Purification and Characterization of DT389GCSF Fusion Protein: A Unique Immunotoxin Against the Human Granulocyte-Colony Stimulating Factor Receptor

  • Maryam Ghodrati Siahmazgi
  • Mohammad Ali Nasiri KhaliliEmail author
  • Mehdi Zeinoddini
  • Fathollah Ahmadpour
  • Sirus Khodadadi


Immunotoxin is a recombinant fusion toxin which has been developed to kill cancer cells selectively. DT389GCSF as a new immunotoxin consists of a truncated diphtheria toxin linked to granulocyte colony stimulating factor (GCSF) via the SerGly4SerMet flexible linker. In this study, DT389GCSF was expressed in inclusion body form in Escherichia coli BL21 (DE3) then it was purified. After refolding, the structure of refolded protein was assessed by Native-PAGE and spectroscopic techniques. In the following, the refolded protein’s nuclease activity and its cytotoxicity toward HL-60 were evaluated. The results showed that the yield of DT389GCSF expression and purification was about 30% and 95% respectively, using CLIQS software. Also structural and functional studies confirmed that DT389GCSF obtained its intact structure and also function. MTT assay revealed that DT389GCSF can induce the death of HL-60 cell line in vitro. IC50 value upon 48 h of exposure of DT389GCSF toward HL-60 was 5.2 × 10−7±0.00011 M. The specific toxicity of DT389GCSF was determined by its disability to kill the K562 cell line. Findings from the present study indicate that DT389GCSF is toxic to human leukemia (HL-60) cells, supporting its use as an effective agent towards the study and treatment of patients with AML.


Immunotoxin DT389GCSF Refold Cytotoxicity HL-60 


Compliance with Ethical Standards

Conflict of interest

The authors declare no conflict of interest, financial or otherwise.

Human and Animal Rights

This article does not contain any studies with human participants or animals performed by any of the authors.


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Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Maryam Ghodrati Siahmazgi
    • 1
  • Mohammad Ali Nasiri Khalili
    • 1
    Email author
  • Mehdi Zeinoddini
    • 1
  • Fathollah Ahmadpour
    • 2
  • Sirus Khodadadi
    • 1
  1. 1.Malek Ashtar University of TechnologyTehranIran
  2. 2.Trauma Research CenterBaqiyatallah University of Medical SciencesTehranIran

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