Streptokinase Gene Research Articles

Detection of Streptokinase Production by Streptococcus pyogen using Real Time PCR

Tonsillitis which is commonly referred to a strep throat is caused by group A β-hemolytic streptococci (GABHS) or Streptococcus pyogenes. The bacteria is consider as one of the major human-specific bacterial pathogen and one of its virulence factors is the streptokinase; for this reason, 100 tonsillitis swabs were taken from Iraqi children suffering from symptoms of sore throat. Using conventional cultural methods, 60 bacterial isolates were characterized and subjected to biochemical tests. As a result, 40 isolates were identified as hemolytic bacteria and only 6 of them were identified as Streptococcus. pyogens using Bacitracin (0.04) and Vitek 2 system. The ability of the 6 isolates for streptokinase production was screened and the results showed that all of them were produce the enzyme with variable degrees. Molecular detection of streptokinase gene using real time PCR technique has been done with specific primers and the amplification results were present with all S.pyogens strains.

Cloning and expression of native streptokinase in E. coli BL21(DE3)

Streptokinase (SK) is a potent plasminogen activator naturally produced by beta-hemolytic streptococcus bacteria. Streptokinase is one of the most important thrombolytic agents that was frequently employed treatment of myocardial infarction. In this study, we amplified the SK gene isolated from Streptococcus pyogenes. The sequence of SK was recorded in the NCBI database. The Accession number of the SK sequence was LC625519.1. The SK gene was digested by EcoR1 and Hindlll, ligated with the linear pGEM®-3Zf (+) vector, and then introduced into an expression host E. coli BL21(DE3). For the production of the SK protein, we induced by 1mM IPTG. SDS-PAGE was used for the evaluation of the streptokinase synthesis with a molecular weight of around 47 kDa. Using the casein lysis method and the in vitro clot lysis assay, the activity of native SK was assessed. The native SK has clot lysis activity and a clear zone in the casein plate.

In silico design and in vitro validation of a novel PCR-RFLP assay for determination of phylogenetic clusters of streptokinase gene alleles in streptococci groups

Streptokinase (SK), a heterogeneous plasminogen (Pg) activator protein secreted by groups A, C and G streptococci (GAS/GCS/GGS) is a virulence factor composed of three structural domains; SKα/SKβ/SKγ. Phylogenetic analysis of the major variable region of SKβ (sk-V1; nucleotides 448–791; 343bp) which classifies the SK alleles into SK1/SK2 clusters and SK2a/SK2b sub-clusters, is an approved assay to categorize clinical/natural streptococcal-isolates into co-related functional/pathogenesis groups. Herein, we describe a novel PCR-RFLP assay that in combination with Numerical Taxonomy and multivariate analysis System (NTSYS) resulted to dendrograms with complete adaption to that of the phylogenetic analysis of sk-V1-based clustering. In silico analyses by 30 restriction enzymes on GenBank-acquired sk-V1 sequences of known streptococcal clusters, resulted to the selection of “BsrI, MseI and Tsp45I″ enzymes that produced proper patterns to construct the expected dendrograms. In vitro analysis of the selected enzymes on clinical isolates of GAS/GCS/GGS validated the production of the same in silico-observed digestion patterns. Comparison of the constructed dendrogram and phylogenetic trees of selected GenBank and clinical isolates of streptococci indicated complete adaptation. Assessment of Pg-activation activity in selected clinical isolates indicated the expected co-related functionalities of the classified SK-clusters by the invented PCR-RFLP/NTSYS method. The simplicity of the assay relieves the need of sequencing/phylogenetic analyses for SK-clustering.

Development of a streptokinase expression platform using the native signal sequence of the protein with internal repeats 1 (PIR1) in P. pastoris: Gene dosage optimization and cell retention strategies

Abstract Streptokinase is a therapeutically important thrombolytic agent which is toxic to its heterologous expression hosts and also prone to proteolytic degradation. In the current study, its secretory expression was targeted in P. pastoris using the native protein with internal repeats 1 (PIR1) secretion signal where the optimization of streptokinase gene dosage resulted in an increase in the volumetric product yield from 339.69 to 1206.48 mg/L. The cell retention strategy was applied during the induction phase to maintain sustained productivity where up to a 2.4 fold enhancement in the overall product yield was achieved at shake flask to a level of 3049.53 mg/L. Moreover, the cell recycling approach prevented the streptokinase proteolytic degradation and also helped in achieving a high specific product yield (YP/X) in a range of 167.65–176.44 mg/g DCW even after five cycles. Similarly, the cell recycling strategy was also employed using S. cerevisiae α-mating factor (α-MF) signal sequence which also resulted in improved product stability and volumetric product concentration confirming its wide applicability in obtaining enhanced product quality and yield. The recombinant streptokinase produced using PIR1 signal sequence was biologically active possessing a specific activity of 62,782.40 IU/mg and was predicted to contain 10.45% α-helix and 29.09% β-sheet.

Optimization and High Level Production of Recombinant Synthetic Streptokinase in E. coli Using Response Surface Methodology.

Streptokinase (SK) is an extracellular protein comprising 414 amino acids with considerable clinical importance as a commonly used thrombolytic agent. Due to its wide spread application and clinical importance designing more efficient SK production platforms worth investigation. In this regard, a synthetic SK gene was optimized and cloned in to pET21b plasmid for periplasmic expression. Response surface methodology was used to design a total of 20 experiments for optimization of IPTG concentration, post-induction period, and cell density of induction (OD600). The optimum levels of the selected parameters were successfully determined to be 0.28 mM for IPTG concentration, 9.889 H for post induction period, and 3.40768 for cell density (OD600). These settings result in 4.14fold increase in SK production rate of optimum expression conditions (7663 IU/mL) in comparison to the primary expression conditions (1853 IU/mL). Achieving higher yields of SK production in shake flask could lead to more cost effective industrial production of this drug which is the ultimate aim of SK production studies.

Point mutation in am-ino acid 263 of streptokinase gene as well as cloning and expression of the cysteine containing mutated protein

Point mutation in amino acid 263 of streptokinase gene as well as cloning and expression of the cysteine containing mutated protein

Cloning, purification and enzymatic assay of streptokinase gene from Streptococcus pyogenes in Escherichia coli

Streptokinase is an important therapeutic enzyme that acts as a fibrinolytic agent and use in the treatment of multi serious and life threatening diseases such as pulmonary embolism and acute myocardial infarction. Cloning and extracellular expression of recombinant streptokinase from Streptococcus pyogenes was done by fusing the gene coding for streptokinase to an efficient vector (pET15b) with NdeI and BamHI as restriction enzymes and T4 DNA ligase. Streptokinase activity was determined using synthetic chromogenic substrate S-2251, and measuring the absorbance at 405 nm in a UV-spectrophotometer; and purification done under N-terminal 6× histidine tag complementation using affinity chromatography. A 1323-bp of the structural streptokinase gene was amplified. The DNA and amino acid sequence alignments resulting from the BLAST search of streptokinase showed high sequence identity with the other strains of S. pyogenes. The recombinant enzyme had the same molecular weight as other reported streptokinase and the E. coli transformants showed high streptokinase activity. Comparative clinical trials and cost-effectiveness considerations between several major available thrombolytic agents suggest that streptokinase is the drug of choice for thrombolytic therapy particularly in the poorer economies.

Site-restricted plasminogen activation mediated by group A streptococcal streptokinase variants

SK (streptokinase) is a secreted plasminogen activator and virulence factor of GAS (group A Streptococcus). Among GAS isolates, SK gene sequences are polymorphic and are grouped into two sequence clusters (cluster type-1 and cluster type-2) with cluster type-2 being further classified into subclusters (type-2a and type-2b). In the present study, we examined the role of bacterial and host-derived cofactors in SK-mediated plasminogen activation. All SK variants, apart from type-2b, can form an activator complex with Glu-Plg (Glu-plasminogen). Specific ligand-binding-induced conformational changes in Glu-Plg mediated by fibrinogen, PAM (plasminogen-binding group A streptococcal M protein), fibrinogen fragment D or fibrin, were required for type-2b SK to form a functional activator complex with Glu-Plg. In contrast with type-1 and type-2a SK, type-2b SK activator complexes were inhibited by α2-antiplasmin unless bound to fibrin or to the GAS cell-surface via PAM in combination with fibrinogen. Taken together, these data suggest that type-2b SK plasminogen activation may be restricted to specific microenvironments within the host such as fibrin deposits or the bacterial cell surface through the action of α2-antiplasmin. We conclude that phenotypic SK variation functionally underpins a pathogenic mechanism whereby SK variants differentially focus plasminogen activation, leading to specific niche adaption within the host.

Cloning and expression of hybrid streptokinase towards clot-specific activity

Streptokinase (SK) is a thrombolytic agent that is widely used to treat myocardial infarction and pulmonary embolism. The lack of fibrin specificity of SK for the clot lysis is one of the limitations of SK. In this study, we have incorporated the finger and Kringle 2 domains from the human tissue type plasminogen activator gene (t-PA) at the 5′ end of the SK gene. These domains are responsible for specific binding to fibrin. We have used the pRSETB vector in an attempt to express the hybrid streptokinase possessing specificity for fibrin. On this regard, three hybrid streptokinase were constructed and expressed in Escherichia coli BL21 (DE3): the finger domain with SK (FSK), the Kringle 2 domain with SK (KSK) and the finger domain+Kringle 2 with SK (FKSK). The activities of the hybrid SKs were assessed by caseinolytic assay and clot lysis assay. All hybrid SKs were found to activate plasminogen in the caseinolytic plate assay. In the clot lysis assay, KSK and FSK were able to dissolute human blood and artificial clots in a fibrin-dependent manner unlike the SK and FKSK proteins.

Screening, Cloning and Expression of Active Streptokinase from an Iranian Isolate of S.equisimilis Group C in E. coli.

Streptokinase (SK) is a fibrinolytic protein secreted by β-hemolytic streptococci (βHS) groups A, C and G. Due to its importance as a thrombolytic drug, national screening programs in different countries for isolation of βHS and especially SK-producing group C (GCS) strains have been conducted. Herein, we provide data of the first screening study on βHS isolates in Iran for the aim of recombinant SK (rSK) production from a local strain. 252 streptococcal samples were collected and characterized using microbial/biochemical assays. The GCS strains were serologically confirmed. Activity of GCS supernatant cultures was determined by caseinolytic assay in comparison with the standard strain GCS9542. The SK gene of the highest producer strain was selected for production of rSK in E.coli system. The rSKs activities were determined using chromogenic assay. βHS were detected in 75 of the collected specimens (29.4%) including groups A (25.8%), C (3.6%) and G (0.4%). Analyses by SDS-PAGE and Western blotting indicated the proper expression of 47 kDa rSK proteins in E. coli for SK genes which were cloned from both the selected (GCS-87) and standard (GCS-9542) strains with the yields of 0.53 and 0.59 mg/ml (of the purified protein), respectively. The calculated activity for rSK 87 was around 90% of rSK9542 activity (0.18x105 IU/mg v/s 0.21x105 IU/mg). RESULTS of the present study for the first time provided the possibility of producing rSK from a local and native source with comparable yields and activities similar to the standard strain.

Pitfalls in screening streptococci for retrieving superior streptokinase (SK) genes: no activity correlation for streptococcal culture supernatant and recombinant SK

Streptokinase (SK), the heterogeneous protein family secreted by some groups of β-hemolytic streptococci (βHS), is a plasminogen activator and well-known drug for thrombolytic therapy. Differences in plasminogen activation property of streptococcal culture supernatants (SCS) have been traditionally used to identify superior producer strains and SK genes (skc) for recombinant SK (rSK) production. However, the role of SK heterogeneity and whether SK activities in SCS correlate with that of their corresponding rSK is a matter of debate. To address these concerns, SCS of nine group C streptococci (GCS) screened among 252 βHS clinical isolates were compared for plasminogen activation using S-2251 chromogenic assay. The GCS (Streptococcus equisimilis) showing the highest (GCS-S87) and lowest (GCS-S131) activities were selected for PCR-based isolation of skc, cloning and rSK production in Escherichia coli. The 6×His-tagged rSK proteins were purified by NI-NTA chromatography, analyzed by SDS-PAGE and Western blotting and their activities were determined. While SCS of GCS-S87 and GCS-S131 showed different plasminogen activations (95 and 35 %, respectively) compared to that of the reference strain (GCS-9542), but interestingly rSK of all three strains showed close specific activities (1.33, 1.70, and 1.55 × 10(4) IU mg(-1)). Accordingly, SKS87 and SKS131 had more than 90 % sequence identity at the amino acids level compared to SK9542. Therefore, SK heterogeneity by itself may not contribute to the differences in plasminogen activation properties of SCS and evaluation of this activity in SCS might not be a proper assay for screening superior skc.

Streptokinase variants from Streptococcus pyogenes isolates display altered plasminogen activation characteristics – implications for pathogenesis

Streptococcus pyogenes (group A streptococcus, GAS) secretes streptokinase, a potent plasminogen activating protein. Among GAS isolates, streptokinase gene sequences (ska) are polymorphic and can be grouped into two distinct sequence clusters (termed cluster type-1 and cluster type-2) with cluster type-2 being further divided into sub-clusters type-2a and type-2b. In this study, far-UV circular dichroism spectroscopy indicated that purified streptokinase variants of each type displayed similar secondary structure. Type-2b streptokinase variants could not generate an active site in Glu-plasminogen through non-proteolytic mechanisms while all other variants had this capability. Furthermore, when compared with other streptokinase variants, type-2b variants displayed a 29- to 35-fold reduction in affinity for Glu-plasminogen. All SK variants could activate Glu-plasminogen when an activator complex was preformed with plasmin; however, type-2b and type-1 complexes were inhibited by α(2) -antiplasmin. Exchanging ska(type-2a) in the M1T1 GAS strain 5448 with ska(type-2b) caused a reduction in virulence while exchanging ska(type-2a) with ska(type-1) into 5448 produced an increase in virulence when using a mouse model of invasive disease. These findings suggest that streptokinase variants produced by GAS isolates utilize distinct plasminogen activation pathways, which directly affects the pathogenesis of this organism.

PCR/RFLP-based allelic variants of streptokinase and their plasminogen activation potencies

PCR-restriction fragment length polymorphism (PCR/RFLP)-based analysis of β-domain variable region of streptokinase genes (sk) has previously identified 14 sk alleles (sk1-sk14) in group A (GAS), C (GCS) and G (GGS) streptococci isolates from a few geographically distinct regions. However, the relation of sk allelic variants to their plasminogen activation potencies remained as a matter of debate. Herein, employing the same PCR/RFLP assay, we analysed sk allelic variants of GAS and GCS/GGS isolates from Iranian patients. In total, 21 sk allelic variants including 14 new alleles (sk14-sk28) were identified. Results implied the horizontal gene transfer of sk fragments between GAS and GCS/GGS strains and did not prove the specificity of particular sk alleles to GCS/GGS or GAS groups. Measurement of streptokinase (SK) activity in streptococcal culture supernatants by colorimetric assay (S2251 substrate) ranged from 9 to 182 IU mL(-1). Although some strains with the highest SK activity were detected in definite variants, no significant correlation between sk alleles and plasminogen activation was detected (P value > 0.05). Analysis of DNA sequences and restriction site mapping of selective sk variants with similar SK activity pointed to the inadequacy of the currently available PCR/RFLP method for differentiation of critical/silent nucleotides to precisely categorize sk alleles for their functional properties.

Inhibitor of streptokinase gene expression improves survival after group A streptococcus infection in mice

The widespread occurrence of antibiotic resistance among human pathogens is a major public health problem. Conventional antibiotics typically target bacterial killing or growth inhibition, resulting in strong selection for the development of antibiotic resistance. Alternative therapeutic approaches targeting microbial pathogenicity without inhibiting growth might minimize selection for resistant organisms. Compounds inhibiting gene expression of streptokinase (SK), a critical group A streptococcal (GAS) virulence factor, were identified through a high-throughput, growth-based screen on a library of 55,000 small molecules. The lead compound [Center for Chemical Genomics 2979 (CCG-2979)] and an analog (CCG-102487) were confirmed to also inhibit the production of active SK protein. Microarray analysis of GAS grown in the presence of CCG-102487 showed down-regulation of a number of important virulence factors in addition to SK, suggesting disruption of a general virulence gene regulatory network. CCG-2979 and CCG-102487 both enhanced granulocyte phagocytosis and killing of GAS in an in vitro assay, and CCG-2979 also protected mice from GAS-induced mortality in vivo. These data suggest that the class of compounds represented by CCG-2979 may be of therapeutic value for the treatment of GAS and potentially other gram-positive infections in humans.

English

Streptokinase (SK) is a therapeutically important thrombolytic agent. Cardiovascular disease is the first cause of adult death worldwide. In Egypt about 13% of the population die every year due to ischemic heart disease. In spite of this fact, there is no local production of cardiovascular therapeutics. We reported for the first time the expression of a recombinant SK from a local Streptococcus strain. When produced on industrial scale this r-SK may substantially contribute to reducing the costs of thrombolytic therapy in developing countries. In this study, a highly purified r-SK from Streptococcus sp. isolated from Egyptian pharyngitis patients was obtained. The isolated strain was partially identified using 16S rDNA sequencing and namedStreptococcus sp. SalMarEg. It was found to be phylogenetically related toStreptococcus pyogenes. Analysis of the obtained sequence showed high similarity with other SK genes. The protein expression in a prokaryotic system obtained a 47-kDa SK protein that could be purified using a single-step his-tagged affinity purification chromatography, with nearly 80% recovery. The clot lytic activities of both recombinant and commercial SK were similar, thus giving the basis to scale up this SK product in order to evaluate the possibilities of its commercialization in local and/or regional markets.   Key words: Streptokinase, Streptococcus SalMarEg, thrombolytic agent,heterologous expression.

Isolation and characterization of β-haemolytic-Streptococci from endometritis in mares

The objective of this manuscript was to validate published PCR-based methods for detection of β-haemolytic Streptococci by comparison with established bacteriological techniques using 85 clinical isolates recovered from uterine swabs of mares with clinical signs of endometritis and to determine the distribution of SeeL/SeeM and SzeL/SzeM superantigens in isolates of Streptococcus equi subsp. equi ( S. equi) and S. equi subsp. zooepidemicus ( S. zooepidemicus). The conventional bacteriological techniques showed the vast majority of these isolates (78) were S. zooepidemicus with just 5 Streptococcus dysgalactiae subsp. equisimilis ( S. equisimilis) and 2 S. equi strains detected. The PCR analyses confirmed the bacteriological results demonstrating the reliability of the 16S rRNA PCR assay for detecting Streptococci, the multiplex PCR for differentiating between S. zooepidemicus, and S. equi, and PCR assays based on streptokinase genes for identification of S. equisimilis. PCRs for genes encoding superantigens revealed seeL and seeM specific amplicons with size of approximately 800 and 810 bp respectively for the S. equi strains and for 2 S. zooepidemicus strains. To our knowledge, this is the first report of s zeL and szeM possession by S. zooepidemicus isolates derived from endometritis in mares.

Identification through Combinatorial Random and Rational Mutagenesis of a Substrate-interacting Exosite in the γ Domain of Streptokinase

To identify new structure-function correlations in the γ domain of streptokinase, mutants were generated by error-prone random mutagenesis of the γ domain and its adjoining region in the β domain followed by functional screening specifically for substrate plasminogen activation. Single-site mutants derived from various multipoint mutation clusters identified the importance of discrete residues in the γ domain that are important for substrate processing. Among the various residues, aspartate at position 328 was identified as critical for substrate human plasminogen activation through extensive mutagenesis of its side chain, namely D328R, D328H, D328N, and D328A. Other mutants found to be important in substrate plasminogen activation were, namely, R319H, N339S, K334A, K334E, and L335Q. When examined for their 1:1 interaction with human plasmin, these mutants were found to retain the native-like high affinity for plasmin and also to generate amidolytic activity with partner plasminogen in a manner similar to wild type streptokinase. Moreover, cofactor activities of the mutants precomplexed with plasmin against microplasminogen as the substrate as well as in silico modeling studies suggested that the region 315-340 of the γ domain interacts with the serine protease domain of the macromolecular substrate. Overall, our results identify the presence of a substrate specific exosite in the γ domain of streptokinase.

Production of Recombinant Streptokinase in E. coli and Reactivity with Immunized Mice

Streptokinase (SK) is a potent plasminogen activator with widespread clinical use as a thrombolytic agent. In this study, we produce high level expression of recombinant streptokinase in E. coli by expression vector pET32a. Genomic DNA of streptokinase gene (SKC) was extracted, then amplified by polymerase chain reaction (PCR) method and sub-cloned to prokaryotic expression vector pET32a. Escherichia coli BL21 (DE3) pLysS were transformed with pET32a-skc and gene expression was induced by IPTG. The expressed protein was purified by affinity chromatography by Ni-NTA resin. High concentration of the recombinant protein obtained from the single-step purification by affinity-chromatography (Ni-NTA). The yield of recombinant streptokinase was nearly 470 mg L(-1) of initial culture. Our data showed that production of recombinant streptokinase improved by pET32a in Escherichia coli.

Studies on cloning and economic production of recombinant Streptokinase in a novel osmotically inducible E. Coli GJ1158

Streptokinase, a 47KDa protein is efficaciously used as a thrombolytic drug. Though it is relatively inexpensive, being a bacterial protein, Streptokinase is antigenic to humans. The low yields obtained in Streptokinase production, and the pathogenic nature of its natural host is the principal reasons to search for a recombinant producer. The report includes successful isolation of the Streptokinase gene from the wild host Streptococcus species MTCC389 and transforming it to E. coli expression hosts BL21(DE3) and GJ1158 through pRSET-A plasmid cloning vector, optimisation of media and analysis of successful expression of recombinant streptokinase in new host.

Enhancement of stability of recombinant streptokinase by intracellular expression and single step purification by hydrophobic interaction chromatography

Maintenance of stability, high biological activity and purity are essential requirements for recombinant therapeutic products and processes must be designed to meet these needs. This work addresses issues related to obtaining a high amount of stable recombinant streptokinase in its soluble form and a method to purify it in a single step with high levels of recovery and purity. The streptokinase gene without its native signal peptide was cloned and expressed in Escherichia coli, using the pRSETB expression vector. A significant amount of the expressed streptokinase was found in the soluble form in the cytoplasm. With an inducer concentration of 0.1 mM IPTG, recombinant streptokinase accumulated to about 20% of the total soluble protein in the cell. The biomass-specific activity of recombinant streptokinase obtained in the intracellular expression system (∼20,000 IU/mg DCW) was two-fold higher than the maximum reported in the literature. Furthermore, the expressed soluble protein was found to be highly stable. A higher concentration of the recombinant protein obtained from the intracellular system enabled single-step purification by hydrophobic interaction chromatography, with nearly 100% purity and 68% recovery. The whole process of expression and purification involved a minimal set of operations and would be very useful for economic production on a large scale of proteins like streptokinase which have therapeutic significance.