Reteplase (recombinant plasminogen activator, rPA) is a mutant non-glycosylated tissue-type plasminogen activator (tPA) containing 355 amino acids with longer half-life and promising thrombolytic activity than its original counterpart, full length tPA. In this study, we aimed to produce and optimize the purification process of recombinant tissue-type plasminogen activator (tPA) known as Reteplase (rPA). Reteplase cDNA synthesized from total mRNA isolated from human placenta was PCR amplified, cloned into a pET-28a(+) E. coli expression vector and expressed in Rosetta-gami 2 E. coli (Novagen^Ⓡ) host. rPA was expressed as an inclusion body in E. coli and its biological activity was achieved after single step solubilization, purification and refolding. We exploited the strategy of Slow Refolding using Gradual Dialysis (SRGD) in which a refolding buffer containing glutathione oxidized (1 mM GSSG) and glutathione reduced (3 mM GSH) and pH 9.0 was used. Using the SRGD method, we were able to successfully obtain the protein in its active form. We obtained 4.26 mg of active refolded protein from a 50 mL culture that was scaled up in a bioreactor. The purity and homogeneity of rPA was evaluated by SDS-PAGE, Western blotting and mass spectrometry. Circular dichroism spectroscopy was conducted to evaluate the refolding and stability of the refolded rPA in comparison to reference standard rPA. The thrombolytic potential of rPA was assessed by fibrin plate assay and In Vitro clot lysis assay. The presented protocol offers a viable approach for enhancing both the yield and refolding efficiency of reteplase, potentially resulting in an increase in yield.

01 Apr 2023·Iranian journal of biotechnology

Response Surface Methodology to Optimize the Expression Efficiency of Recombinant Reteplase.

Article

Author: Mirzaie, Sako ; Farzaneh, Farhad ; Farzaneh, Shirin ; Dehnavi, Ehsan ; Aghaeepoor, Mojtaba ; Pourzardosht, Navid ; Khalili, Saeed

BackgroundOver expression of Reteplase enzyme has already been studies in the periplasmic space of Escherichia coli (E. coli). However, the role different factors in its expresssin rate remained to be elucidated.ObjectivesOptical cell density (OD), IPTG concentration, and expression time are highly effective in the protein expression rates. Therefore, we aimed to determine the optimum levels of these factors for reteplase expression using response surface methodology (RSM).Materials and MethodsThe pET21b plasmid was used to sub-clone the designed reteplase gene. Then, the gene was transformed into E. coli BL21 strain. Induction of expression was done by IPTG and analyzed by the SDS page. experiments were designed using the RMS, while the effects of different conditions were evaluated using the Real time-PCR.ResultsSequence optimization removed all undesirable sequences of the designed gene. Transformation into E. coli BL21 was confirmed with an 1152 bp band on the agarose gel. A 39 kDa expression band on the SDS gel confirmed the gene expression. Performing 20 RSM-designed experiments, the optimum levels for IPTG concentration and OD were determined as 0.34mM and 5.6, respectively. Moreover, the optimum level of expression time was demonstrated to be 11.91 hours. The accuracy of the regression model for reteplase overexpression was confirmed by an F-value equal to 25.31 and a meager probability value [(Prob > F) < 0.0001]. The real-time-PCR results indicated that the performed calculations were highly accurate.ConclusionThe obtained results indicate that IPTG concentration, OD, and expression time are significantly involved in the augmentation of recombinant reteplase expression. To the best of our knowledge, this is the first study to assess the combined effect of these factors on reteplase expression. Further RSM-based experiments would bring about new insights regarding the best conditions for reteplase expression.

01 Jan 2018·Iranian journal of pharmaceutical research : IJPRQ4 · MEDICINE

Determination of Biological Activity of Recombinant Reteplase Using Clot Lysis Time and Activated Partial Thromboplastin Time (APTT) Lysis Methods: A Comparative Study.

Q4 · MEDICINE

Article

Author: Fazeli, Ahmad ; Hashemi-Najafabadi, Sameereh ; Alebouyeh, Mahmoud ; Babaee, Tahereh ; Khoshayand, Mohammad Reza ; Mohammadi, Ali ; Fazeli, Mohammad Reza ; Rastegar, Hosein

Recombinant plasminogen activator (reteplase) is a third generation thrombolytic agent which has been used on coronary artery thrombosis and acute myocardial infarction. Clot lysis assay is usually considered as a unique method to evaluate biological activity of reteplase. In this study biological activity of reteplase was determined by APTT (activated partial thromboplastin time) lysis method. Validity of this method was evaluated in comparison with reference method, clot lysis time assay. Results of APTT lysis test showed good reproducibility (relative standard deviation (RSD) 3-5% for within day analysis and 4-7% for between day analysis), and accuracy (101.3-102.7%). APTT lysis responses were linear in range of 0.001-0.1 mg/mL reteplase. Therefore, APTT lysis method is applicable for biological activity determination of reteplase. Although more comprehensive studies are required to approve this test as a reference method, APTT lysis method seems to be valuable to receive more attention due to advantages of technical simplicity, sensitivity, applicability, and cost efficiency.

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Indication	Highest Phase	Country/Location	Organization	Date
Myocardial Infarction	IND Application	CN	Jiangzhong Pharmaceutical Co., Ltd.	18 Jan 2001

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