Over 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.

Objectives:

Optical 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 Methods:

The 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.

Results:

Sequence 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.

Conclusion:

The 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.

2018-01-01·Iranian journal of pharmaceutical research : IJPR4区 · 医学

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

4区 · 医学

Article

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

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.

2017-09-01·Plant physiology and biochemistry : PPB2区 · 生物学

Biotechnological production of recombinant tissue plasminogen activator protein (reteplase) from transplastomic tobacco cell cultures

2区 · 生物学

Article

作者: Cusidó, Rosa M ; Palazon, Javier ; Hidalgo, Diego ; Bru-Martínez, Roque ; Corchete, Purificación ; Abdoli-Nasab, Maryam ; Jalali-Javaran, Mokhtar

Transplastomic plants are a system of choice for the mass production of biopharmaceuticals due to the polyploidy of the plastid genome and the low risk of pollen-mediated outcrossing because of maternal inheritance. However, as field-grown plants, they can suffer contamination by agrochemicals and fertilizers, as well as fluctuations in yield due to climatic changes and infections. Tissue-type plasminogen activator (tPA), a protein used to treat heart attacks, converts plasminogen into plasmine, which digests fibrin and induces the dissolution of fibrin clots. Recently, we obtained transplastomic tobacco plants carrying the K2S gene encoding truncated human tPA (reteplase) with improved biological activity, and confirmed the presence of the target protein in the transgenic plant leaves. Considering the advantages of plant cell cultures for biopharmaceutical production, we established a cell line derived from the K2S tobacco plants. The active form of reteplase was quantified in cultures grown in light or darkness, with production 3-fold higher in light.

100 项与重组瑞替普酶 (江苏江中集团) 相关的药物交易

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