: 22819936
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pubmed:22819936Le document en format XML
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<profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Amino Acid Sequence</term>
<term>Circular Dichroism</term>
<term>Cloning, Molecular</term>
<term>Electrophoresis, Polyacrylamide Gel</term>
<term>Escherichia coli (genetics)</term>
<term>Lipid Bilayers (chemistry)</term>
<term>Molecular Sequence Data</term>
<term>Mutation</term>
<term>Protein Multimerization</term>
<term>Protein Structure, Secondary</term>
<term>Recombinant Fusion Proteins (chemistry)</term>
<term>Recombinant Fusion Proteins (genetics)</term>
<term>Recombinant Fusion Proteins (isolation & purification)</term>
<term>SARS Virus (chemistry)</term>
<term>SARS Virus (genetics)</term>
<term>Severe Acute Respiratory Syndrome (virology)</term>
<term>Ultracentrifugation</term>
<term>Up-Regulation</term>
<term>Viral Envelope Proteins (chemistry)</term>
<term>Viral Envelope Proteins (genetics)</term>
<term>Viral Envelope Proteins (isolation & purification)</term>
</keywords>
<keywords scheme="KwdFr" xml:lang="fr"><term>Clonage moléculaire</term>
<term>Dichroïsme circulaire</term>
<term>Données de séquences moléculaires</term>
<term>Double couche lipidique ()</term>
<term>Escherichia coli (génétique)</term>
<term>Multimérisation de protéines</term>
<term>Mutation</term>
<term>Protéines de fusion recombinantes ()</term>
<term>Protéines de fusion recombinantes (génétique)</term>
<term>Protéines de fusion recombinantes (isolement et purification)</term>
<term>Protéines de l'enveloppe virale ()</term>
<term>Protéines de l'enveloppe virale (génétique)</term>
<term>Protéines de l'enveloppe virale (isolement et purification)</term>
<term>Régulation positive</term>
<term>Structure secondaire des protéines</term>
<term>Syndrome respiratoire aigu sévère (virologie)</term>
<term>Séquence d'acides aminés</term>
<term>Ultracentrifugation</term>
<term>Virus du SRAS ()</term>
<term>Virus du SRAS (génétique)</term>
<term>Électrophorèse sur gel de polyacrylamide</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Lipid Bilayers</term>
<term>Recombinant Fusion Proteins</term>
<term>Viral Envelope Proteins</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>
<term>Recombinant Fusion Proteins</term>
<term>SARS Virus</term>
<term>Viral Envelope Proteins</term>
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<term>Protéines de fusion recombinantes</term>
<term>Protéines de l'enveloppe virale</term>
<term>Virus du SRAS</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="isolation & purification" xml:lang="en"><term>Recombinant Fusion Proteins</term>
<term>Viral Envelope Proteins</term>
</keywords>
<keywords scheme="MESH" qualifier="isolement et purification" xml:lang="fr"><term>Protéines de fusion recombinantes</term>
<term>Protéines de l'enveloppe virale</term>
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<keywords scheme="MESH" qualifier="virologie" xml:lang="fr"><term>Syndrome respiratoire aigu sévère</term>
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<keywords scheme="MESH" qualifier="virology" xml:lang="en"><term>Severe Acute Respiratory Syndrome</term>
</keywords>
<keywords scheme="MESH" xml:lang="en"><term>Amino Acid Sequence</term>
<term>Circular Dichroism</term>
<term>Cloning, Molecular</term>
<term>Electrophoresis, Polyacrylamide Gel</term>
<term>Molecular Sequence Data</term>
<term>Mutation</term>
<term>Protein Multimerization</term>
<term>Protein Structure, Secondary</term>
<term>Ultracentrifugation</term>
<term>Up-Regulation</term>
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<term>Dichroïsme circulaire</term>
<term>Données de séquences moléculaires</term>
<term>Double couche lipidique</term>
<term>Multimérisation de protéines</term>
<term>Mutation</term>
<term>Protéines de fusion recombinantes</term>
<term>Protéines de l'enveloppe virale</term>
<term>Régulation positive</term>
<term>Structure secondaire des protéines</term>
<term>Séquence d'acides aminés</term>
<term>Ultracentrifugation</term>
<term>Virus du SRAS</term>
<term>Électrophorèse sur gel de polyacrylamide</term>
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<front><div type="abstract" xml:lang="en">Coronavirus envelope (E) proteins are short (~100 residues) polypeptides that contain at least one transmembrane (TM) domain and a cluster of 2-3 juxtamembrane cysteines. These proteins are involved in viral morphogenesis and tropism, and their absence leads in some cases to aberrant virions, or to viral attenuation. In common to other viroporins, coronavirus envelope proteins increase membrane permeability to ions. Although an NMR-based model for the TM domain of the E protein in the severe acute respiratory syndrome virus (SARS-CoV E) has been reported, structural data and biophysical studies of full length E proteins are not available because efficient expression and purification methods for these proteins are lacking. Herein we have used a novel fusion protein consisting of a modified β-barrel to purify both wild type and cysteine-less mutants of two representatives of coronavirus E proteins: the shortest (76 residues), from SARS-CoV E, and one of the longest (109 residues), from the infectious bronchitis virus (IBV E). The fusion construct was subsequently cleaved with cyanogen bromide and all polypeptides were obtained with high purity. This is an approach that can be used in other difficult hydrophobic peptides.</div>
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