Control of streptokinase gene expression in group A & C streptococci by two-component regulators.

BACKGROUND & OBJECTIVES: Group A streptococci (GAS) and human isolates of group C streptococci (GCS) have the stable capacity to produce the plasminogen activator streptokinase, albeit with varying efficiency. This property is subject to control by two two-component regulatory systems, FasCAX and CovRS, which act as activator and repressor, respectively. The present work aims at balancing these opposing activities in GAS and GCS, and at clarifying the phylogenetic position of the FasA response regulator, the less understood regulator of the two systems. METHODS: The GCS strain H46A and GAS strain NZ131 were used. Escherichia coli JM 109 was used as host for plasmid construction. Streptokinase activity of various wild type and mutant strains was measured. Phylogenetic trees of streptococcal FasA homologues were established. RESULTS: The streptokinase activities of the GAS strain NZ131 and the GCS strain H46A were attributable to more efficient CovR repressor action in NZ131 than in H46A. The FasA activator, on the other hand, functioned about equally efficient in the two strains. Phylogenetically, FasA homologues clustered distinctly in the proposed FasA-BlpR-ComE family of streptococcal response regulators and used the LytTR domain for DNA binding. INTERPRETATION & CONCLUSION: Assessing the apparent streptokinase activity of streptoccal strains require the dissection of the activities of the cov and fas systems. Although experimental evidence is still missing, FasA is closely related to a widely distributed family of streptococcal response regulators that is involved in behavioral processes, such as quorum sensing.

Cloning and expression of an actinokinase gene from a thermophilic Streptomyces in Escherechia coli.

A thermophilic Streptomyces megasporus strain SD5, could secrete a new fibrinolytic (actinokinase) at 55 degrees C. The gene (ackS) encoding actinokinase was isolated from the chromosomal DNA of S. megasporus SD5 and cloned in different hosts and vectors. The expression was obtained in E. coli JM109 using Cla I linearized pBR322 as vector (pSR 500). The recombinant E. coli containing pSR 500 expressed active actinokinase but the expression was low and the recombinant was unstable in liquid culture. Deletion analysis revealed that removal of Bam H I-Sal I fragment from down stream and Cla I-EcoRI from upsream enhanced the stability and expression of ackS in both solid and liquid media. For over expresion, the ackS gene was cloned in E. coli C 600 using Bam HI linearized pT7-7. This seemed to be the most suitable host vector system. The recombinant and native form of actinokinase exhibited similar characteristics. Actinokinase was the first thrombolytic enzyme from a thermophile to be cloned and over expressed in a mesophilic heterologous expression system.

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 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.