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Abdi, A., Hosseinpour, M., Mashayekhi, K., Javad Mousavi, M., Elham Badiee Kheirabadi, S., & Sankian, M. (2019). Optimization of Cloning Conditions for High-level Production of Recombinant Mouse Interleukin-2 in Escherichia coli. Research in Molecular Medicine (RMM), 7(1). https://doi.org/10.18502/rmm.v7i1.5256

https://doi.org/10.18502/rmm.v7i1.5256

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authors

  • Arezou Abdi

    Arezou Abdi

    Immuno-Biochemistry lab, Immunology Research Center, Mashhad University of medical Sciences, Mashhad, Iran
  • Mitra Hosseinpour

    Mitra Hosseinpour

    Immuno-Biochemistry lab, Immunology Research Center, Mashhad University of medical Sciences, Mashhad, Iran
  • Kazem Mashayekhi

    Kazem Mashayekhi

    Immuno-Biochemistry lab, Immunology Research Center, Mashhad University of medical Sciences, Mashhad, Iran
  • Mohammad Javad Mousavi

    Mohammad Javad Mousavi

    Department of Hematology, Faculty of Allied Medicine, Bushehr University of Medical Sciences, Bushehr, Iran; Immunology Department, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
  • Seyedeh Elham Badiee Kheirabadi

    Seyedeh Elham Badiee Kheirabadi

    Immuno-Biochemistry lab, Immunology Research Center, Mashhad University of medical Sciences, Mashhad, Iran; Immunology, Torbat Heydariyeh University of Medical Sciences, Torbat Heydariyeh, Iran
  • Mojtaba Sankian

    Mojtaba Sankian

    Immuno-Biochemistry lab, Immunology Research Center, Mashhad University of medical Sciences, Mashhad, Iran

Published Date

  • Sep 30, 2019

Optimization of Cloning Conditions for High-level Production of Recombinant Mouse Interleukin-2 in Escherichia coli

Research in Molecular Medicine (RMM) / Research in Molecular Medicine (RMM): Volume 7, Issue No. 1 /

Abstract

Backgrounds and objectives: Many proteins have been expressed so far in bacterial host. Due to its simple culture conditions, having a short life cycle, and easily genetic manipulation, E.coli have been regarded as a preferable host to produce recombinant proteins, but protein cloning in bacterial host have many challenges. Therefore, we aimed to review some of these problems by an experience from mice IL-2 recombinant. Methods: cDNA synthesis was performed after RNA extraction of mouse splenocytes. PCR product purification carried out after IL-2 coding sequence amplification and was ligated into the pET-21b (+) vector and transformed into the competent BL21 E.coli. Expression and purification of recombinant mouse IL-2 were done using IPTG inducer and metal affinity chromatography respectively. Results: DNA sequencing confirmed the accuracy of the insertion process. A 23 kDa exogenous protein was observed on the SDS-PAGE. Specificity and concentration of produced mouse recombinant IL-2 protein were confirmed by western blotting and BCA methods. Conclusion: Recombinant IL-2 was produced in BL21 and pET-21b (+) expression system at 24∘C in the soluble form.