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 profibrin olytic protein under the control of a tac promoter. Almost all the rec ombinant 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 ph enyl-agarose. Active proteins eluted between 4.5 and 0% ammonium sulfa te, 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 wi th a polyclonal antibody. The 32-kDa protein formed a complex with hum an plasminogen but did not exhibit Glu-plasminogen activator activity, as revealed by a zymographic assay, whereas the 45-kDa protein showed a K-m = 0.70 mu M and k(cat) = 0.82 s(-1), when assayed with a chromo gen-coupled substrate. These results suggest that these proteins are p utative fragments of STK, possibly derived from partial degradation du ring the export pathway or the purification steps. The 47.5-kDa band c orresponded to the native STK, as revealed by peptide sequencing.