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

Kowalski, Michał; Brown, George; Bieniasz, Magdalena; Oszajca, Katarzyna; Chabielska, Ewa; Pietras, Tadeusz; Szemraj, Zofia; Makandjou-Ola, Eusebio; Bartkowiak, Jacek; Szemraj, Janusz

doi:10.18388/abp.2009_2515

Cited by 11 publications

(22 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This reduction in expression might be related to the presence of antithrombin and antiplatelet parts. The level of expression that was observed in this study was higher than that of others studies showing 8.21 mg/L, 33–50 mg/L, and 19 mg/L levels. However a high yield of nearly 1 g/L obtained by Apte‐Deshpnade et al is a notable exception …”

Section: Resultscontrasting

confidence: 87%

“…The TTI not only has intrinsic anticoagulation effects, but also it is a potent inhibitor for thrombin‐induced platelet aggregation . Unlike hirudin, the TTI does not require reshuffling the disulfide bonds to show functional effects . The designed structure was synthetized de novo and adapted to P. pastoris codon usage for expression effectively using pPICZα‐A vector under control of the methanol inducible AOX1 promoter.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Comparison of expression optimization of new derivative of staphylokinase (SAK‐2RGD‐TTI) with the rSAK

Faraji

Ramezani

Mashkani

et al. 2019

Biotechnology Progress

View full text Add to dashboard Cite

Staphylokinase (SAK) is a promising thrombolytic agent for the treatment of patients suffering from blood‐clotting disorders. To increase the potency of SAK and to minimize vessel reocclusion, a new construct bearing SAK motif fused to tsetse thrombin inhibitor (TTI) via a 20‐amino acid linker with 2 RGD (2 × arginine‐glycine‐aspartic acid inhibiting platelet aggregation via attachment to integrin receptors of platelet) was codon optimized and expressed comparatively in Pichia pastoris GS115 as a Mut+ strain and KM71H as a Muts strain. Fusion protein was optimized in terms of best expression condition and fibrinolytic activity and compared with the rSAK. Expression level of the designed construct reached up to 175 mg/L of the culture medium after 72‐hr stimulation with 2.5% methanol and remained steady for 3–4 days. The highest expression was obtained at the range of 2–3% methanol. The SAK‐2RGD‐TT (relative activity >82%) was more active at 25–37 °C than rSAK (relative activity of 93%). Further, it showed relative activity >80% at pH ranges of 7–9. Western blot analysis showed two bands of nearly 27 and 24 kDa at ratio of 5 to 3, respectively. The specific fibrinolytic activity of the SAK‐2RGD‐TTI was measured as 8,269 U/mg, and 19,616 U/mg for the nonpurified and purified proteins, respectively. Deglycosylation by using tunicamycin in culture medium resulted in higher fibrinolytic activity of SAK‐2RGD‐TTI (2.2 fold). Consequently, compared to the rSAK, at the same equimolar proportion, addition of RGD and TTI fragments could increase fibrinolytic activity. Also, P. pastoris can be considered as an efficient host for overexpression of the soluble SAK‐2RGD‐TTI with high activity without requiring a complicated purification procedure.

show abstract

Section: Resultscontrasting

confidence: 87%

Section: Introductionmentioning

confidence: 99%

Comparison of expression optimization of new derivative of staphylokinase (SAK‐2RGD‐TTI) with the rSAK

Faraji

Ramezani

Mashkani

et al. 2019

Biotechnology Progress

View full text Add to dashboard Cite

show abstract

“…A number of plants source especially several fruits and vegetables have been studied for their supplements having anticoagulant, antiplatelet and fibrinolytic activity and there is evidence that consuming such food leads to prevention of coronary events and stroke [26-29]. Some of these plant products are modified further with recombinant technology [30] to make them more effective and site specific.…”

Section: Discussionmentioning

confidence: 99%

Effects of organic extracts of six Bangladeshi plants on in vitro thrombolysis and cytotoxicity

Rahman

Sultana

Emran

et al. 2013

BMC Complement Altern Med

View full text Add to dashboard Cite

Background: Thrombus formed in blood vessels lead to atherothrombotic diseases such as myocardial or cerebral infarction. Thrombolytic agents are used to dissolve the already formed clots in the blood vessels; however, these drugs sometimes cause serious and fatal consequences. Herbal preparations have been used since ancient times for the treatment of several diseases although they show little toxicity in some cases. Aqueous extracts of herbs used in thrombolysis have been reported before with cytotoxic data, however, the organic extracts of herbs have not been documented. This study aims to investigate whether organic extracts possess thrombolytic properties with minimal or no toxicity. Methods: An in vitro thrombolytic model was used to check the clot lysis effect of six Bangladeshi herbal extracts viz., Ageratum conyzoides L., Clausena suffruticosa, Leea indica (Burm.f.) Merr., Leucas aspera Willd., Senna sophera L. Roxb., and Solanum torvum Swartz. using streptokinase as a positive control and water as a negative control. Briefly, venous blood drawn from twenty healthy volunteers was allowed to form clots which were weighed and treated with the test plant materials to disrupt the clots. Weight of clot after and before treatment provided a percentage of clot lysis. Cytotoxicity was screened by brine shrimp lethality bioassay using vincristine sulfate as positive control. Results: Using an in vitro thrombolytic model, Ageratum conyzoides, Clausena suffruticosa, Leea indica, Leucas aspera, Senna sophera and Solanum torvum showed 18.12 ± 2.34%, 48.9 ± 2.44%, 39.30 ± 0.96%, 37.32 ± 2.00%, 31.61 ± 2.97% and 31.51 ± 0.57% and clot lysis respectively. Among the herbs studied Clausena suffruticosa, Leea indica and Leucas aspera showed very significant (p < 0.0001) percentage (%) of clot lysis compared to reference drug streptokinase (75.00 ± 3.04%). In brine shrimp cytotoxic assay, the extracts Ageratum conyzoides, Clausena suffruticosa, Leea indica, Leucas aspera, Senna sophera and Solanum torvum showed LC 50 values 508. 86 ± 6.62,41.16 ± 1.26, 2.65 ± 0.16, 181.67 ± 1.65, 233.37 ± 7.74 and 478.40 ± 3.23 μg/ml, respectively, with reference to vincristine sulfate (LC 50 0.76 ± 0.04). Conclusion: Through our study it was found that Clausena suffruticosa, Leea indica and Leucas aspera possessed effective thrombolytic properties whereas Senna sophera and Solanum torvum showed moderate to mild thrombolytic effects while Ageratum conyzoides showed no significant effect. No extract was found cytoxic compared to positive control. Clausena suffruticosa, Leea indica and Leucas aspera could be incorporated as a thrombolytic agent with in vivo effects to improve the atherothrombotic patients. However, Clausena suffruticosa could be the best one to use in this purpose.

show abstract

“…The antithrombin activity of SAK or SRH was determined as described earlier [18]. The assay measures the amidolytic cleavage of chromogenic substrate Spectrozyme TH (American Diagnostic Inc, USA) in presence of human thrombin.…”

Section: Methodsmentioning

confidence: 99%

“…Fusion proteins SAK-RGD-K2-Hir and SAK-RGD-K2-Hirul were constructed and produced in different eukaryotic expression host, Schizosaccharomyces pombe [17] and Pichia pastoris [18], respectively. Furthermore, a SAK variant (SAK-HV) with 12 amino acids thrombin-binding domain from hirudin was also constructed and expressed in E. coli BL21 (DE3) [19].…”

Section: Introductionmentioning

confidence: 99%

In VitroCharacterization of a Multifunctional Staphylokinase Variant with Reduced Reocclusion, Produced from Salt InducibleE. coliGJ1158

Pulicherla

Kumar

Gadupudi

et al. 2013

BioMed Research International

View full text Add to dashboard Cite

The thrombolytic therapy with clinically approved drugs often ensues with recurrent thrombosis caused by thrombin-induced platelet aggregation from the clot debris. In order to minimize these problems, a staphylokinase (SAK)-based bacterial friendly multifunctional recombinant protein SRH (staphylokinase (SAK) linked with tripeptide RGD and dodecapeptide Hirulog (SRH)) was constructed to have Hirulog as an antithrombin agent and RGD (Arg-Gly-Asp) as an antiplatelet agent in the present study. This multifunctional fusion protein SRH was expressed in osmotically inducible E. coli GJ1158 as soluble form and purified with a yield of 0.27 g/L and functionally characterized in vitro. SRH retained the fibrinolytic activity and plasminogen activation rate comparable to the parental counterpart SAK. The antithrombin activity of SRH was significantly higher than SAK. The platelet rich clot lysis assay indicated that SRH had enhanced platelet binding activity and T 50% and C50 of SRH were significantly lower than that of SAK. Furthermore, SRH inhibited the ADP-induced platelet aggregation in dose-dependent manner while SAK had no significant effect on platelet aggregation. Thus, the current study suggests that the SAK variant produced from osmotically inducible GJ1158 is more potent thrombolytic agent with antithrombin and antiplatelet aggregation activities for reduction of reocclusion in thrombolytic therapy.

show abstract

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

Cited by 11 publications

References 34 publications

Comparison of expression optimization of new derivative of staphylokinase (SAK‐2RGD‐TTI) with the rSAK

Comparison of expression optimization of new derivative of staphylokinase (SAK‐2RGD‐TTI) with the rSAK

Effects of organic extracts of six Bangladeshi plants on in vitro thrombolysis and cytotoxicity

In VitroCharacterization of a Multifunctional Staphylokinase Variant with Reduced Reocclusion, Produced from Salt InducibleE. coliGJ1158

Contact Info

Product

Resources

About