Production Of Streptokinase Research Articles

Recombinant streptokinase production by fed-batch cultivation of Escherichia coli

Streptokinase (SK) is a specific effective medicine for thrombolytic therapy of acute myocardial infarction. This study established a process for the pilot production of recombinant streptokinase (r-SK). Engineering bacteria were fermented in a 20-l fermentor to produce r-SK. After simple renaturation and purification, 12.9 g of r-SK with 97.8% of purity and about 10 5 IU mg −1 of specific activity was obtained, the yield of protein and the recovery of activity were 44.9% and 51%, respectively. Finally, the r-SK was made into about 700 doses of injections for clinical applications.

Streptokinase as a mediator of acute post-streptococcal glomerulonephritis in an experimental mouse model.

Group A streptococcal infections are sometimes followed by the inflammatory kidney disease acute post-streptococcal glomerulonephritis (APSGN). To test the importance of streptokinase in the pathogenesis of this disease, isogenic strains of the nephritis isolate NZ131, differing only in the ability to produce streptokinase of the nephritis-associated ska1 genotype, were used for infection in a mouse tissue cage model for APSGN. Streptokinase production was found to be a prerequisite for the capacity of the strain to induce APSGN in mice. In addition, streptokinase was demonstrated in the kidneys of mice infected with the nephritogenic NZ131 and EF514 strains. After infection with the nonnephritogenic strain S84, neither streptokinase nor C3 deposition were observed. Deposition of streptokinase in the glomeruli was detected as soon as 4 days after infection. These findings provide support for the hypothesis that streptokinase initiates the nephritis process by glomerular deposition, which leads to local activation of the complement cascade. Detection of streptokinase in kidney tissue increased with the degree of glomerular hypercellularity. Thus, the severity of the pathological process may be a reflection of the degree of streptokinase deposition.

Enhanced production and secretion of streptokinase into extracellular medium in Escherichia coli by removal of 13 N-terminal amino acids

The production and secretion of streptokinase using OmpA signal sequence in E. coli was enhanced by removing the 13 N-terminal amino acids (SK(N13). The secretion level of SK(N13 protein into the extracellular medium was two times higher than that of wild-type streptokinase. About 4500 IU of SK(N13 protein per 1 ml LB-ampicillin medium was secreted into extracellular medium at 12 hours after induction. Fully active and enhanced extracellular preparation of the mutant streptokinase may be a potential alternative source for the simple downstream processing

Incomplete mixing in large bioreactors ? a study of its role in the fermentative production of streptokinase

The production of streptokinase in a batch fermentation has been analysed for the role of incomplete macromixing of the broth. The analysis is based on a kinetic model exhibiting inhibition by the substrate and a primary metabolite (lactic acid), and a mixing model comprising two continuous flow reactors (CFRs) with closed-loop recycle. The inoculum is introduced into one region (one CFR) and the mixing process determines its distribution, growth and reactivity. By varying the dilution rates of the CFRs, any degree of macromixing can be simulated. For dilution rates larger than 1.0 h−1 almost complete macromixing is achieved, for which an analogy has been drawn with micromixing. Increasing the volume of the inoculated region relative to the noninoculated region improves the maximum attainable activity of streptokinase and shortens the time for this. In such a situation an imperfectly mixed bioreactor is superior to a perfectly mixed one, implying that good productivity requires a large inoculated region and incomplete macromixing. These inferences are supported by earlier studies of fluid mixing and relaxation times in bioreactors.

Incomplete mixing in large bioreactors – a study of its role in the fermentative production of streptokinase

Incomplete mixing in large bioreactors – a study of its role in the fermentative production of streptokinase

Streptokinase activity among group A streptococci in relation to streptokinase genotype, plasminogen binding, and disease manifestations

Certain genotypic variants of streptokinase (ska) of beta-hemolytic streptococci group A have been associated with acute post-streptococcal glomerulonephritis (APSGN). In our earlier studies on strains isolated from Ethiopian children with various streptococcal disease manifestation, we reported an even distribution of streptokinase genotypes with no association to disease patterns. Considering the possibility that strains could differ in their ability to secrete the protein, levels of streptokinase activity in culture supernatants of these strains were determined by a plasminogen activation assay using a synthetic tripeptide, H-D-valyl-leucyl-lysin-p-nitroaniline, as a substrate. Of the 53 streptococcal group A strains, ten (19%), which belonged to genotype ska4 and ska8, did not activate human plasminogen. These strains did not activate bovine, sheep, horse, rabbit or porcine plasminogens either. They represented at least five M protein and non-typeable serotypes, and were characterized by high human plasminogen binding activity. Six of the 53 strains (11%) harbouring genotype ska3 and ska7 showed low levels of human plasminogen activation. Strains of ska1 and ska2, 37/53, activated human plasminogen at a higher level (p < 0.005). Levels of plasminogen activation were not significantly different among the ska1 and ska2 strains associated with various streptococcal disease manifestations. Antibody levels against streptokinase were higher (p < 0.05) in convalescent sera from acute rheumatic fever and APSGN patients in comparison with sera from other patient categories and healthy controls. Streptokinase genotype and in vitro streptokinase production do not correlate directly to streptococcal disease manifestation, indicating a probable significance of additional streptococcal and/or host factors in the initiation of APSGN.

Streptokinase activity among group a streptococci in relation to streptokinase genotype, plasminogen binding, and disease manifestations

Certain genotypic variants of streptokinase (ska) of beta-hemolytic streptococci group A have been associated with acute post-streptococcal glomerulonephritis (APSGN). In our earlier studies on strains isolated from Ethiopian children with various streptococcal disease manifestation, we reported an even distribution of streptokinase genotypes with no association to disease patterns. Considering the possibility that strains could differ in their ability to secrete the protein, levels of streptokinase activity in culture supernatants of these strains were determined by a plasminogen activation assay using a synthetic tripeptide, H-D-valyl-leucyl-lysin-p-nitroaniline, as a substrate. Of the 53 streptococcal group A strains, ten (19%), which belonged to genotype ska4 and ska8, did not activate human plasminogen. These strains did not activate bovine, sheep, horse, rabbit or porcine plasminogens either. They represented at least five M protein and non-typeable serotypes, and were characterized by high human plasminogen binding activity. Six of the 53 strains (11%) harbouring genotype ska3 and ska7 showed low levels of human plasminogen activation. Strains of ska1 and ska2, 37/53, activated human plasminogen at a higher level (p < 0.005). Levels of plasminogen activation were not significantly different among the ska1 and ska2 strains associated with various streptococcal disease manifestations. Antibody levels against streptokinase were higher (p < 0.05) in convalescent sera from acute rheumatic fever and APSGN patients in comparison with sera from other patient categories and healthy controls. Streptokinase genotype and in vitro streptokinase production do not correlate directly to streptococcal disease manifestation, indicating a probable significance of additional streptococcal and/or host factors in the initiation of APSGN.

Polymorphism of the Streptokinase Gene: Implications for the Pathogenesis of Post-Streptococcal Glomerulonephritis

Recent studies of streptokinase genes from epidemiologically and clinically defined streptococci of groups A, C and G have provided evidence of the polymorphism of the streptokinase locus in the chromosome of pathogenic streptococci. This review considers genetic and pathogenetic data suggesting that there exists a causal relationship between nephritis strain-associated streptokinase production and the initial stages of post-streptococcal glomerulonephritis (PSGN). Currently available sequence information allows to recognize, in the middle of the streptokinase molecule, a major variable region, V1, of about 70 amino acid residues in which sequence identity drops to below 50% when the proteins from nephritogenic and non-nephritogenic strains are compared. The V1 regions, although showing microheterogeneity within either protein category, appear to be more hydrophobic and possess a higher content of ordered secondary structures in the "nephritogenic" molecules. As a working hypothesis, they may be considered the nephrotropic domain(s) with which streptokinases from nephritogenic strains bind to glomerular structures and activate plasminogen in situ, thus triggering the cascade of proteolytic processes leading to PSGN.

Sunflower peptones: use as nitrogen source for the formulation of fermentation media

Sunflower peptones were used as a nitrogen source for the growth of Streptococcus equisimilis H-46 on defined fermentation media for the production of streptokinase (SK). The growth of S. equisimilis H-46 on PC-peptone, obtained from sunflower protein concentrate (SF-PC) with a reduced content of polyphenols, is similar to that obtained in the media formulated with NZ-Amine R, usually used as the nitrogen source for the production of SK. However, SK production is higher in the medium formulated with PC-peptone than in that formulated with NZ-Amine R.

Duplication of the streptokinase gene in the chromosome ofStreptococcus equisimilisH46A

The erythromycin resistance plasmid pSM752 carrying the cloned streptokinase gene, skc, was introduced by protoplast transformation into Streptococcus equisimilis H46A from which skc was originally cloned. Cells transiently supporting the replication of pSM752 gave rise to an erythromycin-resistant clone designated H46SM which was plasmid free and produced streptokinase at levels approximately twice as high as the wild type. Southern hybridization of total cell DNA with an skc-containing probe provided evidence for the duplication of the skc gene in the H46SM chromosome. The results, which have some bearing on industrial streptokinase production, can be best explained by a single cross-over event between the chromosome and the plasmid in the region of shared homology leading to the integration of pSM752 in a Campbell-like manner.

Duplication of the streptokinase gene in the chromosome of Streptococcus equisimilis H46A

The erythromycin resistance plasmid pSM752 carrying the cloned streptokinase gene, skc, was introduced by protoplast transformation into Streptococcus equisimilis H46A from which skc was originally cloned. Cells transiently supporting the replication of pSM752 gave rise to an erythromycin-resistant clone designated H46SM which was plasmid free and produced streptokinase at levels approximately twice as high as the wild type. Southern hybridization of total cell DNA with an skc-containing probe provided evidence for the duplication of the skc gene in the H46SM chromosome. The results, which have some bearing on industrial streptokinase production, can be best explained by a single cross-over event between the chromosome and the plasmid in the region of shared homology leading to the integration of pSM752 in a Campbell-like manner.

Streptokinase: cloning, expression, and excretion by Escherichia coli.

Genomic DNA from Streptococcus equisimilis strain H46A was cloned in Escherichia coli by using the bacteriophage lambda replacement vector L47 and an in vitro packaging system. A casein/plasminogen overlay technique was used to screen the phage bank for recombinants carrying the streptokinase gene ( skc ). The gene was present with a frequency of 1 in 836 recombinants, and 10 independent clones containing skc were isolated and physically characterized. One recombinant clone was used to subclone skc in E. coli plasmid vectors. Plasmid pMF2 [10.4 kilobases (kb)] consisting of pACYC184 with a 6.4-kb H46A DNA fragment in the EcoRI site and pMF5 (6.9 kb) carrying a 2.5-kb fragment in the Pst I site of pBR322 were among the recombinant plasmids determining streptokinase production in three different E. coli host strains. Expression of skc was independent of its orientation in either vector, indicating that its own promoter was present and functional in E. coli. However, expression in pBR322 was more efficient in one orientation than in the other, suggesting that one or both of the bla gene promoters contributed to skc expression. Several lines of evidence, including proof obtained by the immunodiffusion technique, established the identity of E. coli streptokinase. Testing cell-free culture supernatant fluids, osmotic shock fluids, and sonicates of osmotically shocked cells for streptokinase activity revealed the substance to be present in all three principal locations, indicating that E. coli cells were capable of releasing substantial amounts of streptokinase into the culture medium.

Production of streptokinase in continuous culture.

A method for continuous cultivation of a beta-hemolytic streptococcus, strain H 64, is described. The production of cells and streptokinase at various dilution rates, pH, and temperature were studied in a complex medium supplied with excess glucose. At pH 7.0, productivity of cells and streptokinase, as well as the yield constant with respect to glucose, all increased with increasing dilution rate in the range of 0.1 to 0.5 hr(-1). The production of streptokinase was found to be a function of both growth rate and cell concentration. Although higher concentrations of streptokinase were obtained in experiments with batch cultures, the production of streptokinase in continuous cultures was found to be 2.3 times higher. The possible industrial application of a continuous production method is considered.

Production of Streptokinase in Continuous Culture

A method for continuous cultivation of a beta-hemolytic streptococcus, strain H 64, is described. The production of cells and streptokinase at various dilution rates, pH, and temperature were studied in a complex medium supplied with excess glucose. At pH 7.0, productivity of cells and streptokinase, as well as the yield constant with respect to glucose, all increased with increasing dilution rate in the range of 0.1 to 0.5 hr(-1). The production of streptokinase was found to be a function of both growth rate and cell concentration. Although higher concentrations of streptokinase were obtained in experiments with batch cultures, the production of streptokinase in continuous cultures was found to be 2.3 times higher. The possible industrial application of a continuous production method is considered.

The streptokinase-plasminogen system. II. Its effect on the development of local streptococcal infections in rabbit skin.

Streptokinase, an extracellular product of the majority of group A streptococcal strains, activates plasminogen, a proenzyme present in the euglobulin fraction of human blood, to the proteolytic enzyme plasmin, which is able to lyse fibrin and other protein substrates. Streptokinase does not act directly upon plasminogen but rather through a proactivator to form an activator which in turn catalyzes the conversion of plasminogen to plasmin (M ullertz, 1957; Troll and Sherry, 1955). Its fibrinolytic activity may play a significant part in the pathogenesis of streptococcal infections by interfering with the formation of a localizing fibrin barrier. Attempts to correlate streptokinase production in vitro with streptococcal virulence have led to conflicting results (Stewart, 1936; Madison, 1934; Sherwood et al, 1952; Rantz and Boisvert, 1948). Krasner and Young (1959) reported that streptokinase contributes to the pathogenicity of group A streptococci by an i n vivo interaction with plasminogen. Their work demonstrated that the pathogenicity of several group A streptokinase-positive streptococcal strains was significantly enhanced in terms of mouse mortality by injecting the organisms suspended in small amounts of human plasminogen. With streptokinase-negative organisms the combination of plasminogen and streptokinase similarly enhanced virulence, although either product alone was without effect. The biological system employed in the investigation did not permit observation of the mechanism of