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

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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References

  1. Allahyari H, Heidari S, Ghamgosha M, Saffarian P, Amani J (2017) Immunotoxin: a new tool for cancer therapy. Tumor Biol 39:1010428317692226

    Article  Google 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 26:523

    CAS  Article  Google Scholar 

  3. Anderlini P, Przepiorka D, Champlin R, Korbling M (1996) Biologic and clinical effects of granulocyte colony-stimulating factor in normal individuals. Blood 88:2819–2825

    CAS  Article  Google 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 401:713

    CAS  Article  Google 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 46:1–5

    Google 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

    Article  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 72:248–254

    CAS  Article  Google Scholar 

  8. Carson DA, Seto S, Wasson DB, Carrera CJ (1986) DNA strand breaks, NAD metabolism, and programmed cell death. Exp Cell Res 164:273–281

    CAS  Article  Google 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 11:249–262

    CAS  Article  Google Scholar 

  10. Chang MP, Baldwin RL, Bruce C, Wisnieski BJ (1989) Second cytotoxic pathway of diphtheria toxin suggested by nuclease activity. Science 246:1165–1168

    CAS  Article  Google Scholar 

  11. Chaudhary VK, FitzGerald DJ, Pastan I (1991) A proper amino terminus of diphtheria toxin is important for cytotoxicity. Biochem Biophys Res Commun 180:545–551

    CAS  Article  Google Scholar 

  12. Chen X, Zaro JL, Shen W-C (2013) Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev 65:1357–1369

    CAS  Article  Google Scholar 

  13. Choe W-S, Nian R, Lai W-B (2006) Recent advances in biomolecular process intensification. Chem Eng Sci 61:886–906

    CAS  Article  Google 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 217–236

  15. Dranoff G (2004) Cytokines in cancer pathogenesis and cancer therapy. Nat Rev Cancer 4:11

    CAS  Article  Google Scholar 

  16. Figgitt DP, Lamb HM, Goa KL (2000) Denileukin diftitox. Am J Clin Dermatol 1:67–72

    CAS  Article  Google Scholar 

  17. Foss FM (2000) DAB389IL-2 (denileukin diftitox, ONTAK): a new fusion protein technology. Clin Lymphoma 1:S27–S31

    Article  Google 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 16:190–201

    CAS  Article  Google 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 8:1004–1013

    CAS  PubMed  Google 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 54:713–733

    CAS  Article  Google Scholar 

  21. George RA, Heringa J (2002) An analysis of protein domain linkers: their classification and role in protein folding. Protein Eng Des Sel 15:871–879

    CAS  Article  Google Scholar 

  22. Guise AD, West SM, Chaudhuri JB (1996) Protein folding in vivo and renaturation of recombinant proteins from inclusion bodies. Mol Biotechnol 6:53–64

    CAS  Article  Google Scholar 

  23. Healthcare G, Healthcare FG (2007) Purifying challenging proteins principles and methods

  24. Kawakami K, Aggarwal BB, Puri RK (2004) Cytotoxins and immunotoxins for cancer therapy. CRC Press, Boca Raton

    Google 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 4:463–468

    CAS  Article  Google Scholar 

  26. Kreitman RJ (2006) Immunotoxins for targeted cancer therapy. AAPS J 8:E532–E551

    CAS  Article  Google Scholar 

  27. Ladokhin AS (2013) pH-triggered conformational switching along the membrane insertion pathway of the diphtheria toxin T-domain. Toxins 5:1362–1380

    CAS  Article  Google 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) 1747:121–131

    CAS  Article  Google Scholar 

  29. Moghaddas M, Zeinoddini M, Saeedinia A, Bayat S (2018) Structural and functional assessment of diphtheria fusion toxin: DT389GCSF. J Bionanosci 12:240–244

    CAS  Article  Google Scholar 

  30. Nakamura L, Wisnieski B (1990) Characterization of the deoxyribonuclease activity of diphtheria toxin. J Biol Chem 265:5237–5241

    CAS  PubMed  Google 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 10:19–26

    Article  Google Scholar 

  32. Pastan I, Hassan R, FitzGerald DJ, Kreitman RJ (2007) Immunotoxin treatment of cancer. Annu Rev Med 58:221–237

    CAS  Article  Google Scholar 

  33. Potala S, Verma RS (2010) Modified DT–IL2 fusion toxin targeting uniquely IL2Rα expressing leukemia cell lines—construction and characterization. J Biotechnol 148:147–155

    CAS  Article  Google Scholar 

  34. Potala S, Sahoo SK, Verma RS (2008) Targeted therapy of cancer using diphtheria toxin-derived immunotoxins. Drug Discov Today 13:807–815

    CAS  Article  Google 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 50:77–87

    Article  Google 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

  37. Singh SM, Panda AK (2005) Solubilization and refolding of bacterial inclusion body proteins. J Biosci Bioeng 99:303–310

    CAS  Article  Google 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 75:1489

    CAS  Article  Google 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 103:3135–3140

    CAS  Article  Google 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 33:123–133

    CAS  Article  Google 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 11:25–38

    Google 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

    Article  Google Scholar 

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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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Keywords

  • Immunotoxin
  • DT389GCSF
  • Refold
  • Cytotoxicity
  • HL-60