• Assessing the Cloning and Expression Efficiency of Recombinant Reteplase by Response Surface Methodology
  • farhad farzaneh,1,* Sako Mirzaie,2 Mojtaba Aghaeepoor ,3 Shirin Farzaneh,4
    1. Department of Biochemistry, Faculty of Science, Sanandaj Branch, Islamic Azad University, Sanandaj, Iran.
    2. Department of Biochemistry, Faculty of Science, Sanandaj Branch, Islamic Azad University, Sanandaj, Iran.
    3. Gene Transfer Pioneers (GTP) Research Group, Shahid Beheshti University of Medical Sciences. Tehran,Iran
    4. Pharmaceutical Science Research Centre, Tehran medical Science, Islamic Azad University, Tehran, Iran.


  • Introduction: Reteplase is a thrombolytic enzyme which is derived from Tissue plasminogen activator (t-PA) with better thrombolytic effects. Although Reteplase has been previously expressed in periplasmic space of Escherichia coli (E. coli), the extent of effects exerted by each expression factor remained to be determined. In this regard, we have used the Response surface Methodology (RSM) to determine the effects of IPTG concentration, optical cell density (OD) and expression time on Reteplase expression and their optimum levels.
  • Methods: the Reteplase gene was designed and chemically synthesized. The gene was sub-cloned into PET21b plasmid and transformed by CaCl2 method into E. coli BL21 strain. The expression was done by IPTG induction and analysed by SDS page. The RMS was used to design experiments and the Real time-PCR was used to evaluate the effects of conditions.
  • Results: the recombinant plasmid PET21b containing the designed Reteplase gene was constructed and transformed into E. coli BL21. The existence of a 39 kDA Reteplase band on the SDS gel confirmed the gene expression. The RSM has designed 20 experiments to evaluation the effects of IPTG concentration, OD and expression time of Reteplase expression levels. The real time-PCR results indicated that the performed calculations were highly accurate and the optimum levels for IPTG concentration is 0.34mM, OD (5.6) and expression time is 11.91 hourse.
  • Conclusion: The obtained results indicate that RSM is an amenable approach to optimize the expression levels of recombinant proteins. This method would help to determine the optimum levels of each factor. Further experiments and RSM designs on other contributing factors would bring new insights about the best conditions for periplasmic expression of Reteplase.
  • Keywords: Reteplase, Tissue plasminogen activator (t-PA), RSM.