Molecular cloning of Reteplase and its expression in E. coli using tac promoter

Aghaabdollahian, Safieh; Rabbani, Mohammad; Ghaedi, Kamran; Sadeghi, Hamid Mir Mohammad

doi:10.4103/2277-9175.140622

Cited by 16 publications

(4 citation statements)

References 21 publications

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“…As a recombinant thrombolytic peptide, r-PA is a structurally modified type of native t-PA, thus has better qualities in half-life and fibrin specificity and play an important role in the treatment [ 16 ]. Besides, it has lower side effects and is easier for administration, and has good effects in treatments for diseases like heart and brain occlusive disorders [ 17 ]. In a research about cerebral embolic rabbits, rPA is found to improve the return of cerebral perfusion faster than rtPA [ 15 ], but the effectiveness and safety of rRA for the treatment of CI in humans remain to be studied.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of efficacy and safety of Reteplase and Alteplase in the treatment of hyper-acute cerebral infarction

et al. 2018

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Objective: The present study aimed to investigate the efficacy and safety of Reteplase (rPA) and Alteplase (rt-PA) in the treatment of hyper-acute cerebral infarction (CI).Methods: Six hundred and eleven patients with hyper-acute CI selected from September 2014 to September 2016 were assigned into the aspirin, rt-PA, rPA, rt-PA + aspirin, and rPA + aspirin groups based on their willingness. The difference of efficacy in five groups were evaluated with National Institute of Health Stroke Scale (NIHSS), modified rankin scale (mRS), and Barthel Index (BI). Coagulation function, blood lipid, and hemodynamics were analyzed. The safety differences were compared by observing the adverse reactions.Results: Compared with the rt-PA, rPA, and aspirin groups, NIHSS score, mRS score, the incidence of non- and symptomatic cerebral hemorrhage as well as the rate of adverse reactions were decreased, while BI were increased in the rt-PA + aspirin and rPA + aspirin groups after treatment. Compared with the rt-PA and rPA groups, total cholesterol (TC), triacylglycerol (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) levels were lower, whereas the hematocrit, whole blood high shear viscosity, whole blood low shear viscosity, plasma viscosity, erythrocyte electrophoresis time, fibrinogen, erythrocyte sedimentation rate (ESR), K value in blood sedimentation equation, and the comprehensive abnormality degree of blood rheology were higher in the rt-PA + aspirin and rPA + aspirin groups.Conclusion: The efficacy and safety of rt-PA or rPA combined with aspirin in the treatment of hyper-acute CI were better than those of rPA or rt-PA monotherapy.

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Section: Introductionmentioning

confidence: 99%

Evaluation of efficacy and safety of Reteplase and Alteplase in the treatment of hyper-acute cerebral infarction

et al. 2018

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“…In addition, hybrid tac promoter, derived from the trp and lacUV5 promoters, is well-known for the 2–7 folds higher strength than the parent promoters [ 46 ]. The tac promoter has been popularly employed in metabolic engineering [ 47 , 48 ]. Thus, two strong constitutive promoters BBa - J23100 and tac were chosen for expression of the hpaBC and d - ldh Y52A genes as bicistron.…”

Section: Resultsmentioning

confidence: 99%

Chromosome engineering of Escherichia coli for constitutive production of salvianic acid A

et al. 2017

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BackgroundSalvianic acid A (SAA), a valuable natural product from herbal plant Salvia miltiorrhiza, exhibits excellent antioxidant activities on food industries and efficacious therapeutic potential on cardiovascular diseases. Recently, production of SAA in engineered Escherichia coli was established via the artificial biosynthetic pathway of SAA on the multiple plasmids in our previous work. However, the plasmid-mediated system required to supplement expensive inducers and antibiotics during the fermentation process, restricting scale-up production of SAA. Microbial cell factory would be an attractive approach for constitutive production of SAA by chromosome engineering.ResultsThe limited enzymatic reactions in SAA biosynthetic pathway from glucose were grouped into three modules, which were sequentially integrated into chromosome of engineered E. coli by λ Red homologous recombination method. With starting strain E. coli BAK5, in which the ptsG, pykF, pykA, pheA and tyrR genes were previously deleted, chassis strain BAK11 was constructed for constitutive production of precursor l-tyrosine by replacing the 17.7-kb mao-paa cluster with module 1 (PlacUV5-aroG fbr-tyrA fbr-aroE) and the lacI gene with module 2 (Ptrc-glk-tktA-ppsA). The synthetic 5tacs promoter demonstrated the optimal strength to drive the expression of hpaBC-d-ldh Y52A in module 3, which then was inserted at the position between nupG and speC on the chromosome of strain BAK11. The final strain BKD13 produced 5.6 g/L of SAA by fed-batch fermentation in 60 h from glucose without any antibiotics and inducers supplemented.ConclusionsThe plasmid-free and inducer-free strain for SAA production was developed by targeted integration of the constitutive expression of SAA biosynthetic genes into E. coli chromosome. Our work provides the industrial potential for constitutive production of SAA by the indel microbial cell factory and also sets an example of further producing other valuable natural and unnatural products.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0700-2) contains supplementary material, which is available to authorized users.

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“…Common strategies to improve solubility during protein expression include the use of weak promoters, low IPTG concentration, low temperature (24), modified growth media (25), co-expression with molecular chaperons (26), fusion with solubility enhancing tags (27), and use of various expression hosts (28). Unfortunately, these experimental studies have had low yield of protein solubility and require denaturation/refolding processes, hence being cost-intensive and time-consuming.…”

Section: Discussionmentioning

confidence: 99%

Improving the solubility, activity, and stability of reteplase using in silico design of new variants

et al. 2019

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Reteplase (recombinant plasminogen activator, r-PA) is a thrombolytic agent recombined from tissue-type plasminogen activator (t-PA), which has several prominent features such as strong thrombolytic ability and E. coli expressibility. Despite these outstanding features, it demonstrates reduced fibrin binding affinity, reduced stimulation of protease activity, and lower solubility, hence higher aggregation propensity, compared to t-PA. The present study was devoted to design r-PA variants with comparable structural stability, enhanced biological activity, and high solubility. For this purpose, computational molecular modeling techniques were utilized. The supercharging technique was applied for r-PA to designing new species of the protein. Based on the results from in silico evaluation of selected mutations in comparison to the wild-type r-PA, the designed supercharged mutant (S7 variant) exhibited augmented stability, decreased solvation energy, as well as enhanced binding affinity to fibrin. The data also implied increased plasminogen cleavage activity of the new variant. These findings have implications to therapies which involve removal of intravascular blood clots, including the treatment of acute myocardial infarction.

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Molecular cloning of Reteplase and its expression in E. coli using tac promoter

Cited by 16 publications

References 21 publications

Evaluation of efficacy and safety of Reteplase and Alteplase in the treatment of hyper-acute cerebral infarction

Evaluation of efficacy and safety of Reteplase and Alteplase in the treatment of hyper-acute cerebral infarction

Chromosome engineering of Escherichia coli for constitutive production of salvianic acid A

Improving the solubility, activity, and stability of reteplase using in silico design of new variants

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