A high-copy T7 Escherichia coli expression vector for the production of recombinant proteins with a minimal N-terminal His-tagged fusion peptide

Detalhes bibliográficos
Autor(a) principal: Ramos,C.R.R.
Data de Publicação: 2004
Outros Autores: Abreu,P.A.E., Nascimento,A.L.T.O., Ho,P.L.
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Brazilian Journal of Medical and Biological Research
Texto Completo: http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2004000800001
Resumo: We report here the construction of a vector derived from pET3-His and pRSET plasmids for the expression and purification of recombinant proteins in Escherichia coli based on T7 phage RNA polymerase. The resulting pAE plasmid combined the advantages of both vectors: small size (pRSET), expression of a short 6XHis tag at N-terminus (pET3-His) and a high copy number of plasmid (pRSET). The small size of the vector (2.8 kb) and the high copy number/cell (200-250 copies) facilitate the subcloning and sequencing procedures when compared to the pET system (pET3-His, 4.6 kb and 40-50 copies) and also result in high level expression of recombinant proteins (20 mg purified protein/liter of culture). In addition, the vector pAE enables the expression of a fusion protein with a minimal amino-terminal hexa-histidine affinity tag (a tag of 9 amino acids using XhoI restriction enzyme for the 5'cloning site) as in the case of pET3-His plasmid and in contrast to proteins expressed by pRSET plasmids (a tag of 36 amino acids using BamHI restriction enzyme for the 5'cloning site). Thus, although proteins expressed by pRSET plasmids also have a hexa-histidine tag, the fusion peptide is much longer and may represent a problem for some recombinant proteins.
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spelling A high-copy T7 Escherichia coli expression vector for the production of recombinant proteins with a minimal N-terminal His-tagged fusion peptideEscherichia coliExpression vectorImmobilized metal affinity chromatographyProtein purificationWe report here the construction of a vector derived from pET3-His and pRSET plasmids for the expression and purification of recombinant proteins in Escherichia coli based on T7 phage RNA polymerase. The resulting pAE plasmid combined the advantages of both vectors: small size (pRSET), expression of a short 6XHis tag at N-terminus (pET3-His) and a high copy number of plasmid (pRSET). The small size of the vector (2.8 kb) and the high copy number/cell (200-250 copies) facilitate the subcloning and sequencing procedures when compared to the pET system (pET3-His, 4.6 kb and 40-50 copies) and also result in high level expression of recombinant proteins (20 mg purified protein/liter of culture). In addition, the vector pAE enables the expression of a fusion protein with a minimal amino-terminal hexa-histidine affinity tag (a tag of 9 amino acids using XhoI restriction enzyme for the 5'cloning site) as in the case of pET3-His plasmid and in contrast to proteins expressed by pRSET plasmids (a tag of 36 amino acids using BamHI restriction enzyme for the 5'cloning site). Thus, although proteins expressed by pRSET plasmids also have a hexa-histidine tag, the fusion peptide is much longer and may represent a problem for some recombinant proteins.Associação Brasileira de Divulgação Científica2004-08-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersiontext/htmlhttp://old.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2004000800001Brazilian Journal of Medical and Biological Research v.37 n.8 2004reponame:Brazilian Journal of Medical and Biological Researchinstname:Associação Brasileira de Divulgação Científica (ABDC)instacron:ABDC10.1590/S0100-879X2004000800001info:eu-repo/semantics/openAccessRamos,C.R.R.Abreu,P.A.E.Nascimento,A.L.T.O.Ho,P.L.eng2004-07-20T00:00:00Zoai:scielo:S0100-879X2004000800001Revistahttps://www.bjournal.org/https://old.scielo.br/oai/scielo-oai.phpbjournal@terra.com.br||bjournal@terra.com.br1414-431X0100-879Xopendoar:2004-07-20T00:00Brazilian Journal of Medical and Biological Research - Associação Brasileira de Divulgação Científica (ABDC)false
dc.title.none.fl_str_mv A high-copy T7 Escherichia coli expression vector for the production of recombinant proteins with a minimal N-terminal His-tagged fusion peptide
title A high-copy T7 Escherichia coli expression vector for the production of recombinant proteins with a minimal N-terminal His-tagged fusion peptide
spellingShingle A high-copy T7 Escherichia coli expression vector for the production of recombinant proteins with a minimal N-terminal His-tagged fusion peptide
Ramos,C.R.R.
Escherichia coli
Expression vector
Immobilized metal affinity chromatography
Protein purification
title_short A high-copy T7 Escherichia coli expression vector for the production of recombinant proteins with a minimal N-terminal His-tagged fusion peptide
title_full A high-copy T7 Escherichia coli expression vector for the production of recombinant proteins with a minimal N-terminal His-tagged fusion peptide
title_fullStr A high-copy T7 Escherichia coli expression vector for the production of recombinant proteins with a minimal N-terminal His-tagged fusion peptide
title_full_unstemmed A high-copy T7 Escherichia coli expression vector for the production of recombinant proteins with a minimal N-terminal His-tagged fusion peptide
title_sort A high-copy T7 Escherichia coli expression vector for the production of recombinant proteins with a minimal N-terminal His-tagged fusion peptide
author Ramos,C.R.R.
author_facet Ramos,C.R.R.
Abreu,P.A.E.
Nascimento,A.L.T.O.
Ho,P.L.
author_role author
author2 Abreu,P.A.E.
Nascimento,A.L.T.O.
Ho,P.L.
author2_role author
author
author
dc.contributor.author.fl_str_mv Ramos,C.R.R.
Abreu,P.A.E.
Nascimento,A.L.T.O.
Ho,P.L.
dc.subject.por.fl_str_mv Escherichia coli
Expression vector
Immobilized metal affinity chromatography
Protein purification
topic Escherichia coli
Expression vector
Immobilized metal affinity chromatography
Protein purification
description We report here the construction of a vector derived from pET3-His and pRSET plasmids for the expression and purification of recombinant proteins in Escherichia coli based on T7 phage RNA polymerase. The resulting pAE plasmid combined the advantages of both vectors: small size (pRSET), expression of a short 6XHis tag at N-terminus (pET3-His) and a high copy number of plasmid (pRSET). The small size of the vector (2.8 kb) and the high copy number/cell (200-250 copies) facilitate the subcloning and sequencing procedures when compared to the pET system (pET3-His, 4.6 kb and 40-50 copies) and also result in high level expression of recombinant proteins (20 mg purified protein/liter of culture). In addition, the vector pAE enables the expression of a fusion protein with a minimal amino-terminal hexa-histidine affinity tag (a tag of 9 amino acids using XhoI restriction enzyme for the 5'cloning site) as in the case of pET3-His plasmid and in contrast to proteins expressed by pRSET plasmids (a tag of 36 amino acids using BamHI restriction enzyme for the 5'cloning site). Thus, although proteins expressed by pRSET plasmids also have a hexa-histidine tag, the fusion peptide is much longer and may represent a problem for some recombinant proteins.
publishDate 2004
dc.date.none.fl_str_mv 2004-08-01
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2004000800001
url http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2004000800001
dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv 10.1590/S0100-879X2004000800001
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv text/html
dc.publisher.none.fl_str_mv Associação Brasileira de Divulgação Científica
publisher.none.fl_str_mv Associação Brasileira de Divulgação Científica
dc.source.none.fl_str_mv Brazilian Journal of Medical and Biological Research v.37 n.8 2004
reponame:Brazilian Journal of Medical and Biological Research
instname:Associação Brasileira de Divulgação Científica (ABDC)
instacron:ABDC
instname_str Associação Brasileira de Divulgação Científica (ABDC)
instacron_str ABDC
institution ABDC
reponame_str Brazilian Journal of Medical and Biological Research
collection Brazilian Journal of Medical and Biological Research
repository.name.fl_str_mv Brazilian Journal of Medical and Biological Research - Associação Brasileira de Divulgação Científica (ABDC)
repository.mail.fl_str_mv bjournal@terra.com.br||bjournal@terra.com.br
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