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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
Article

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.

Keywords

Immunotoxin DT389GCSF Refold Cytotoxicity HL-60 

Notes

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.

References

  1. Allahyari H, Heidari S, Ghamgosha M, Saffarian P, Amani J (2017) Immunotoxin: a new tool for cancer therapy. Tumor Biol 39:1010428317692226CrossRefGoogle Scholar
  2. Amet N, Lee H-F, Shen W-C (2009) Insertion of the designed helical linker led to increased expression of tf-based fusion proteins. Pharm Res 26:523CrossRefGoogle Scholar
  3. Anderlini P, Przepiorka D, Champlin R, Korbling M (1996) Biologic and clinical effects of granulocyte colony-stimulating factor in normal individuals. Blood 88:2819–2825Google Scholar
  4. Aritomi M, Kunishima N, Okamoto T, Kuroki R, Ota Y, Morikawa K (1999) Atomic structure of the GCSF-receptor complex showing a new cytokine-receptor recognition scheme. Nature 401:713CrossRefGoogle Scholar
  5. Babavalian E, Zeinoddini M, Saeedinia A, Mohammadi R, Xodadadi N (2018) Design of a recombinant immunotoxin against the human granulocyte-colony stimulating factor receptor. Mol Biol Rep 46:1–5Google Scholar
  6. Bayat S, Zeinoddini M, Azizi A, Khalili MN (2019) Co-solvents effects on the stability of recombinant immunotoxin denileukin diftitox: structure and function assessment. Iran J Sci Technol Trans A.  https://doi.org/10.1007/s40995-019-00676-7 Google Scholar
  7. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  8. Carson DA, Seto S, Wasson DB, Carrera CJ (1986) DNA strand breaks, NAD metabolism, and programmed cell death. Exp Cell Res 164:273–281CrossRefGoogle Scholar
  9. Chadwick D, Williams D, Niho Y, Murphy J, Minden M (1993) Cytotoxicity of a recombinant diphtheria toxin-granulocyte colony-stimulating factor fusion protein on human leukemic blast cells. Leukemia Lymphoma 11:249–262CrossRefGoogle Scholar
  10. Chang MP, Baldwin RL, Bruce C, Wisnieski BJ (1989) Second cytotoxic pathway of diphtheria toxin suggested by nuclease activity. Science 246:1165–1168CrossRefGoogle Scholar
  11. Chaudhary VK, FitzGerald DJ, Pastan I (1991) A proper amino terminus of diphtheria toxin is important for cytotoxicity. Biochem Biophys Res Commun 180:545–551CrossRefGoogle Scholar
  12. Chen X, Zaro JL, Shen W-C (2013) Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev 65:1357–1369CrossRefGoogle Scholar
  13. Choe W-S, Nian R, Lai W-B (2006) Recent advances in biomolecular process intensification. Chem Eng Sci 61:886–906CrossRefGoogle Scholar
  14. Clark EDB, Schwarz E, Rudolph R (1999) Inhibition of aggregation side reactions during in vitro protein folding. In: Methods in enzymology, Elsevier, pp 217–236Google Scholar
  15. Dranoff G (2004) Cytokines in cancer pathogenesis and cancer therapy. Nat Rev Cancer 4:11CrossRefGoogle Scholar
  16. Figgitt DP, Lamb HM, Goa KL (2000) Denileukin diftitox. Am J Clin Dermatol 1:67–72CrossRefGoogle Scholar
  17. Foss FM (2000) DAB389IL-2 (denileukin diftitox, ONTAK): a new fusion protein technology. Clin Lymphoma 1:S27–S31CrossRefGoogle Scholar
  18. Frankel AE, Ramage J, Latimer A, Feely T, Delatte S, Hall P, Tagge E, Kreitman R, Willingham M (1999) High-level expression and purification of the recombinant diphtheria fusion toxin DTGM for PHASE I clinical trials. Protein Expr Purif 16:190–201CrossRefGoogle Scholar
  19. Frankel AE, Powell BL, Hall PD, Case LD, Kreitman RJ (2002) Phase I trial of a novel diphtheria toxin/granulocyte macrophage colony-stimulating factor fusion protein (DT388GMCSF) for refractory or relapsed acute myeloid leukemia. Clin Cancer Res 8:1004–1013Google Scholar
  20. Gallagher R, Collins S, Trujillo J, McCredie K, Ahearn M, Tsai S, Metzgar R, Aulakh G, Ting R, Ruscetti F (1979) Characterization of the continuous, differentiating myeloid cell line (HL-60) from a patient with acute promyelocytic leukemia. Blood 54:713–733Google Scholar
  21. George RA, Heringa J (2002) An analysis of protein domain linkers: their classification and role in protein folding. Protein Eng Des Sel 15:871–879CrossRefGoogle Scholar
  22. Guise AD, West SM, Chaudhuri JB (1996) Protein folding in vivo and renaturation of recombinant proteins from inclusion bodies. Mol Biotechnol 6:53–64CrossRefGoogle Scholar
  23. Healthcare G, Healthcare FG (2007) Purifying challenging proteins principles and methodsGoogle Scholar
  24. Kawakami K, Aggarwal BB, Puri RK (2004) Cytotoxins and immunotoxins for cancer therapy. CRC Press, Boca RatonCrossRefGoogle Scholar
  25. Kiyokawa T, Williams DP, Snider CE, Strom TB, Murphy JR (1991) Protein engineering of diphtheria-toxin-related interleukin-2 fusion toxins to increase cytotoxic potency for high-affinity IL-2-receptor-bearing target cells. Protein Eng Des Sel 4:463–468CrossRefGoogle Scholar
  26. Kreitman RJ (2006) Immunotoxins for targeted cancer therapy. AAPS J 8:E532–E551CrossRefGoogle Scholar
  27. Ladokhin AS (2013) pH-triggered conformational switching along the membrane insertion pathway of the diphtheria toxin T-domain. Toxins 5:1362–1380CrossRefGoogle Scholar
  28. Lee JW, Nakamura LT, Chang MP, Wisnieski BJ (2005) Mechanistic aspects of the deoxyribonuclease activity of diphtheria toxin. Biochim et Biophys Acta (BBA) 1747:121–131CrossRefGoogle Scholar
  29. Moghaddas M, Zeinoddini M, Saeedinia A, Bayat S (2018) Structural and functional assessment of diphtheria fusion toxin: DT389GCSF. J Bionanosci 12:240–244CrossRefGoogle Scholar
  30. Nakamura L, Wisnieski B (1990) Characterization of the deoxyribonuclease activity of diphtheria toxin. J Biol Chem 265:5237–5241Google Scholar
  31. Ninci E, Brandstetter T, Meinhold-Heerlein I, Bettendorf H, Sellin D, Bauknecht T (2000) G-CSF receptor expression in ovarian cancer. Int J Gynecol Cancer 10:19–26CrossRefGoogle Scholar
  32. Pastan I, Hassan R, FitzGerald DJ, Kreitman RJ (2007) Immunotoxin treatment of cancer. Annu Rev Med 58:221–237CrossRefGoogle Scholar
  33. Potala S, Verma RS (2010) Modified DT–IL2 fusion toxin targeting uniquely IL2Rα expressing leukemia cell lines—construction and characterization. J Biotechnol 148:147–155CrossRefGoogle Scholar
  34. Potala S, Sahoo SK, Verma RS (2008) Targeted therapy of cancer using diphtheria toxin-derived immunotoxins. Drug Discov Today 13:807–815CrossRefGoogle Scholar
  35. Rao DVK, Narasu ML, Rao AKSB (2008) A purification method for improving the process yield and quality of recombinant human granulocyte colony-stimulating factor expressed in Escherichia coli and its characterization. Biotechnol Appl Biochem 50:77–87CrossRefGoogle Scholar
  36. Siahmazgi, M., M. Khalili, F. Ahmadpour, S. Khodadadi & M. Zeinoddini (2018) In silico design of fusion toxin DT389GCSF and comparison of interaction it with GCSF receptor rather than DT486GCSF. Current Computer-Aided Drug Design Google Scholar
  37. Singh SM, Panda AK (2005) Solubilization and refolding of bacterial inclusion body proteins. J Biosci Bioeng 99:303–310CrossRefGoogle Scholar
  38. Tachibana M, Miyakawa A, Uchida A, Murai M, Eguchi K, Nakamura K, Kubo A, Hata J (1997) Granulocyte colony-stimulating factor receptor expression on human transitional cell carcinoma of the bladder. Br J Cancer 75:1489CrossRefGoogle Scholar
  39. Tamada T, Honjo E, Maeda Y, Okamoto T, Ishibashi M, Tokunaga M, Kuroki R (2006) Homodimeric cross-over structure of the human granulocyte colony-stimulating factor (GCSF) receptor signaling complex. Proc Natl Acad Sci USA 103:3135–3140CrossRefGoogle Scholar
  40. Urieto JO, Liu T, Black JH, Cohen KA, Hall PD, Willingham MC, Pennell LK, Hogge DE, Kreitman RJ, Frankel AE (2004) Expression and purification of the recombinant diphtheria fusion toxin DT388IL3 for phase I clinical trials. Protein Expr Purif 33:123–133CrossRefGoogle Scholar
  41. Weidle UH, Tiefenthaler G, Schiller C, Weiss EH, Georges G, Brinkmann U (2014) Prospects of bacterial and plant protein-based immunotoxins for treatment of cancer. Cancer Genom Proteom 11:25–38Google Scholar
  42. Zarkar N, Khalili M, Ahmadpour F, Khodadadi S, Zeinoddini M (2019) In silico and in vitro evaluation of deamidation effects on the stability of the fusion toxin DAB389IL-2. Curr Proteom.  https://doi.org/10.2174/1570164616666190131150033 Google Scholar

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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