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

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Abstract

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.

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Authors and Affiliations

  1. Malek Ashtar University of Technology, Tehran, Iran

    Maryam Ghodrati Siahmazgi, Mohammad Ali Nasiri Khalili, Mehdi Zeinoddini & Sirus Khodadadi

  2. Trauma Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran

    Fathollah Ahmadpour

Authors
  1. Maryam Ghodrati Siahmazgi
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  2. Mohammad Ali Nasiri Khalili
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  3. Mehdi Zeinoddini
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  4. Fathollah Ahmadpour
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  5. Sirus Khodadadi
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Correspondence to Mohammad Ali Nasiri Khalili.

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Ghodrati Siahmazgi, M., Nasiri Khalili, M.A., Zeinoddini, M. et al. Purification and Characterization of DT389GCSF Fusion Protein: A Unique Immunotoxin Against the Human Granulocyte-Colony Stimulating Factor Receptor. Int J Pept Res Ther 26, 767–774 (2020). https://doi.org/10.1007/s10989-019-09884-6

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  • DOI: https://doi.org/10.1007/s10989-019-09884-6

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