L. Avilan et al., CLONING, EXPRESSION AND PURIFICATION OF RECOMBINANT STREPTOKINASE - PARTIAL CHARACTERIZATION OF THE PROTEIN EXPRESSED IN ESCHERICHIA-COLI, Brazilian journal of medical and biological research, 30(12), 1997, pp. 1427-1430
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.