Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coli
Autor(a) principal: | |
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Data de Publicação: | 1997 |
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=S0100-879X1997001200007 |
Resumo: | We cloned the streptokinase (STK) gene of Streptococcus equisimilis in an expression vector of Escherichia coli to overexpress the profibrinolytic protein under the control of a tac promoter. Almost all the recombinant STK was exported to the periplasmic space and recovered after gentle lysozyme digestion of induced cells. The periplasmic fraction was chromatographed on DEAE Sepharose followed by chromatography on phenyl-agarose. Active proteins eluted between 4.5 and 0% ammonium sulfate, when a linear gradient was applied. Three major STK derivatives of 47.5 kDa, 45 kDa and 32 kDa were detected by Western blot analysis with a polyclonal antibody. The 32-kDa protein formed a complex with human plasminogen but did not exhibit Glu-plasminogen activator activity, as revealed by a zymographic assay, whereas the 45-kDa protein showed a Km = 0.70 µM and kcat = 0.82 s-1, when assayed with a chromogen-coupled substrate. These results suggest that these proteins are putative fragments of STK, possibly derived from partial degradation during the export pathway or the purification steps. The 47.5-kDa band corresponded to the native STK, as revealed by peptide sequencing |
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Brazilian Journal of Medical and Biological Research |
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Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia colirecombinant streptokinaseplasminogen activatorsprotein purificationWe cloned the streptokinase (STK) gene of Streptococcus equisimilis in an expression vector of Escherichia coli to overexpress the profibrinolytic protein under the control of a tac promoter. Almost all the recombinant STK was exported to the periplasmic space and recovered after gentle lysozyme digestion of induced cells. The periplasmic fraction was chromatographed on DEAE Sepharose followed by chromatography on phenyl-agarose. Active proteins eluted between 4.5 and 0% ammonium sulfate, when a linear gradient was applied. Three major STK derivatives of 47.5 kDa, 45 kDa and 32 kDa were detected by Western blot analysis with a polyclonal antibody. The 32-kDa protein formed a complex with human plasminogen but did not exhibit Glu-plasminogen activator activity, as revealed by a zymographic assay, whereas the 45-kDa protein showed a Km = 0.70 µM and kcat = 0.82 s-1, when assayed with a chromogen-coupled substrate. These results suggest that these proteins are putative fragments of STK, possibly derived from partial degradation during the export pathway or the purification steps. The 47.5-kDa band corresponded to the native STK, as revealed by peptide sequencingAssociação Brasileira de Divulgação Científica1997-12-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersiontext/htmlhttp://old.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X1997001200007Brazilian Journal of Medical and Biological Research v.30 n.12 1997reponame:Brazilian Journal of Medical and Biological Researchinstname:Associação Brasileira de Divulgação Científica (ABDC)instacron:ABDC10.1590/S0100-879X1997001200007info:eu-repo/semantics/openAccessAvilán,L.Yarzábal,A.Jürgensen,C.Bastidas,M.Cruz,J.Puig,J.eng1998-10-07T00:00:00Zoai:scielo:S0100-879X1997001200007Revistahttps://www.bjournal.org/https://old.scielo.br/oai/scielo-oai.phpbjournal@terra.com.br||bjournal@terra.com.br1414-431X0100-879Xopendoar:1998-10-07T00:00Brazilian Journal of Medical and Biological Research - Associação Brasileira de Divulgação Científica (ABDC)false |
dc.title.none.fl_str_mv |
Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coli |
title |
Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coli |
spellingShingle |
Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coli Avilán,L. recombinant streptokinase plasminogen activators protein purification |
title_short |
Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coli |
title_full |
Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coli |
title_fullStr |
Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coli |
title_full_unstemmed |
Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coli |
title_sort |
Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coli |
author |
Avilán,L. |
author_facet |
Avilán,L. Yarzábal,A. Jürgensen,C. Bastidas,M. Cruz,J. Puig,J. |
author_role |
author |
author2 |
Yarzábal,A. Jürgensen,C. Bastidas,M. Cruz,J. Puig,J. |
author2_role |
author author author author author |
dc.contributor.author.fl_str_mv |
Avilán,L. Yarzábal,A. Jürgensen,C. Bastidas,M. Cruz,J. Puig,J. |
dc.subject.por.fl_str_mv |
recombinant streptokinase plasminogen activators protein purification |
topic |
recombinant streptokinase plasminogen activators protein purification |
description |
We cloned the streptokinase (STK) gene of Streptococcus equisimilis in an expression vector of Escherichia coli to overexpress the profibrinolytic protein under the control of a tac promoter. Almost all the recombinant STK was exported to the periplasmic space and recovered after gentle lysozyme digestion of induced cells. The periplasmic fraction was chromatographed on DEAE Sepharose followed by chromatography on phenyl-agarose. Active proteins eluted between 4.5 and 0% ammonium sulfate, when a linear gradient was applied. Three major STK derivatives of 47.5 kDa, 45 kDa and 32 kDa were detected by Western blot analysis with a polyclonal antibody. The 32-kDa protein formed a complex with human plasminogen but did not exhibit Glu-plasminogen activator activity, as revealed by a zymographic assay, whereas the 45-kDa protein showed a Km = 0.70 µM and kcat = 0.82 s-1, when assayed with a chromogen-coupled substrate. These results suggest that these proteins are putative fragments of STK, possibly derived from partial degradation during the export pathway or the purification steps. The 47.5-kDa band corresponded to the native STK, as revealed by peptide sequencing |
publishDate |
1997 |
dc.date.none.fl_str_mv |
1997-12-01 |
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=S0100-879X1997001200007 |
url |
http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X1997001200007 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
10.1590/S0100-879X1997001200007 |
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.30 n.12 1997 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_ |
1754302928884072448 |