Repository of Research and Investigative Information

Repository of Research and Investigative Information

Baqiyatallah University of Medical Sciences

Purification and Characterization of DT(389)GCSF Fusion Protein: A Unique Immunotoxin Against the Human Granulocyte-Colony Stimulating Factor Receptor

(2020) Purification and Characterization of DT(389)GCSF Fusion Protein: A Unique Immunotoxin Against the Human Granulocyte-Colony Stimulating Factor Receptor. International Journal of Peptide Research and Therapeutics. pp. 767-774. ISSN 1573-3149

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Abstract

Immunotoxin is a recombinant fusion toxin which has been developed to kill cancer cells selectively. DT(389)GCSF 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, DT(389)GCSF 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 DT(389)GCSF expression and purification was about 30 and 95 respectively, using CLIQS software. Also structural and functional studies confirmed that DT(389)GCSF obtained its intact structure and also function. MTT assay revealed that DT(389)GCSF can induce the death of HL-60 cell line in vitro. IC50 value upon 48 h of exposure of DT(389)GCSF toward HL-60 was 5.2 x 10(-7)+/- 0.00011 M. The specific toxicity of DT(389)GCSF was determined by its disability to kill the K562 cell line. Findings from the present study indicate that DT(389)GCSF is toxic to human leukemia (HL-60) cells, supporting its use as an effective agent towards the study and treatment of patients with AML.

Item Type: Article
Keywords: Immunotoxin DT(389)GCSF Refold Cytotoxicity HL-60 diphtheria-toxin deoxyribonuclease activity expression stability insertion bacterial linkers pathway Biochemistry & Molecular Biology
Page Range: pp. 767-774
Journal or Publication Title: International Journal of Peptide Research and Therapeutics
Journal Index: ISI
Volume: 26
Number: 2
Identification Number: https://doi.org/10.1007/s10989-019-09884-6
ISSN: 1573-3149
Depositing User: مهندس مهدی شریفی
URI: http://eprints.bmsu.ac.ir/id/eprint/8804

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