Activity Of Staphylokinase Research Articles

Split intein-mediated backbone cyclization enhances the stability and activity of staphylokinase, a potent fibrin-selective plasminogen activator

Staphylokinase (Sak), a small 15 kDa globular protein that is secreted by certain strains of Staphylococcus aureus, shows a potent fibrin-selective thrombolytic activity. Earlier work has shown that Sak could potentially become a low-cost alternative to currently used thrombolytic agents, such as tissue plasminogen activator (tPA). In attempts to improve its potential for clinical applications, numerous modifications of Sak have already been investigated. Here, we have characterized a novel Sak modification, cyclized Sak (cyc-Sak), which was prepared through split-intein mediated protein backbone cyclization. We have characterized the structure, stability and the activity of cyc-Sak using biophysical techniques, limited proteolysis studies and plasminogen (PG)-activation assays. Our results show that cyc-Sak possesses an identical structure, enhanced stability, resistance to proteolysis by exoproteases and improved PG-activation properties compared to its linear counterpart. It can be over-expressed with high yield in the cytoplasm of Escherichia coli and is easily purified in a two-step process. The intein-mediated cyclization occurs spontaneously in vivo during protein expression and does not necessitate further modification steps after purification of the protein. Furthermore, covalent Sak cyclization could be readily combined with other Sak modifications previously proposed, to generate an effective thrombolytic agent with lower immunogenicity and improved stability and activity.

Extended Mechanism of the Plasminogen Activator Staphylokinase Revealed by Global Kinetic Analysis: 1000-fold Higher Catalytic Activity than That of Clinically Used Alteplase

The plasminogen activator staphylokinase is a fibrin-specific thrombolytic biomolecule and an attractive target for the development of effective myocardial infarction and stroke therapy. To engineer the protein rationally, a detailed understanding of the biochemical mechanism and limiting steps is essential. Conventional fitting to equations derived on the basis of simplifying approximations may be inaccurate for complex mechanisms such as that of staphylokinase. We employed a modern numerical approach of global kinetic data analysis whereby steady-state kinetics and binding affinity data sets were analyzed in parallel. Our approach provided an extended, revised understanding of the staphylokinase mechanism without simplifying approximations and determined the value of turnover number kcat of 117 s–1 that was 10000-fold higher than that reported in the literature. The model further showed that the rate-limiting step of the catalytic cycle is binding of staphylokinase to plasmin molecules, which occurs via an induced-fit mechanism. The overall staphylokinase effectivity is further influenced by the formation of an inactive staphylokinase·plasminogen complex. Here, we describe a quick and simplified guide for obtaining reliable estimates of key parameters whose determination is critical to fully understand the staphylokinase catalytic functionality and define rational strategies for its engineering. Our study provides an interesting example of how a global numerical analysis of kinetic data can be used to better understand the mechanism and limiting factors of complex biochemical processes. The high catalytic activity of staphylokinase (more than 1000-fold higher than that of the clinically used drug alteplase) determined herein makes this thrombolytic agent a very attractive target for further engineering.

Production and Characterization of Staphylokinase Enzyme from Staphylococcusaureus ASIA4

Six clinical Staphylococcus aureus strains isolated from different clinical samples. Isolates ASIA1 and ASIA2 isolated from urine samples of urinary tract infected patients; ASIA3 isolated from swab samples of burn abscess patients at Assiut University hospital as well as ASIA4, ASIA5 and ASIA6 obtained from blood samples of different cancer patients at South Egypt Cancer Institute. All isolates showed varied abilities to produce halo zones of hydrolysis with different diameters on blood agar, heated plasma agar, casein agar and skim milk agar plates along with different clot lyses percent. Staphylococcus aureus ASIA3, ASIA4 and ASIA6 produced 4.83, 5.98 and 2.08 U/mL of staphylokinase on tryptone soy broth reduced to 1.95, 2.08 and 1.70 U/mL on casein hydrolysate yeast extract broth,respectively. On the other hand, Staphylococcus aureus ASIA1, ASIA2 and ASIA5 gave 2.20, 2.93 and 3.65 U/mL on CYEB compared to 2.10, 1.88 and 3.41 U/mL on TSB as production medium. The staphylokinase yielded from the hyperactive producer Staphylococcus aureus ASIA4 was increased for 7.64-fold (from 2.08 U/mL to 15.88 U/mL) on the optimized fermentation medium composed of 5.0 g sucrose as carbon source, 10.0 g soy bean as nitrogen source, 5.0 g NaCl, K2HPO4 5.0 g and pH 7.0 that inoculated with isolate ASIA4 and incubated for 24 h at 35°C. Moreover, Staphylokinase activity reached its peak at the optimal enzymatic reaction conditions which were reaction time 25 min, casein as substrate, reaction pH 8.0, reaction temperature 40°C. In addition it retained 100% of its activity at temperature ranged between 15 and 45°C and pH ranged from pH 6.0 to 9.0 EDTA inhibited the enzyme activity by 3.0% to 32.2% with increasing its values from 30.0 to 90.0 mM. MgCl2 at a concentration of 30 mM increased the enzyme activity by 4% and then slightly decreased at higher concentrations but NaCl was potent staphylokinase activator at concentrations lower than 90 mM.

Production and characterization of staphylokinase enzyme from Staphylococcus aureus ASIA4

Six clinical Staphylococcus aureus strains isolated from different clinical samples. Isolates ASIA1 and ASIA2 isolated from urine samples of urinary tract infected patients; ASIA3 isolated from swab samples of burn abscess patients at Assiut University hospital as well as ASIA4, ASIA5 and ASIA6 obtained from blood samples of different cancer patients at South Egypt Cancer Institute. All isolates showed varied abilities to produce halo zones of hydrolysis with different diameters on blood agar, heated plasma agar, casein agar and skim milk agar plates along with different clot lyses percent. Staphylococcus aureus ASIA3, ASIA4 and ASIA6 produced 4.83, 5.98 and 2.08 U/mL of staphylokinase on tryptone soy broth reduced to 1.95, 2.08 and 1.70 U/mL on casein hydrolysate yeast extract broth, respectively. On the other hand, Staphylococcus aureus ASIA1, ASIA2 and ASIA5 gave 2.20, 2.93 and 3.65 U/mL on CYEB compared to 2.10, 1.88 and 3.41 U/mL on TSB as production medium. The staphylokinase yielded from the hyperactive producer Staphylococcus aureus ASIA4 was increased for 7.64-fold (from 2.08 U/mL to 15.88 U/mL) on the optimized fermentation medium composed of 5.0 g sucrose as carbon source, 10.0 g soy bean as nitrogen source, 5.0 g NaCl, K2HPO4 5.0 g and pH 7.0 that inoculated with isolate ASIA4 and incubated for 24 h at 35 °C. Moreover, Staphylokinase activity reached its peak at the optimal enzymatic reaction conditions which were reaction time 25 min, casein as substrate, reaction pH 8.0, reaction temperature 40 °C. In addition it retained 100% of its activity at temperature ranged between 15 and 45 °C and pH ranged from pH 6.0 to 9.0. EDTA inhibited the enzyme activity by 3.0% to 32.2% with increasing its values from 30.0 to 90.0 mM. MgCl2 at a concentration of 30 mM increased the enzyme activity by 4% and then slightly decreased at higher concentrations but NaCl was potent staphylokinase activator at concentrations lower than 90 mM.

PURIFICATION, CHARACTERIZATION, AND EVALUATION OF FIBRINOLYTIC ACTIVITY OF STAPHYLOKINASE FROM LOCALLY ISOLATED STAPHYLOCOCCUS AUREUS GH38

This study was aimed to purify, characteristic, and fibrinolytic activity of staphylokinase (SAK), is an enzyme activates plasminogen to form plasmin, which digest fibrin clots that cause thrombosis clot. Staphylokinase was purified from local isolate Staphylococcus aureus GH38 by ammonium sulfate precipitation at 70% saturation followed by ion exchange chromatography (CM-Cellulose) with a purification fold 2.73, and 1.53, and recovery 72.1, and 33.11% in wash and elution steps respectively. The partially purified enzyme was high activity at 40°C with pH 7 and the enzyme retained 100% of its activity at 35°C with pH 7. The activity of an enzyme increased by its treatment with calcium and sodium chloride, while the activity affected when incubated with mercury, silver, and iron chloride. The enzyme have high effective against thrombus (blood clot), which encourage the use of enzyme in the treatment as therapeutic agent to remove clots formed in the human body.

Detection of Methicillin Resistance, Staphylokinase Activity and Biofilm Formation in S. aureus Isolated from Clinical Samples

Present study was conducted for the isolation, identification, molecular detection and antibiotic sensitivity patterns of Staphylococcus aureus obtained from Hospitals in Erbil City-Kurdistan Region-Iraq. From 630 specimens collected, ninety eight isolates of S. aureus were obtained. Some isolates were further confirmed by PCR with amplification of nuc and spa genes, the results showed that nuc gene was amplified in all chosen isolates except one isolate, whereas spa amplicons showed the polymorphism in two of chosen isolates, and not amplified in three of them. There was a relationship between the virulence factors where 82.65% of coagulase positive isolates were staphylokinase (Sak) producers; and a relationship between Sak production, biofilm formation and methecillin resistance S. aureus (MRSA), where 68 (83.95%) from 81 of Sak producers isolates were MRSA while from 84 MRSA isolates 68 (80.95%) were Sak producers; 75 (89.28%) of MRSA were biofilm formers and vice versa. A negative correlation was seen between Sak production and biofilm formation. Antimicrobial sensitivity screening showed variable susceptibility of isolates with low resistance in all isolates (1.02%, 4.08%, 6.12%, 14.28%, 18.36% and 39.79% for Gen, Amx and Cip, Van, Azm, Ery, and PenG respectively), and the highest resistance rate was 98.97% for Nit and Sul. All 98 S. aureus isolates were distributed in 38 groups as an anti-biogram ranging from the most multi-resistant group to the most sensitive group. Most isolates were methicillin resistant (85.71%) and it was shown that 31 isolates of 98 isolates carried the mecA gene

The pharmaceutics from the foreign empire: the molecular pharming of the prokaryotic staphylokinase in Arabidopsis thaliana plants.

Here, we present the application of microbiology and biotechnology for the production of recombinant pharmaceutical proteins in plant cells. To the best of our knowledge and belief it is one of few examples of the expression of the prokaryotic staphylokinase (SAK) in the eukaryotic system. Despite the tremendous progress made in the plant biotechnology, most of the heterologous proteins still accumulate to low concentrations in plant tissues. Therefore, the composition of expression cassettes to assure economically feasible level of protein production in plants remains crucial. The aim of our research was obtaining a high concentration of the bacterial anticoagulant factor—staphylokinase, in Arabidopsis thaliana seeds. The coding sequence of staphylokinase was placed under control of the β-phaseolin promoter and cloned between the signal sequence of the seed storage protein 2S2 and the carboxy-terminal KDEL signal sequence. The engineered binary vector pATAG-sak was introduced into Arabidopsis thaliana plants via Agrobacterium tumefaciens-mediated transformation. Analysis of the subsequent generations of Arabidopsis seeds revealed both presence of the sak and nptII transgenes, and the SAK protein. Moreover, a plasminogen activator activity of staphylokinase was observed in the protein extracts from seeds, while such a reaction was not observed in the leaf extracts showing seed-specific activity of the β-phaseolin promoter.

Construction of novel chimeric proteins through the truncation of SEC2 and Sak from Staphylococcus aureus.

It is an usual clinical phenomenon that cancer patients are prone to thrombosis. Until now, there have been no efficient methods or appropriate drugs to prevent and cure tumor thrombus. Therefore, the construction of a bifunctional chimeric protein for the treatment of cancer, complicated with thrombosis, is of great significance. Utilizing the superantigenic activity of staphylococcal enterotoxin C2 (SEC2) and the thrombolytic activity of staphylokinase (Sak), Sak-linker-SEC2 and SEC2-linker-Sak were constructed which had good anti-tumor and thrombolytic activities at the same time. Due to the intrinsic emetic activity of SEC2 and high molecular weight (MW) of chimeric proteins (44 kDa), their clinical applications will be restricted. In this study, novel chimeric proteins including ΔSEC2-ΔSak and ΔSak-ΔSEC2 were constructed through the truncation of SEC2 and Sak without 9-Ala linker and His-tag. Compared with the former, both the truncated proteins preserved nearly the same anti-tumor and thrombolytic activities. In addition, their MWs were only 29 kDa and their immunoreactivities were slightly lower than that of Sak-linker-SEC2 and SEC2-linker-Sak, respectively. Therefore, the novel chimeric proteins possessed merits and characteristics, such as low MS, low immunogenicity, and difunctionality which the former had not. It will be of great interest if the above-mentioned proteins can be used to cure Trousseau syndrome in clinic.

Cloning and expression of a new recombinant thrombolytic and anthithrombotic agent - a staphylokinase variant.

To develop a more potent antithrombin agent with thrombolytic and antiplatelet properties, a new staphylokinase (SAK) variant was constructed. The kringle 2 domain (K2) of tissue type-plasminogen activator (t-PA) containing a fibrin-specific binding site (i), the RGD sequence (Arg-Gly-Asp) for the prevention of platelet aggregation (ii) and the antithrombotic agent - hirulog (iii) was assembled to the C-terminal part of recombinant staphylokinase (r-SAK). cDNA for the hybrid protein SAK-RGD-K2-Hirul was cloned into Pichia pastoris pPIC9K yeast expression vector. The introduction of K2 t-PA, the RGD sequence and hirulog into the C-terminus of r-SAK did not alter the staphylokinase activity. We observed a higher clot lysis potency of SAK-RGD-K2-Hirul as evidenced by a faster and more profound lysis of (125)I-labeled human fibrin clots. The potency of thrombin inhibition by the hirulog C-terminal part of the recombinant fusion protein was almost identical to that of r-Hir alone. These results suggest that the SAK-RGD-K2-Hirul construct can be a more potent and faster-acting thrombolytic agent with better antithrombin and antiplatelet properties compared to r-SAK and SAK-RGD-K2-Hir.

Enhanced plasminogen activation by staphylokinase in the presence of streptokinase β/βγ domains: Plasminogen kringles play a role

Presence of isolated β or βγ domains of streptokinase (SK) increased the catalytic activity of staphylokinase (SAK)–plasmin (Pm) complex up to 60%. In contrast, fusion of SK β or βγ domains with the C-terminal end of SAK drastically reduced the catalytic activity of the activator complex. The enhancement effect mediated by β or βγ domain on Pg activator activity of SAK–Pm complex was reduced greatly (45%) in the presence of isolated kringles of Pg, whereas, kringles did not change cofactor activity of SAK fusion proteins (carrying β or βγ domains) significantly. When catalytic activity of SAK–microPm (catalytic domain of Pm lacking kringle domains) complex was examined in the presence of isolated β and βγ domains, no enhancement effect on Pg activation was observed, whereas, enzyme complex formed between microplasmin and SAK fusion proteins (SAKβ and SAKβγ) displayed 50–70% reduction in their catalytic activity. The present study, thus, suggests that the exogenously present β and βγ interact with Pg/Pm via kringle domains and elevate catalytic activity of SAK–Pm activator complex resulting in enhanced substrate Pg activation. Fusion of β or βγ domains with SAK might alter these intermolecular interactions resulting in attenuated functional activity of SAK.

The effect of argatroban on injured endothelial cells by thrombin.

When endothelial cells are exposed to thrombin, they become perturbed and acquire thrombogenic properties. Argatroban is an arginine derivative, synthetic small molecule that binds to the active site of thrombin and inhibits its catalytic activity. Therefore, the effects of argatroban on endothelial cells, which had been injured by thrombin, were investigated. The established endothelial cell line, TKM-33, which had been cloned from human umbilical vein endothelial cells, was used. Endothelial cells produce plasminogen activator (PA) to prevent thrombosis and maintain the blood flow. When the endothelial cells were injured by thrombin, secretion of plasminogen activator inhibitor-1 (PAI-1) increased and then the PA activity proportionally decreased. The treatment of endothelial cells with argatroban after thrombin injury did not restore their reduced PA activity. However, the treatment of endothelial cells with argatroban prior to thrombin injury resulted in inhibiting the induction of PAI-1 secretion. Thus, pretreatment of endothelial cells with argatroban suppresses the inhibition of their PA activity by thrombin. Since the effect of thrombolytic agent may be modified by the fibrinolytic factors produced by the endothelial cells, the activity of staphylokinase (SAK) was measured in the presence of endothelial cells that had been injured by thrombin. SAK is a newly developed thrombolytic agent. SAK activity in the presence of injured endothelial cells by thrombin was lower than that in the presence of endothelial cells without thrombin injury. However, treatment of endothelial cells with argatroban prior to thrombin injury revealed higher SAK activity than that after thrombin injury. These findings indicate that argatroban pretreatment prevents thrombin injury of endothelial cells, which may then maintain their physiological function.

Analysis of plasminogen activation by the plasmin-staphylokinase complex in plasma of alpha2-antiplasmin-deficient mice.

Staphylokinase (SAK) expresses plasminogen activator (PA) activity by forming a complex with plasmin; this PA activity is inhibited by alpha2-antiplasmin (alpha2-AP) in plasma. However, SAK's activity is protected against inhibition by alpha2-AP in the presence of fibrin because the plasmin-SAK complex binds to fibrin. In the present study, the interaction between SAK and murine plasminogen was investigated in the plasma of alpha2-AP-deficient (alpha2-AP-/-) mice or plasminogen-deficient (Plg-/-) mice. Although the human plasmin-SAK complex was formed in equimolar mixtures of plasmin and SAK, the murine plasmin-SAK complex was not formed. Human plasminogen was activated by the human plasmin-SAK complex, although equimolar mixtures of murine plasmin and SAK did not activate murine plasminogen. These findings suggest that SAK does not react with murine plasmin. However, the murine plasminogen was activated by the human plasmin-SAK complex, although this activation was approximately 100-fold weaker than human plasminogen. Human and wild-type mouse plasminogens were not activated by the human plasmin-SAK complex in their plasma. In alpha2-AP-/- mouse plasma, murine plasminogen was activated by the human plasmin-SAK complex. Human or murine plasminogen, which had been added to Plg-/- mouse plasma, was not activated by the human plasmin-SAK complex. However, plasma clot lysis by the human plasmin-SAK complex was observed in both human and murine plasma. These findings indicate that: (1) murine plasmin does not react with SAK, (2) human plasmin-SAK complex activates murine plasminogen, (3) this activation is inhibited by murine alpha2-AP, but (4) this activation is not inhibited by murine alpha2-AP in the presence of fibrin.

114 The effect of argatroban on staphylokinase activity in the presence of endothelial cell injured by thrombin

When Glu-plasminogen (Glu-plg) was incubated with urokinase (UK) in the presence of various concentrations of tranexamic acid and S-2251, the hydrolysis of S-2251 increased in the presence of 1 mM of tranexamic acid, but decreased in the presence of more than 10 mM of tranexamic acid. Use of SDS-polyacrylamide gel electrophoresis indicated: 1) Glu-plg was converted to plasmin better in the presence of 1 mM of tranexamic acid, but in the presence of more than 10 mM of tranexamic acid it was less converted. 2) Plg I (of higher molecular weight) was more easily converted than plg II by UK, but binding of tranexamic acid with lysine binding sites of plasminogen caused almost equal rate of activation of plg I and II.

Production of a Recombinant Antithrombotic and Fibrinolytic Protein, PLATSAK, in Escherichia coli

The three main components involved in thrombosis and haemostasis are thrombin, platelets, and plasmin. Almost all inhibitors of thrombosis are focused either on the inhibition of thrombin or on the inhibition of platelets. We designed a construct using the fibrinolytic activity of staphylokinase, fused via a cleavable linker to an antithrombotic peptide of 29 amino acids. The peptide was designed to include three inhibitory regions: (1) the Arg-Gly-Asp (RGD) amino acid sequence to prevent fibrinogen binding to platelets; (2) a part of fibrinopeptide A, an inhibitor of thrombin; and (3) the tail of hirudin, a potent direct antithrombin. The amino acid sequence of the 29 amino acid peptide was reverse translated, and the gene was chemically synthesised and cloned into an expression vector as a 3′ fusion to the staphylokinase gene. Gene expression was induced in E. coli Top 10 cells and the fusion protein, designated PLATSAK, was purified using metal affinity chromatography. The purified fusion protein significantly lengthened the activated partial thromboplastin time and thrombin time and inhibited the amidolytic activity of thrombin. The fibrinolytic activity was almost equal to that of recombinant staphylokinase as measured with a thrombelastograph. Platelet aggregation was not markedly inhibited by PLATSAK, probably due to the unfavourable three dimensional structure, with the Arg-Gly-Asp sequence buried inside. Our results confirm that it is feasible to design and produce a hybrid multifunctional protein that targets various components of the haemostatic process.

A NOVEL VARIANT OF STAPHYLOKINASE GENE FROM STAPHYLOCOCCUS AUREUS ATCC 29213

The processes of hemostasis and thrombolysis are elegantly regulated in order to ensure normal functions of vascular system. A search for new plasminogen activators as thrombolytic agents has been carried out for the purpose of clinical applications to modulate thrombolytic processes. In the current work, several strains and clinical isolates of Staphylococcus aureus were screened for the fibrinolytic activity. The DNA sequences of staphylokinase gene in the strains expressing 15 kDa protein with staphylokinase activity were determined and subsequently compared with three known staphylokinase gene sequences. From the sequence comparison a new variant of staphylokinase gene has been identified in ATCC 29213 strain. The gene product needs to be further characterized and tested for the therapeutic potential. © 1997 Elsevier Science Ltd

Effects of fibrin and alpha2-antiplasmin on plasminogen activation by staphylokinase.

Staphylokinase obtains plasminogen activating activity by forming a complex with plasminogen. Although the enzymatic activity of staphylokinase is enhanced by fibrin, how fibrin enhances enzymatic activity has not been determined yet. The effects of fibrin, or fibrinogen fragments, on the activation of plasminogen by staphylokinase was investigated using CNBr-digested fibrinogen fragments (FCB-2 and FCB-5) and plasmin-degraded cross-linked fibrin fragments ((DD)E complex, DD fragments and E fragments). Kinetic analysis of the activity of staphylokinase revealed that its plasminogen activating activity, which was expressed as kcat/Km, was enhanced by FCB-2 (10-fold) and FCB-5 (5-fold). These fibrin fragments caused 38-, 30-, and 8.5-fold increases in activity for the DD fragment, (DD)E complex and E fragment, respectively. Although alpha2-antiplasmin inhibited the activation of plasminogen by staphylokinase, FCB-2 abolished its inhibitory effects, and the plasminogen activating activity of staphylokinase was restored. The inhibitory effects of alpha2-antiplasmin on the activation of mini-plasminogen by staphylokinase were less than for Glu- or Lys-plasminogen, and the inhibitory effect of alpha2-antiplasmin was not altered by fibrin or EACA. These findings indicate that the staphylokinase/plasmin(ogen) complex reacts with fibrin even in the presence of alpha2-antiplasmin, and efficient plasminogen activation takes place on the surface of fibrin.

Effects of endothelial cells on activity of staphylokinase.

Staphylokinase (SAK), produced by Staphylococcus aureus, induces fibrinolytic activity in circulation without systemic fibrinolytic activation. Since the effect of blood vessels on the activity of SAK has not yet been clarified, plasminogen activator (PA) activity of SAK in the presence or absence of endothelial cells was analyzed. The endothelial cells used in this experiment were of a cloned established cell line (TKM-33). In the expression of PA activity by SAK or streptokinase (SK), the kinetic constants revealed as Vmax/km were increased about 1.5-fold in the presence of endothelial cells. Furthermore, an initial lag phase which was observed during the plasminogen activation by SAK was markedly shortened in the presence of endothelial cells. In the case of SK, an initial lag phase was not observed in the absence or presence of endothelial cells. Although PA activity of SAK was inhibited by alpha 2-antiplasmin (alpha 2-AP), the inhibitory effect of alpha 2-AP in the presence of endothelial cells was weaker than in the absence of endothelial cells. The cyanogen bromide digested fibrinogen fragment-2 (FCB-2) distinctly enhanced the PA activity of SAK in the absence and the presence of endothelial cells. However, alpha 2-AP and FCB-2 did not cause a significant alteration of PA activity of SK even in the absence or presence of endothelial cells. These findings suggest that PA activity of SAK is enhanced by endothelial cells, but inhibited by alpha 2-AP. Moreover, PA activity of SAK is further enhanced by fibrin clot in the presence of endothelial cells.

23 Effect of mutagenized NH 2-terminal amino acid region on plasminogen activator activity of staphylokinase

23 Effect of mutagenized NH 2-terminal amino acid region on plasminogen activator activity of staphylokinase

Primary structure and gene structure of staphylokinase

The nucleotide sequence was determined of the 2.9 kb HindIII restriction fragment, obtained from the genomic DNA of a selected Staphylococcus aureus strain (strain no. 23) which directs high level expression of staphylokinase (STA) in E. coli JM83 cells transformed with the recombinant plasmid pUCSTAHH, consisting of pUC19 containing this 2.9 kb insert. The fragment contained an open reading frame of 489 base pairs (base pairs 996–1484) encoding 163 amino acids, with amino acids 28, 34 and 38 corresponding to the NH 2-terminal residue of the three variants (STA-M, STA-Δ6 and STA-Δ10) of recombinant STA (STAR) recovered from culture broth conditioned by transformed E. coli (Collen et al, Fibrinolysis, 1992; 6: 203–213). This coding sequence is preceded upstream by canonical Shine-Dalgarno, -10 and -35 prokaryotic promoter sequences and in addition by an open reading frame spanning nucleotides 50–802 which encodes an unknown protein of 251 amino acids. The sequence encoding STA is very similar to that previously cloned from bacteriophages Sø-c and 42D. Recombinant plasmids with a l.7kb AccI- EcoRI fragment insert (base pairs 683–2168) containing only the sequence encoding STA, directed excretion of STA activity to an extent comparable to that obtained with the recombinant plasmid containing the 2.9 kb HindIII restriction fragment insert.

Isolation and characterisation of natural and recombinant staphylokinase

Staphylokinase (STA), a M r 18000 protein produced by Staphylococcus aureus has profibrinolytic properties (Lack CH, Nature 161, 559–560, 1948), but its potential for thrombolytic therapy has not been thoroughly investigated. Therefore we have elaborated procedures for the large scale production of natural (STAN) and recombinant (STAR) staphylokinase. A strain of Staphylococcus aureus (strain no. 23), selected from 100 consecutive cultures at our Bacteriology Laboratory, was found to secrete up to 300 μg staphylokinase per litre culture broth within 18h. The material, purified by batch adsorption on SP-Sephadex and chromatography on insolubilised active site-blocked plasmin with a yield of approximately 40 μg per l, migrated as a single band on SDS gel electrophoresis with M r ≈ 16 500 and had NHZ-terminal sequence Lys-Gly-Asp-AspAla-. Restriction enzyme digested genomic DNA from this strain was hybridised with a degenerate 14-mer deoxyoligonucleotide encoding the NH 2-terminal sequence, hybridising fragments were isolated and ligated into pUC19, competent E. coli cells were transformed, recombinant clones isolated and culture medium assayed for STA activity. A clone (subclone 159-2) transformed with recombinant pUC19 containing a 2.9 kb insert obtained by HindIII restriction enzyme digestion was found to secrete STAR without further manipulation (up to 20 mg/l into the culture broth and 30 mg/l into the periplasmic space). The material, purified by chromatography on insolubilised active-site blocked plasmin with a yield of 50 and 25%, respectively, contained three STAR variants with NH 2-terminal sequence Ser-Ser-Ser-Phe-Asp-Lys-Gly-Lys-Tyr-Lys-Lys-Gly-Asp-Asp-Ala- (STA-M), with Gly-Lys-Tyr-Lys-Lys-Gly-Asp-Asp-Ala- (STA-Δ6) and with Lys-Gly-Asp-Asp-Ala- (STA-Δ10). Chromatography on CM-Sephadex yielded two peaks, STA-CM-I containing STA-Δ10 and STA-CM-11 containing STA-M and STA-Δ6. The specific activities of STAN, of STA-Δ10 obtained from STAR and of mixtures of STA-M and STA-Δ6 from STAR were indistinguishable by clot lysis and plasminogen coupled chromogenic substrate assays. Thus, highly purified preparations of natural (STAN) and recombinant (STAR) staphylokinase can be obtained by simple and straightforward techniques in sufficiently large amounts to allow detailed investigation of their biochemical and thrombolytic properties.