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

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 K m = 0.70 µM and k cat = 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.

Recombinant production of Streptococcus equisimilis streptokinase by Streptomyces lividans

“…Similar degradation of recombinant SK has been also reported to occur in heterologous hosts such as Streptococcus sanguis [23] and E. coli [26]. Chymotrypsin-like activity and several genes encoding chymotrypsin-like serine proteases have been reported in S. lividans 66 [27,28].…”

Purification and Characterization of Fibrinolytic Enzyme from Pseudoalteromonas sp., IND11 and its in vitro Activity on Blood Clot

“…The matrix such as cationic exchanger is helpful for the separation of proteases by pH gradient or increasing salt concentration (Tsuchiya et al, 1992). From the earlier studies the fibrinolytic enzymes purified from the earthworm Lumbricus rubellus (Mihara et al, 1991); Streptococcus aureus (Avilan et al, 1997) and from the fruiting body of mushrooms, namely Armillaria mellea, Tricholoma saponaceum and Cardyceps militaris Kim, 1999, 2001;Kim et al, 2006).…”

Renaturation, purification and characterization of streptokinase expressed as inclusion body in recombinant E. coli

“…Subsequently streptokinase fragment from the genome of S. equisimilis H46A encoding the SK gene was sequenced [12]. The SK gene was then cloned in several Gram-negative and Gram-positive bacteria [13][14][15][16][17]. Several methods of recovery and purification of SK have been described previously which includes purification of wild strain as well as recombinant SK [15,16,[19][20][21][22].…”