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

L. Avilán, A. Yarzábal, C. Jürgensen, M. Bastidas, J. Cruz, J. Puig

Research output: Contribution to journalArticlepeer-review

Abstract

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 microM 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.

Original languageEnglish
Pages (from-to)1427-30
Number of pages4
JournalBrazilian Journal of Medical and Biological Research
Volume30
Issue number12
DOIs
Publication statusPublished - Dec 1997

Keywords

  • Cloning, Molecular
  • Escherichia coli
  • Recombinant Proteins
  • Streptococcus/genetics
  • Streptokinase/genetics

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