Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coli
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 dig...
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| Format: | Article |
| Language: | English |
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Associação Brasileira de Divulgação Científica
1997-12-01
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| Series: | Brazilian Journal of Medical and Biological Research |
| Subjects: | |
| Online Access: | http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X1997001200007 |
| _version_ | 1819140083324813312 |
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| author | L. Avilán A. Yarzábal C. Jürgensen M. Bastidas J. Cruz J. Puig |
| author_facet | L. Avilán A. Yarzábal C. Jürgensen M. Bastidas J. Cruz J. Puig |
| author_sort | L. Avilán |
| collection | DOAJ |
| 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 |
| first_indexed | 2024-12-22T11:32:55Z |
| format | Article |
| id | doaj.art-0781712de672405facb7ae83c13b699f |
| institution | Directory Open Access Journal |
| issn | 0100-879X 1414-431X |
| language | English |
| last_indexed | 2024-12-22T11:32:55Z |
| publishDate | 1997-12-01 |
| publisher | Associação Brasileira de Divulgação Científica |
| record_format | Article |
| series | Brazilian Journal of Medical and Biological Research |
| spelling | doaj.art-0781712de672405facb7ae83c13b699f2022-12-21T18:27:32ZengAssociação Brasileira de Divulgação CientíficaBrazilian Journal of Medical and Biological Research0100-879X1414-431X1997-12-013012142710.1590/S0100-879X1997001200007Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coliL. AvilánA. YarzábalC. JürgensenM. BastidasJ. CruzJ. PuigWe 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 sequencinghttp://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X1997001200007recombinant streptokinaseplasminogen activatorsprotein purification |
| spellingShingle | L. Avilán A. Yarzábal C. Jürgensen M. Bastidas J. Cruz J. Puig Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coli Brazilian Journal of Medical and Biological Research recombinant streptokinase plasminogen activators protein purification |
| title | 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_short | 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 |
| topic | recombinant streptokinase plasminogen activators protein purification |
| url | http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X1997001200007 |
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