The Experts below are selected from a list of 1920 Experts worldwide ranked by ideXlab platform
Christoph Bode - One of the best experts on this subject based on the ideXlab platform.
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Platelet Function During and After Thrombolytic Therapy for Acute Myocardial Infarction With Reteplase, Alteplase, or Streptokinase
Circulation, 1999Co-Authors: Martin Moser, Thomas K. Nordt, Benedikt Kohler, Marc Schmittner, Richard W. Smalling, Karlheinz Peter, Wolfgang Kübler, Johannes Ruef, Christoph BodeAbstract:Background—Changes in platelet aggregation (PA) and platelet surface receptor expression induced by thrombolytic therapy for acute myocardial infarction may influence the rate of initial reperfusion and early reocclusion. Methods and Results—In the RAPID-1 (Reteplase Angiographic Phase II International Dose-finding study), RAPID-2 (Reteplase vs Alteplase Patency Investigation During myocardial infarction), INJECT (INternational Joint Efficacy Comparison of Thrombolytics), and GUSTO-3 (Global Use of Strategies To Open occluded coronary arteries) trials, 126 patients were enrolled in a single center. Patients were treated with either conventional alteplase (100 mg/180 min; n=15), accelerated alteplase (100 mg/90 min; n=21), Reteplase 10+10-U double bolus (n=50), Reteplase 10+5-U double bolus (n=15), Reteplase 15-U single bolus (n=15), or streptokinase (1.5 MU/60 min; n=10). PA (after stimulation with ADP), P-selectin expression and fibrinogen binding to glycoprotein (GP) IIb/IIIa (determined by flow cytomet...
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Recombinant Plasminogen Activators
BioDrugs, 1998Co-Authors: Martin Moser, Benedikt Kohler, Marc Schmittner, Christoph BodeAbstract:Thrombolytic therapy has been shown to reduce mortality after acute myocardial infarction. Great efforts have been undertaken in the past decade to develop more efficient thrombolytic regimens. Novel recombinant thrombolytic substances have been engineered. Reteplase, a deletion mutant of wild-type tissue plasminogen activator with a longer half-life, has been evaluated in clinical trials and is now available for clinical use. In the randomised Reteplase Angiographic Phase II International Dose-Finding (RAPID-1) trial, involving 606 patients with acute myocardial infarction, a double-bolus regimen of Reteplase (10 + 10U given 30 minutes apart) achieved significantly higher patency rates at 90 minutes after initiation of thrombolytic therapy than a 10 + 5U double-bolus Reteplase regimen, a 15U single bolus Reteplase regimen, or a conventional alteplase regimen (100mg in 180 minutes). In the Reteplase versus Alteplase Patency Investigation During Acute Myocardial Infarction (RAPID-2) trial, the Reteplase 10 + 10U double-bolus regimen was more effective with regard to patency at 60 and 90 minutes than accelerated alteplase (100mg in 90 minutes). The International Joint Efficacy Comparison of Thrombolytics (INJECT) trial showed that a double-bolus regimen of Reteplase 10 + 10U was at least equivalent to streptokinase in terms of 35-day mortality rate. The third Global Utilization of Strategies to Open Occluded Coronary Arteries (GUSTO-III) trial resulted in similar 30-day mortality after therapy with double-bolus Reteplase 10 + 10U (7.47%) or accelerated alteplase (7.24%). Hence, the higher early patency rates achieved with Reteplase treatment did not translate into improved survival. Consequently, there is still some uncertainty as to whether or not these drugs are equivalent.
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Reteplase (r-PA): a new plasminogen activator.
Expert opinion on investigational drugs, 1997Co-Authors: Christoph Bode, Benedikt Kohler, Marc Schmittner, Richard W. Smalling, Martin Moser, Ruth H StrasserAbstract:Reteplase (r-PA) is a genetically engineered deletion mutant of wild-type tissue-type plasminogen activator. The structural differences lead to different functional properties, such as a prolonged half-life. The compound demonstrated good thrombolytic efficacy in in vitro as well as in animal studies. In angiographically controlled patency studies (GRECO, GRECO-2 RAPID-1, RAPID-2), the double-bolus application scheme was established, and a superior patency profile for Reteplase in comparison to alteplase was demonstrated. Mortality studies established Reteplase as a safe drug with a 30-day mortality at least equivalent to streptokinase (INJECT) and very similar to alteplase (GUSTO-3). A possible advantage may be the double-bolus application without a need for weight adjustment, especially in a prehospital setting. Thus, Reteplase can be regarded as an excellent alternative to streptokinase or alteplase for thrombolytic therapy in acute myocardial infarction.
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patency trials with Reteplase r pa what do they tell us
American Journal of Cardiology, 1996Co-Authors: Christoph Bode, Thomas K. Nordt, Richard W. Smalling, Karlheinz Peter, M S Runge, W KüblerAbstract:Abstract Thrombolytic therapy has been shown to reduce mortality and morbidity after acute myocardial infarction. Therapeutic benefit seems to be directly correlated with completeness of reperfusion (Thrombolysis in Myocardial Infarction [TIMI] grade 3 flow) of the infarct-related coronary artery, as well as the timeliness of reperfusion. To determine which regimen of Reteplase (r-PA), a deletion mutant of wild-type tissue plasminogen activator (t-PA), is most effective for clinical thrombolysis, several Reteplase regimens were compared with the most successful standard regimens of recombinant t-PA (alteplase) in 2 large-scale, randomized studies. All patients received aspirin and intravenous heparin. In the Reteplase Angiographic Phase II International Dose Finding Trial (RAPID-1), results in 606 randomized patients showed that a 10 + 10 U double bolus of Reteplase was more effective than a 15 U single bolus, a 10 + 5 double bolus, or conventional alteplase (100 mg over 3 hours). In the Reteplase versus Alteplase Patency Investigation During Acute Myocardial Infarction (RAPID-2) trial, results in 324 patients showed that significantly more patients achieved patency of the infarct-related artery (TIMI grade 2 or 3 flow) at 90 minutes with Reteplase (10 + 10 U double bolus) than with accelerated alteplase (100 mg over 90 minutes): 83.4% versus 73.3%, respectively (p = 0.03). The incidence of complete patency (TIMI grade 3 flow) at 90 minutes was likewise greater with Reteplase than with accelerated alteplase (59.9% vs 45.2%, respectively; p=0.01). At 60 minutes, the incidence of TIMI grade 2 or 3 flow was also significantly higher with Reteplase than with alteplase (81.8% vs 66.1%, respectively; p=0.01), as was the incidence of TIMI grade 3 flow (51.2% vs 37.4%, respectively; p 1996 by Excerpta Medica, Inc . Am J Cardiol 1996;78(suppl 12A):16–19
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Patency trials with Reteplase (r-PA): what do they tell us?
The American Journal of Cardiology, 1996Co-Authors: Christoph Bode, Thomas K. Nordt, Richard W. Smalling, Karlheinz Peter, M S Runge, Wolfgang KüblerAbstract:Abstract Thrombolytic therapy has been shown to reduce mortality and morbidity after acute myocardial infarction. Therapeutic benefit seems to be directly correlated with completeness of reperfusion (Thrombolysis in Myocardial Infarction [TIMI] grade 3 flow) of the infarct-related coronary artery, as well as the timeliness of reperfusion. To determine which regimen of Reteplase (r-PA), a deletion mutant of wild-type tissue plasminogen activator (t-PA), is most effective for clinical thrombolysis, several Reteplase regimens were compared with the most successful standard regimens of recombinant t-PA (alteplase) in 2 large-scale, randomized studies. All patients received aspirin and intravenous heparin. In the Reteplase Angiographic Phase II International Dose Finding Trial (RAPID-1), results in 606 randomized patients showed that a 10 + 10 U double bolus of Reteplase was more effective than a 15 U single bolus, a 10 + 5 double bolus, or conventional alteplase (100 mg over 3 hours). In the Reteplase versus Alteplase Patency Investigation During Acute Myocardial Infarction (RAPID-2) trial, results in 324 patients showed that significantly more patients achieved patency of the infarct-related artery (TIMI grade 2 or 3 flow) at 90 minutes with Reteplase (10 + 10 U double bolus) than with accelerated alteplase (100 mg over 90 minutes): 83.4% versus 73.3%, respectively (p = 0.03). The incidence of complete patency (TIMI grade 3 flow) at 90 minutes was likewise greater with Reteplase than with accelerated alteplase (59.9% vs 45.2%, respectively; p=0.01). At 60 minutes, the incidence of TIMI grade 2 or 3 flow was also significantly higher with Reteplase than with alteplase (81.8% vs 66.1%, respectively; p=0.01), as was the incidence of TIMI grade 3 flow (51.2% vs 37.4%, respectively; p 1996 by Excerpta Medica, Inc . Am J Cardiol 1996;78(suppl 12A):16–19
Martin Moser - One of the best experts on this subject based on the ideXlab platform.
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Platelet Function During and After Thrombolytic Therapy for Acute Myocardial Infarction With Reteplase, Alteplase, or Streptokinase
Circulation, 1999Co-Authors: Martin Moser, Thomas K. Nordt, Benedikt Kohler, Marc Schmittner, Richard W. Smalling, Karlheinz Peter, Wolfgang Kübler, Johannes Ruef, Christoph BodeAbstract:Background—Changes in platelet aggregation (PA) and platelet surface receptor expression induced by thrombolytic therapy for acute myocardial infarction may influence the rate of initial reperfusion and early reocclusion. Methods and Results—In the RAPID-1 (Reteplase Angiographic Phase II International Dose-finding study), RAPID-2 (Reteplase vs Alteplase Patency Investigation During myocardial infarction), INJECT (INternational Joint Efficacy Comparison of Thrombolytics), and GUSTO-3 (Global Use of Strategies To Open occluded coronary arteries) trials, 126 patients were enrolled in a single center. Patients were treated with either conventional alteplase (100 mg/180 min; n=15), accelerated alteplase (100 mg/90 min; n=21), Reteplase 10+10-U double bolus (n=50), Reteplase 10+5-U double bolus (n=15), Reteplase 15-U single bolus (n=15), or streptokinase (1.5 MU/60 min; n=10). PA (after stimulation with ADP), P-selectin expression and fibrinogen binding to glycoprotein (GP) IIb/IIIa (determined by flow cytomet...
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Recombinant Plasminogen Activators
BioDrugs, 1998Co-Authors: Martin Moser, Benedikt Kohler, Marc Schmittner, Christoph BodeAbstract:Thrombolytic therapy has been shown to reduce mortality after acute myocardial infarction. Great efforts have been undertaken in the past decade to develop more efficient thrombolytic regimens. Novel recombinant thrombolytic substances have been engineered. Reteplase, a deletion mutant of wild-type tissue plasminogen activator with a longer half-life, has been evaluated in clinical trials and is now available for clinical use. In the randomised Reteplase Angiographic Phase II International Dose-Finding (RAPID-1) trial, involving 606 patients with acute myocardial infarction, a double-bolus regimen of Reteplase (10 + 10U given 30 minutes apart) achieved significantly higher patency rates at 90 minutes after initiation of thrombolytic therapy than a 10 + 5U double-bolus Reteplase regimen, a 15U single bolus Reteplase regimen, or a conventional alteplase regimen (100mg in 180 minutes). In the Reteplase versus Alteplase Patency Investigation During Acute Myocardial Infarction (RAPID-2) trial, the Reteplase 10 + 10U double-bolus regimen was more effective with regard to patency at 60 and 90 minutes than accelerated alteplase (100mg in 90 minutes). The International Joint Efficacy Comparison of Thrombolytics (INJECT) trial showed that a double-bolus regimen of Reteplase 10 + 10U was at least equivalent to streptokinase in terms of 35-day mortality rate. The third Global Utilization of Strategies to Open Occluded Coronary Arteries (GUSTO-III) trial resulted in similar 30-day mortality after therapy with double-bolus Reteplase 10 + 10U (7.47%) or accelerated alteplase (7.24%). Hence, the higher early patency rates achieved with Reteplase treatment did not translate into improved survival. Consequently, there is still some uncertainty as to whether or not these drugs are equivalent.
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Reteplase (r-PA): a new plasminogen activator.
Expert opinion on investigational drugs, 1997Co-Authors: Christoph Bode, Benedikt Kohler, Marc Schmittner, Richard W. Smalling, Martin Moser, Ruth H StrasserAbstract:Reteplase (r-PA) is a genetically engineered deletion mutant of wild-type tissue-type plasminogen activator. The structural differences lead to different functional properties, such as a prolonged half-life. The compound demonstrated good thrombolytic efficacy in in vitro as well as in animal studies. In angiographically controlled patency studies (GRECO, GRECO-2 RAPID-1, RAPID-2), the double-bolus application scheme was established, and a superior patency profile for Reteplase in comparison to alteplase was demonstrated. Mortality studies established Reteplase as a safe drug with a 30-day mortality at least equivalent to streptokinase (INJECT) and very similar to alteplase (GUSTO-3). A possible advantage may be the double-bolus application without a need for weight adjustment, especially in a prehospital setting. Thus, Reteplase can be regarded as an excellent alternative to streptokinase or alteplase for thrombolytic therapy in acute myocardial infarction.
Heydar Sadeghi - One of the best experts on this subject based on the ideXlab platform.
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Investigation of Supercharging as a Strategy to Enhance the Solubility and Plasminogen Cleavage Activity of Reteplase
Iranian Journal of Biotechnology, 2020Co-Authors: Hooria Seyedhosseini Ghaheh, Mohamad Reza Ganjalikhany, Parichehreh Yaghmaei, Morteza Pourfarzam, Heydar SadeghiAbstract:Background: Reteplase, the recombinant form of tissue plasminogen activator, is a thrombolytic drug with outstanding characteristics, while demonstrating limited solubility and reduced plasminogen activation. Previously, we in silico designed a variant of Reteplase with positively supercharged surface, which showed promising stability, solubility and activity. This study was devoted to evaluation of the utility of supercharging technique for enhancing these characteristics in Reteplase. Objective: To test the hypothesis that reinforced surface charge of a rationally-designed Reteplase variant will not compromise its stability, will increase its solubility, and will enhance its plasminogen cleavage activity. Materials and Methods: Supercharged Reteplase coding sequence was cloned in pDest527 vector and expressed in E. coli BL21 (DE3). The expressed protein was extracted by cell disruption. Inclusion bodies were solubilized using guanidine hydrochloride, followed by dialysis for protein refolding. After confirmation with SDS-PAGE and western blotting, extracted proteins were assayed for solubility and tested for bioactivity. Results: SDS-PAGE and western blot analysis confirmed the successful expression of Reteplase. Western blot experiments showed most of Reteplase expressed in the insoluble form. Plasminogen cleavage assay showed significantly higher activity of the supercharged variant than the wild type protein ( P < 0.001). The stability of the supercharged variant was also comparable to the wild type. Conclusion: Our findings, i.e. the contribution of the surface supercharging technique to retained stability, enhanced plasminogen cleavage activity, while inefficiently changed solubility of Reteplase, contain implications for future designs of soluble variants of this fibrinolytic protein drug.
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Improvement of Soluble Production of Reteplase in Escherichia coli by Optimization of Chemical Chaperones in Lysis Buffer
Advanced biomedical research, 2019Co-Authors: Behnaz Fazeli, Vajihe Akbari, Asgar Barkhordari, Heydar SadeghiAbstract:Background: Reteplase is a nonglycosylated derivative of recombinant tissue plasminogen activator, a thrombolytic agent, which can be easily expressed in Escherichia coli. However, overexpression of Reteplase in E. coli usually leads to accumulation of insoluble and inactive aggregates and inclusion bodies. In the present study, we aimed to optimize chemical additives of lysis buffer to avoid the initial aggregation and formation of inclusion bodies of Reteplase at cell disruption step. Materials and Methods: After protein expression in E. coli BL21 (DE3), the bacterial cells were disrupted in different lysis buffers using microsmashing. Eleven chemical additives at two concentration levels were combined based on a Plackett–Burman design to prepare 12 different lysis buffers used at cell disruption stage. Then, three additives with the most positive effect on improvement of solubility of Reteplase were chosen and used for the second screening based on Box–Behnken model. Results: The primary screening results showed that among 11 additives, arginine, K2PO4,and cetyltrimethylammonium bromide (CTAB) had the most positive effect on solubility of Reteplase. Our final results based on 14 runs of Box–Behnken design showed that the optimum buffer additive condition is 0.005 mg/ml CTAB, 0.065 mg/ml arginine, and 0.026 mg/ml K2PO4. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis and Western blotting of soluble and total fraction of samples confirmed that these additives significantly improved soluble production of Reteplase compared with control. Conclusion: Our study indicates that the application of chemical additives in cell lysis can improve the solubility of Reteplase. Further studies are still required to understand the exact mechanism of chemical additives as a chemical chaperone during cell lysis.
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Effect of buffer additives on solubilization and refolding of Reteplase inclusion bodies
Research in pharmaceutical sciences, 2018Co-Authors: Iman Esmaili, Heydar Sadeghi, Vajihe AkbariAbstract:Reteplase is a non-glycosylated and recombinant form of tissue type plasminogen activator, which is produced in Escherichia coli. However, its overexpression usually leads to formation of inactive aggregates or inclusion bodies. In the present study, we report on the development of optimized processes for isolation, solubilization, and refolding of Reteplase inclusion bodies to recover active protein. After protein overexpression in E. coli BL21 (DE3) inclusion bodies were isolated by cell disruption and repeated wash of pellet with buffer containing Triton X-100. To solubilize the inclusion bodies, different types, concentrations, pHs, and additives of denaturing agents were used. Rapid micro dilution method was applied for refolding of solubilized Reteplase. Different chemical additives including sugars, alcohols, polymers, detergents, amino acids, kosmotropic, and chaotropic salts, reducing agents, and buffering agents were used in the refolding buffer. To evaluate the biological activity of refolded Reteplase, an indirect chromogenic assay was performed. The best solubilizing agent for dissolving Reteplase inclusion bodies was 6 M urea at pH 12. The optimized buffer for refolding of solubilized Reteplase was found to be 1.15 M glucose, 9.16 mM imidazole, and 0.16 M sorbitol which resulted in high yield of biologically active protein. Our results indicate type, concentration, and pH of solvent and type, concentration, and combination of chemical additives can significantly influence the yield of inclusion bodies solubilization and refolding.
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Expression and activity evaluation of Reteplase in Escherichia coli TOP10
Journal of paramedical sciences, 2015Co-Authors: Fatemeh Shafiee, Fatemeh Moazen, M. Rabbani, Heydar SadeghiAbstract:Reteplase is a part of tissue Plasminogen Activator (t-PA) used for theremoval of thrombi in blood vessels. In the present study we express the Reteplase genein Escherichia coli TOP10 and then its thrombolytic activity was measured. The recombinant plasmid pBADgIIIA was transformed into the competent Escherichia coli TOP10 and then transformed bacteria was seeded into bioreactor containing 1.5 L LB medium and induced by 0.02% L-Arabinoseat 37°C, pH 7, and 180 rpm until OD 600 of 0.6 was reached.Samples were analyzed by SDS-PAGE and western blotting andthe expression of Reteplase was examined. Finally the activity of this recombinant protein was evaluated using Chromogenic Activity Assay Kit. The presence of Reteplase in transformed Escherichia coli TOP10 wasexamined by western blotting which revealed that the target protein in form inclusion body was expressed as a unique band at39 and the refolded Reteplase was 66KDa. The amount of protein produced was 90.5µg/mL and its activity was determined as 0.8 units. In this study, the expression of Reteplase in Escherichia coli TOP10 wasscaled up under optimum condition. Furthermore we earned Reteplase with partially suitable thrombolytic activity.
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Optimization of the Expression of Reteplase in Escherichia coli TOP10 Using Arabinose Promoter.
Jundishapur journal of natural pharmaceutical products, 2015Co-Authors: Fatemeh Shafiee, Fatemeh Moazen, M. Rabbani, Heydar SadeghiAbstract:Background: Reteplase is a mutant version of t-PA (tissue plasminogen activator) with prolonged half-life. In the present study, E. coli Top 10 bacteria were utilized in the production of Reteplase, which is the nonglycosylated active domain of t-PA. Reteplase gene was ligated into pBAD/gIII plasmid which, allows secretion of this protein in periplasmic space. It would allow the correct formation of disulfide bonds in protein structure. Objectives: This study aimed at expression of Reteplase in optimum condition. In this study, the Reteplase gene was cloned and expressed in Escherichia coli top 10 as a suitable host cell and its expression was optimized. Materials and Methods: The recombinant plasmid, pET15b/Reteplase was digested by NcoI and BamHI restriction enzymes; while pBAD/ gIIIA vector was digested by NcoI and BglII. Then the insert and vector were ligated and used for transformation of E. coli Top10 cells by heat shock method. Overnight culture of transformed bacteria was induced by L-arabinose in various concentrations (0.2, 0.02, 0.002, and 0.0002%) and at various temperatures. Results: The obtained recombinant plasmid was sequenced to confirm the presence and correct framing of Reteplase gene regarding the expression of Reteplase. Maximum production of this enzyme was obtained under the following condition: 0.0002% L-arabinose at 37°C for 2 hours incubation. The purified protein was detected on SDS-PAGE (sodium dodecyl sulfate Polyacrylamide gel electrophoresis) as a 66 kDa band. The concentration of t-PA standard was 1 unit which is equal to 12 µg/mL. The enzymatic activity of samples was measured as 0.8 units compared to the standards. Conclusions: Reteplase was expressed in E. coli Top 10 after activation of pBAD/gIIIA promoter region by arabinose and optimized.
H. Mir Mohammad Sadeghi - One of the best experts on this subject based on the ideXlab platform.
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Expression of Reteplase in Escherichia coli top10 using arabinose promoter
Research in Pharmaceutical Sciences, 2012Co-Authors: Fatemeh Khodabakhsh, Fatemeh Moazen, H. Mir Mohammad Sadeghi, M. RabbaniAbstract:Background and Aims: Production of tissue plasminogen activator protein (t -PA) in prokaryotes systems has many problems such as lack of active protein production, multiple purification steps, and refolding process which has been shown to be costly and time consuming. In the present study TOP 10 E. coli bacteria was utilized for the production of Reteplase which is the nonglycosylated active domain of t -PA. Retplase gene was ligated into pBAD/gIII plasmid which allows secretion of this protein in periplasmic space which would allow the correct formation of disulfide bonds in protein structure. Methods: Recombinant pET15b/Reteplase plasmid was digested by NcoI and BamHI restriction enzymes while pBAD/gIII vector was digested by NcoI and BglII. Then the insert and vector were ligated and used for transformation E.coli Top10 cells by heat shock method. Overnight culture of transformed bacteria was induced by L-Arabinose in various concentrations (0/2, 0/02, 0/002 and 0/0002%) at various temperatures. Results: The obtained recombinant plasmid was sequenced to confirm the presence and correct framing of Reteplase gene regarding the expression of Reteplase. Maximum production of this enzyme was obtained under the following condition: 0/0002% L-Arabinose (as inducer), at 37°C (tempreture of incubation). The production of Reteplase was confirmed by Western blotting using Anti-TpA antibody. Conclusions: Reteplase was expressed in E. coli after activation of pBAD/gIII promoter region by arabinose.
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Expression of Reteplase in Escherichia coli BL21 (DE3) using T7 promoter
Research in Pharmaceutical Sciences, 2012Co-Authors: Fatemeh Khodabakhsh, Fatemeh Moazen, H. Mir Mohammad Sadeghi, M. RabbaniAbstract:Background and Aims: Reteplase is a segment of tissue Plasminogen Activator (t-PA) used for the removal of thrombi in blood vessels. In the present study the cloned Reteplase gene was used for its expression in E. coli strain BL21 (DE3) cells. Methods: The recombinant plasmid, pET 15b/Reteplase, was transformed into competent E.Coli strain BL21 (DE3) cells. Overnight culture of transformed bacteria was induced by addition of IPTG to the final concentrations of 0.25, 0.5, 1 and 1.5 mM. Also, the effects of temperature, shaking speeds and glucose concentration on the expression of Reteplase were examined (25, 30, 37 and 39 °C; 100, 170 and 190 rpm and 0.25, 0.5, 0.75 and 1 mM, respectively). Samples were analyzed by SDS-PAGE and the expression of Reteplase was examined. Results: After obtaining recombinant E. coli cells, the presence of Reteplase in these cells was examined by western blotting which revealed that the target protein was expressed as a unique band at 39kD. The purpose of this investigation was to find conditions which produce high levels of Reteplase. Maximum amount of protein expression was obtained by the addition of 1mM IPTG, at 37°C, 100rpm of shaking speed and the absence of glucose in the media. Conclusions: In this study, expression of Reteplase in E.coil BL21 (DE3) was optimized.
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Molecular cloning and expression of Reteplase in E. coli using tac promoter
Research in Pharmaceutical Sciences, 2012Co-Authors: Safieh Aghaabdollahian, M. Rabbani, Kamran Ghaedi, H. Mir Mohammad SadeghiAbstract:Background and Aims: This study was aimed to clone and express the Reteplase gene, a thrombolytic agent used for the treatment of acute myocardial infarction and stroke, under controlling of tac promoter in E.coli. Methods: Reteplase gene was amplified using polymerase chain reaction (PCR) with designed primers. The product was then cloned into pTZ57 plasmid. The cloned gene was digested out and ligated into pGEX-5x-1 expression vector. The presence of the insert was confirmed by restriction digestion and determination of the nucleotide sequence. By using 0.2, 0.5 and 1mM isopropyl beta-D thiogalactopyranoside (IPTG), Reteplase was induced in E. coli TOP10 cells and analyzed by SDS-PAGE. Results: Electrophoresis of PCR product and also double digested recombinant pTZ57 plasmid (rptz), showed a 1068bp band of Reteplase. SDS-PAGE analysis showed a 60 KDa band of protein product induced with all the concentrations of IPTG. Conclusions: in the present study Reteplase gene was successfully cloned and expressed under controlling tac promoter. The induced vector can be used for future analysis of expressed protein, Reteplase.
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Expression of Reteplase by a non-viral insect cell expression system
Research in Pharmaceutical Sciences, 2012Co-Authors: S. Aflakiyan, H. Mir Mohammad Sadeghi, M. Rabbani, Mohammad Ali Shokrgozar, Saeid Bouzari, Ali Jahanian-najafabadiAbstract:Background and Aims: Thrombolysis is the first choice of therapy for acute myocardial infarction (AMI) and Reteplase is a thrombolytic agent often used for the treatment of acute myocardial infarction. In the present study we evaluated the expression of Reteplase by a non-viral insect cell expression system. Methods: Coding sequence of the Reteplase was cloned into the pMIB/V5-His plasmid. The recombinant plasmid was used to transfect Sf9 insect cells. Stable cell lines were produced at the presence of 80μg/ml Blasticidin S HCl. Finally, the stable cells were screened for the expression of the Reteplase protein by dot-blot and Western blot analysis using HRP conjugated anti His-tag and anti-V5 tag antibodies. Results: DNA sequencing confirmed the fidelity of the cloned sequence. Screening of the transfected cells using Blasticidin S HCl resulted in some stable clones. Some producing cell lines were selected following dotblot analysis of the concentrated culture medium of the stable cells. The producer stable cells were propagated into larger cell culture monolayer. Western blot analysis confirmed the expression of the Reteplase protein by showing bands of about 45 kD. Conclusions: These results confirmed the ability of the non-viral insect cell expression system to produce Reteplase. Therefore, the obtained stable cell lines could be used for further expression of the recombinant protein in order to evaluate its biological activity and also for large scale protein production.
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Optimization of the expression of Reteplase in Escherichia coli.
Research in pharmaceutical sciences, 2011Co-Authors: H. Mir Mohammad Sadeghi, Fatemeh Khodabakhsh, Fatemeh Moazen, M. Rabbani, E Rismani, K Dormiani, Y KhazaeiAbstract:Reteplase is a segment of tissue plasminogen activator used for the removal of thrombi in blood vessels. In the present study the cloned Reteplase gene was used for its expression in competent E. coli. The recombinant plasmid, pET15b/Reteplase (rpET-BL21), was transformed into competent E. coli strain BL21 (DE3) cells. Overnight culture of the transformed bacteria was induced by the addition of isopropylthio-s-Dgalactoside (IPTG) to the final concentrations of 0.25, 0.5, 1 and 1.5 mM. Also, the effects of different temperatures(25, 30, 37 and 39°C), shaking speeds (100, 170 and 190 rpm), and various glucose concentrations (0.25, 0.5, 0.75 and 1 mM) on the expression of Reteplase were examined. Samples were analyzed by SDS-PAGE. Maximum amount of protein production was obtained by the addition of 1 mM IPTG at 37°C, 100 rpm of shaking speed in the absence of glucose.
M. Rabbani - One of the best experts on this subject based on the ideXlab platform.
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Expression and activity evaluation of Reteplase in Escherichia coli TOP10
Journal of paramedical sciences, 2015Co-Authors: Fatemeh Shafiee, Fatemeh Moazen, M. Rabbani, Heydar SadeghiAbstract:Reteplase is a part of tissue Plasminogen Activator (t-PA) used for theremoval of thrombi in blood vessels. In the present study we express the Reteplase genein Escherichia coli TOP10 and then its thrombolytic activity was measured. The recombinant plasmid pBADgIIIA was transformed into the competent Escherichia coli TOP10 and then transformed bacteria was seeded into bioreactor containing 1.5 L LB medium and induced by 0.02% L-Arabinoseat 37°C, pH 7, and 180 rpm until OD 600 of 0.6 was reached.Samples were analyzed by SDS-PAGE and western blotting andthe expression of Reteplase was examined. Finally the activity of this recombinant protein was evaluated using Chromogenic Activity Assay Kit. The presence of Reteplase in transformed Escherichia coli TOP10 wasexamined by western blotting which revealed that the target protein in form inclusion body was expressed as a unique band at39 and the refolded Reteplase was 66KDa. The amount of protein produced was 90.5µg/mL and its activity was determined as 0.8 units. In this study, the expression of Reteplase in Escherichia coli TOP10 wasscaled up under optimum condition. Furthermore we earned Reteplase with partially suitable thrombolytic activity.
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Optimization of the Expression of Reteplase in Escherichia coli TOP10 Using Arabinose Promoter.
Jundishapur journal of natural pharmaceutical products, 2015Co-Authors: Fatemeh Shafiee, Fatemeh Moazen, M. Rabbani, Heydar SadeghiAbstract:Background: Reteplase is a mutant version of t-PA (tissue plasminogen activator) with prolonged half-life. In the present study, E. coli Top 10 bacteria were utilized in the production of Reteplase, which is the nonglycosylated active domain of t-PA. Reteplase gene was ligated into pBAD/gIII plasmid which, allows secretion of this protein in periplasmic space. It would allow the correct formation of disulfide bonds in protein structure. Objectives: This study aimed at expression of Reteplase in optimum condition. In this study, the Reteplase gene was cloned and expressed in Escherichia coli top 10 as a suitable host cell and its expression was optimized. Materials and Methods: The recombinant plasmid, pET15b/Reteplase was digested by NcoI and BamHI restriction enzymes; while pBAD/ gIIIA vector was digested by NcoI and BglII. Then the insert and vector were ligated and used for transformation of E. coli Top10 cells by heat shock method. Overnight culture of transformed bacteria was induced by L-arabinose in various concentrations (0.2, 0.02, 0.002, and 0.0002%) and at various temperatures. Results: The obtained recombinant plasmid was sequenced to confirm the presence and correct framing of Reteplase gene regarding the expression of Reteplase. Maximum production of this enzyme was obtained under the following condition: 0.0002% L-arabinose at 37°C for 2 hours incubation. The purified protein was detected on SDS-PAGE (sodium dodecyl sulfate Polyacrylamide gel electrophoresis) as a 66 kDa band. The concentration of t-PA standard was 1 unit which is equal to 12 µg/mL. The enzymatic activity of samples was measured as 0.8 units compared to the standards. Conclusions: Reteplase was expressed in E. coli Top 10 after activation of pBAD/gIIIA promoter region by arabinose and optimized.
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Expression of Reteplase in Escherichia coli top10 using arabinose promoter
Research in Pharmaceutical Sciences, 2012Co-Authors: Fatemeh Khodabakhsh, Fatemeh Moazen, H. Mir Mohammad Sadeghi, M. RabbaniAbstract:Background and Aims: Production of tissue plasminogen activator protein (t -PA) in prokaryotes systems has many problems such as lack of active protein production, multiple purification steps, and refolding process which has been shown to be costly and time consuming. In the present study TOP 10 E. coli bacteria was utilized for the production of Reteplase which is the nonglycosylated active domain of t -PA. Retplase gene was ligated into pBAD/gIII plasmid which allows secretion of this protein in periplasmic space which would allow the correct formation of disulfide bonds in protein structure. Methods: Recombinant pET15b/Reteplase plasmid was digested by NcoI and BamHI restriction enzymes while pBAD/gIII vector was digested by NcoI and BglII. Then the insert and vector were ligated and used for transformation E.coli Top10 cells by heat shock method. Overnight culture of transformed bacteria was induced by L-Arabinose in various concentrations (0/2, 0/02, 0/002 and 0/0002%) at various temperatures. Results: The obtained recombinant plasmid was sequenced to confirm the presence and correct framing of Reteplase gene regarding the expression of Reteplase. Maximum production of this enzyme was obtained under the following condition: 0/0002% L-Arabinose (as inducer), at 37°C (tempreture of incubation). The production of Reteplase was confirmed by Western blotting using Anti-TpA antibody. Conclusions: Reteplase was expressed in E. coli after activation of pBAD/gIII promoter region by arabinose.
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Expression of Reteplase in Escherichia coli BL21 (DE3) using T7 promoter
Research in Pharmaceutical Sciences, 2012Co-Authors: Fatemeh Khodabakhsh, Fatemeh Moazen, H. Mir Mohammad Sadeghi, M. RabbaniAbstract:Background and Aims: Reteplase is a segment of tissue Plasminogen Activator (t-PA) used for the removal of thrombi in blood vessels. In the present study the cloned Reteplase gene was used for its expression in E. coli strain BL21 (DE3) cells. Methods: The recombinant plasmid, pET 15b/Reteplase, was transformed into competent E.Coli strain BL21 (DE3) cells. Overnight culture of transformed bacteria was induced by addition of IPTG to the final concentrations of 0.25, 0.5, 1 and 1.5 mM. Also, the effects of temperature, shaking speeds and glucose concentration on the expression of Reteplase were examined (25, 30, 37 and 39 °C; 100, 170 and 190 rpm and 0.25, 0.5, 0.75 and 1 mM, respectively). Samples were analyzed by SDS-PAGE and the expression of Reteplase was examined. Results: After obtaining recombinant E. coli cells, the presence of Reteplase in these cells was examined by western blotting which revealed that the target protein was expressed as a unique band at 39kD. The purpose of this investigation was to find conditions which produce high levels of Reteplase. Maximum amount of protein expression was obtained by the addition of 1mM IPTG, at 37°C, 100rpm of shaking speed and the absence of glucose in the media. Conclusions: In this study, expression of Reteplase in E.coil BL21 (DE3) was optimized.
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Molecular cloning and expression of Reteplase in E. coli using tac promoter
Research in Pharmaceutical Sciences, 2012Co-Authors: Safieh Aghaabdollahian, M. Rabbani, Kamran Ghaedi, H. Mir Mohammad SadeghiAbstract:Background and Aims: This study was aimed to clone and express the Reteplase gene, a thrombolytic agent used for the treatment of acute myocardial infarction and stroke, under controlling of tac promoter in E.coli. Methods: Reteplase gene was amplified using polymerase chain reaction (PCR) with designed primers. The product was then cloned into pTZ57 plasmid. The cloned gene was digested out and ligated into pGEX-5x-1 expression vector. The presence of the insert was confirmed by restriction digestion and determination of the nucleotide sequence. By using 0.2, 0.5 and 1mM isopropyl beta-D thiogalactopyranoside (IPTG), Reteplase was induced in E. coli TOP10 cells and analyzed by SDS-PAGE. Results: Electrophoresis of PCR product and also double digested recombinant pTZ57 plasmid (rptz), showed a 1068bp band of Reteplase. SDS-PAGE analysis showed a 60 KDa band of protein product induced with all the concentrations of IPTG. Conclusions: in the present study Reteplase gene was successfully cloned and expressed under controlling tac promoter. The induced vector can be used for future analysis of expressed protein, Reteplase.