A high-copy T7 Escherichia coli expression vector for the production of recombinant proteins with a minimal N-terminal His-tagged fusion peptide
Autor(a) principal: | |
---|---|
Data de Publicação: | 2004 |
Outros Autores: | , , |
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. |
id |
ABDC-1_806b319a6045773b75572e5a35fa4793 |
---|---|
oai_identifier_str |
oai:scielo:S0100-879X2004000800001 |
network_acronym_str |
ABDC-1 |
network_name_str |
Brazilian Journal of Medical and Biological Research |
repository_id_str |
|
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 |
_version_ |
1754302933073133568 |