Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coli

Detalhes bibliográficos
Autor(a) principal: Avilán,L.
Data de Publicação: 1997
Outros Autores: Yarzábal,A., Jürgensen,C., Bastidas,M., Cruz,J., Puig,J.
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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spelling 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
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