Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coli
Braz. j. med. biol. res
;
30(12): 1427-30, Dec. 1997. ilus, graf
Article
Dans Anglais
| LILACS
| ID: lil-212586
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 periplasmatic fraction was chromatographed on DEAE Sepharose followed by chromatography on phenyl-agarose. Active proteins eluted between 4.5 and 0 percent ammonium sulfate, when a linear grandient was applied. Theree 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 muM and kcat = 0.82 s(-1), when assayed with a chromogen-coupled subtrate. 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.
Texte intégral:
Disponible
Indice:
LILAS (Amériques)
Sujet Principal:
Streptococcus
/
Streptokinase
/
Protéines recombinantes
/
Clonage moléculaire
/
Escherichia coli
langue:
Anglais
Texte intégral:
Braz. j. med. biol. res
Thème du journal:
Biologie
/
Médicament
Année:
1997
Type:
Article
/
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